WO2009088022A1 - Novel cancer marker, and diagnosis using the same - Google Patents

Abstract

Disclosed is a test method for diagnosing pancreatic cancer in a subject, which is characterized by measuring the quantity of at least one peptide selected from the group consisting of peptides comprising the amino acid sequences depicted in SEQ ID NOs:1, 2 and 3, respectively, in a biological sample (preferably a body fluid, more preferably a serum, a plasma or the like) collected from the subject (preferably by mass spectrometry or by using an antibody capable of specifically recognizing the peptide).

Description

Novel cancer marker and diagnosis using it

The present invention relates to a novel diagnostic marker for various cancers including pancreatic cancer, and a diagnostic method for the disease using the same.

Pancreatic cancer has the lowest 5-year survival rate among common solid cancers, with approximately 20,000 and 30,000 deaths each year in Japan and the United States due to pancreatic cancer. Early detection is essential because pancreatic cancer is characterized by massive local invasion and early metastasis to the liver and lymph nodes, and surgical resection is the only credible cure. However, as often described as "silent organs", except for obstructive jaundice, the clinical symptoms of pancreatic cancer often do not become noticeable until the stage progresses, and the pancreas is located in the back of the abdomen Therefore, it is difficult to find pancreatic cancer with simple and inexpensive inspection means such as echo. As a result, only 20-40% of pancreatic cancer patients are surgically indicated.
Therefore, development of a new test method that is inexpensive, simple, highly sensitive, and highly specific that enables early detection of pancreatic cancer is eagerly desired.

With the progress of proteomics research that comprehensively analyzes protein expression in vivo, search for new biomarkers using proteomics has been energetically performed. For example, Honda et al. (Non-Patent Document 1) analyzed a plasma sample of a pancreatic cancer patient using SELDI-TOFMS technology, and found four mass peaks (m / z7668,766) with significantly different peak intensities compared to healthy individuals. , 17,272, 28,080, 14,779) have been identified and reported as pancreatic cancer markers. However, it is unclear what protein these mass peaks originate from.

When thrombin acts on fibrinogen, it first hydrolyzes between the 16th Arg and the 17th Gly from the N-terminus of the Aα chain to cut out fibrinopeptide A (FPA). Next, fibrinopeptide B (FPB) is cut out by cutting between the 14th Arg and 15th Gly from the N-terminus of the Bβ chain. The function of these excised fibrinopeptides is not well understood. Recently, mass spectrometry was used to examine proteolytic product patterns in the serum of several cancer patients, and FPA or fragments thereof have been reported to vary in prostate cancer, bladder cancer, and breast cancer (non-patented) Reference 2). However, there is no report on the association between FPA or a fragment thereof and pancreatic cancer.

On the other hand, the fibrin monomer produced by cutting out FPA and FPB plays a main role in blood coagulation by polymerizing and insolubilizing. The existence of a C-terminal fragment of fibrin α (positions 496-629 of the fibrinogen Aα chain precursor polypeptide) has been reported, but its physiological significance has not been elucidated at all.

Apolipoprotein E (apoE) is known as a causative gene for familial type III hyperlipidemia (E2) and a risk factor for Alzheimer's disease (E4). The relationship between apoE or its fragments and pancreatic cancer Is not reported at all.
Honda et al., "Cancer Res.", Volume 65 (No. 22), pp. 10613-10622 (2005) Villanueva et al., "The Journal of Clinical Investigation", Volume 116 (No. 1), pp. 271-284 (2006)

An object of the present invention is to provide a novel diagnostic marker for pancreatic cancer and an effective diagnostic method for cancer using the same.

In order to achieve the above object, the present inventors examined serum collected from various cancer patients by mass spectrometry, and as a result, in pancreatic cancer patients, there was a marked increase compared to healthy individuals and cancer patients in other organs. Peptides having molecular weights of about 1616, about 2410 and about 2930 were found. As a result of analyzing the amino acid sequence, these peptides were found to be a peptide in which the Ser residue at position 3 of FPA was phosphorylated, a peptide consisting of a partial amino acid sequence at positions 170-192 of the apoE protein, and one of the fibrin α chain C-terminal fragment. It was found to be a peptide consisting of the amino acid sequence of the part (corresponding to positions 571-601 of the fibrinogen Aα chain precursor polypeptide). Moreover, as a result of measuring and comparing the peak intensities of these peptides for each tissue type for pancreatic cancer patients, these peptides were detected with a significant difference from those in healthy subjects even in the earliest cancer in the epithelium, and derived from fibrinogen The two peptides showed higher levels of histopathological microinvasion more clearly in stage II of the disease classification that could not be diagnosed by imaging (MRI, CT, etc.).
Based on these findings, the present inventors have identified the peptide as an early diagnostic marker for pancreatic cancer, and have completed the present invention.

That is, the present invention
[1] In the subject, the amount of one or more peptides selected from the peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 in a biological sample collected from the subject is measured. Testing method for diagnosis of pancreatic cancer;
[2] The method according to [1] above, wherein the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.
[3] The method according to [1] or [2] above, wherein the biological sample is a body fluid;
[4] The body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid [3] ] The method according to
[5] The method according to any one of [1] to [4] above, comprising subjecting the biological sample to mass spectrometry;
[6] One or more antibodies selected from the group of antibodies specifically recognizing each peptide consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 are used. ] The method in any one of
[7] Collecting a biological sample from a patient in time series, and examining the temporal change in the amount of one or more peptides selected from the peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 in the sample The method according to any one of [1] to [6] above, characterized by:
[8] A method for evaluating a therapeutic effect in a patient with pancreatic cancer, comprising a peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 in a biological sample collected from the patient before and after the treatment. Examining a change in the amount of one or more selected peptides;
[9] The method according to [8] above, wherein the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.
[10] a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2; and [11] a peptide consisting of the amino acid sequence shown in SEQ ID NO: 3;
Etc.

According to the present invention, pancreatic cancer can be determined quickly and accurately, so that early detection and early treatment of the disease are possible. In addition, cancers in other organs can be diagnosed by appropriately combining the marker peptides of the present invention.

The present invention provides three new and useful diagnostic marker peptides for pancreatic cancer (hereinafter, the three peptides may be collectively referred to as “the peptide of the present invention”).

The first peptide of the present invention (also referred to as “peptide 1”) consists of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence corresponds to a fragment from the N-terminal Ala residue to the 16th Arg residue of the mature fibrinogen Aα chain. Peptide 1 is characterized in that the third Ser residue in the amino acid sequence is phosphorylated.
As described above, it is well known that fibrinopeptide A (FPA) is produced by cleaving between the 16th Arg residue and the 17th Gly residue from the N-terminus of fibrinogen Aα chain by thrombin. In addition, it has already been reported that the serum level of the peptide significantly decreases in the serum of prostate cancer, bladder cancer and breast cancer patients (Non-patent Document 2). However, it has not been known so far that peptide 1 which is a phosphorylated FPA exhibits a very high value in the serum of pancreatic cancer patients, as opposed to the above cancer.
In addition, although the serum level of peptide 1 is not as prominent as in pancreatic cancer patients, it is significantly increased in lung cancer, hematopoietic tumors, and gallbladder / bile duct cancer patients as compared with healthy individuals. On the other hand, in the same way as FPA (unphosphorylated), this peptide has a significantly lower serum level in prostate cancer and breast cancer patients than in healthy individuals, as well as endometrial cancer, colorectal cancer, esophagus. It is also significantly reduced in patients with cancer, cervical cancer and gastric cancer. Therefore, peptide 1 can also be used as a diagnostic marker for these cancers.

The second peptide of the present invention (also referred to as “peptide 2”) consists of the amino acid sequence shown in SEQ ID NO: 2. The amino acid sequence corresponds to a fragment from the 170th Ala residue to the 192nd Leu residue from the N-terminus of apolipoprotein E (apoE).
The serum level of peptide 2 is significantly higher not only in patients with pancreatic cancer but also in patients with gallbladder / bile duct cancer as compared with cancer patients in healthy subjects and other organs. In addition, although not as remarkable as pancreatic cancer and gallbladder / bile duct cancer patients, serum levels are significantly increased in ovarian cancer, esophageal cancer, and hematopoietic tumor patients as compared with healthy individuals. Therefore, peptide 2 can also be used as a diagnostic marker for these cancers.

The third peptide of the present invention (also referred to as “peptide 3”) consists of the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence corresponds to a fragment from the 571th Ser residue to the 601st Tyr residue from the N-terminus of the fibrinogen Aα chain precursor polypeptide.
Serum levels of peptide 3 are not as prominent as in pancreatic cancer patients, but cervical cancer, colorectal cancer, ovarian cancer, esophageal cancer, gallbladder / bile duct cancer, prostate cancer, endometrial cancer, stomach cancer, breast cancer and lung cancer In patients, it is significantly reduced compared to healthy individuals. Therefore, peptide 3 can also be used as a diagnostic marker for these cancers.

As described above, peptides 1 and 3 are fibrinogen Aα chain fragments (degradation products), and peptide 2 is an apoE protein fragment. Therefore, the patient to be tested has a polymorphism or substitution comprising one or more amino acid substitutions, deletions, insertions or additions or combinations thereof within the partial amino acid sequence corresponding to these peptides of fibrinogen Aα or apoE protein. In the case of having an allelic mutation, the amino acid sequence of the peptide to be detected should be understood as “the amino acid sequence having the polymorphism or allelic mutation in each of the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3.” It will be obvious to those skilled in the art.

The invention also provides a test for the diagnosis of pancreatic cancer in a patient by measuring the amount of one or more peptides of the invention in a biological sample from a patient suspected of suffering from pancreatic cancer. Provide a method. Here, “test for diagnosis” means measurement of the amount of the peptide and, if necessary, comparison with the measured value in a control sample. The “patient suspected of having pancreatic cancer” may be subjectively suspected by the patient or based on some objective basis, but is preferably publicly known As a result of clinical examinations and / or examinations, patients who have been judged by a doctor to have a reasonable possibility of those diseases, or humans with an equivalent condition.
In the treatment of pancreatic cancer, early detection and early treatment are the major principles, but the peptide of the present invention is an intraepithelial cancer that is positioned at stage 0, which is the earliest of all six stages of disease classification (UICC, 1997). Even (Tis) is detected with a significant difference from that of a healthy person, and by adopting a periodic diagnosis using the peptide of the present invention as an index, there is a possibility that it can contribute to early detection and early treatment of pancreatic cancer.

The patient-derived biological sample to be the test sample is not particularly limited, but is preferably one that is less invasive to the patient, for example, blood, plasma, serum, saliva, urine, tears that can be easily collected from the living body And those collected relatively easily such as cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, aqueous humor, and vitreous humor.
When serum or plasma is used, it can be prepared by collecting blood from a patient according to a conventional method and separating the liquid component. The peptide of the present invention as a detection target can be separated and removed in advance using a spin column or the like, if necessary, such as a high molecular weight protein fraction.

The detection of the peptide of the present invention in a biological sample can be performed, for example, by measuring various molecular weights of the biological sample, for example, gel electrophoresis or various separation and purification methods (eg, ion exchange chromatography, hydrophobic chromatography, affinity). Chromatography, reverse phase chromatography, etc.), ionization methods (eg, electron impact ionization, field desorption, secondary ionization, fast atom bombardment, matrix-assisted laser desorption ionization (MALDI), electrospray ionization Etc.), mass spectrometers (eg double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass spectrometer, ion cyclotron mass spectrometer, etc.) Detect bands, spots or peaks that match the molecular weight of the peptide Can be performed by the, not limited thereto. Since the amino acid sequence of the peptide of the present invention is known, a method of preparing an antibody that recognizes the amino acid sequence and detecting the peptide by Western blotting or various immunoassays can be used more preferably. Furthermore, the hybrid detection method of the above method is also effective.

Peptides 1, 2 and 3 of the present invention have molecular weights (calculated values) of 1615.65, 2409.69 and 2930.28, respectively, but it goes without saying that the actual values may vary slightly depending on the measurement method / measurement equipment used. Absent. For example, in the case of a method using a mass spectrometer, it is preferable to measure the peak intensity appearing at the position of the calculated value ± 0.5% (preferably ± 0.3%, more preferably ± 0.1%).

One of the particularly preferable measurement methods in the inspection method of the present invention is that a test sample is brought into contact with the surface of a plate used for time-of-flight mass spectrometry, and the mass of a component captured on the plate surface is measured using a time-of-flight mass spectrometer. The method of measuring by is mentioned.
The plate that can be adapted to the time-of-flight mass spectrometer may be any plate as long as it has a surface structure that can efficiently adsorb the peptide of the present invention to be detected. Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicon, a wide range of gels or polymers (eg (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof). Examples of surface structures having a plurality of monomer or polymer sequences include, for example, linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having α-, β- or ω-amino acids, gel surfaces used in chromatography Carriers (anionic / cationic compounds, hydrophobic compounds composed of carbon chains 1-18, hydrophilic compounds (eg, carriers cross-linked with silica, nitrocellulose, cellulose acetate, agarose, etc.), artificial homopolymers) (For example, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, etc.) Any known drug or natural compound is bound to any of the above compounds (covalent and non-covalent bonds). ) Coated with heteropolymer etc. Packaging and the like.

In a preferred embodiment, the support used as a plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF [usually a plate for mass spectrometry For example, insulators (glass, ceramics, plastics, resins, etc.), metals (aluminum, stainless steel, etc.), conductive polymers, composites thereof, etc. Although an aluminum plate is preferably used, refer to WO 2004/031759). The shape of the support can be appropriately devised into a shape suitable for the sample analyzer, in particular, the sample introduction port, but is not limited thereto. As such a plate for mass spectrometry coated with a thin layer with PVDF, a blot chip (registered trademark) manufactured by Protocera is preferably used.

Preferably, the coating refers to a thin layer formed by depositing on a support in a state where coating molecules are dispersed, instead of overlaying a pre-formed structure like a membrane on the support. The manner in which the coating molecules are deposited is not particularly limited, but means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
The thickness of the thin layer can be appropriately selected within a range that does not adversely affect the transfer efficiency of the molecules contained in the tissue or cells and the measurement sensitivity of mass spectrometry, etc., for example, about 0.001 to about 100 μm, Preferably, it is about 0.01 to about 30 μm.

Although the plate for mass spectrometry (support) can be prepared by a method known per se, for example, the above preferred mass spectrometry plate is prepared by thinly coating the surface of the support with a coating molecule such as PVDF. The Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
In the case of “application”, the coating molecule is dissolved in an appropriate solvent, for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (for example, about 1 to about 100 mg / mL) ( The coating molecule-containing solution) can be applied to the substrate using a suitable tool such as a brush.
In the case of “spraying”, the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.
In the case of “evaporation”, the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the substrate using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer of molecules can be formed.
In the case of “immersing”, the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.
In the case of “printing”, various printing techniques that can be normally used depending on the material of the substrate can be appropriately selected and used. For example, screen printing or the like is preferably used.
In the case of “sputtering”, for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas is caused to collide with the molecule. The repelled coating molecules can be deposited on the substrate to form a thin layer.
The coating may be applied to the entire surface of the substrate, or may be applied only to the surface (fraction) subjected to mass spectrometry.

The coating molecule can be used in a suitable form depending on the coating means. For example, the coating molecule can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form. “Apply” refers to bringing a coating molecule into contact with the support so that the coating molecules remain and deposit on the support after contact. The amount of application is not particularly limited, and examples of the coating molecular weight include about 10 to about 100,000 μg / cm ² , preferably about 50 to about 5,000 μg / cm ² . After the application, the solvent is removed by natural drying, vacuum drying or the like.

The surface of the substrate in the mass spectrometry plate may be modified (processed) in advance by an appropriate physical or chemical technique before coating with a coating molecule. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.

Transfer of the test sample to the mass spectrometric plate (support) can be performed by leaving the patient-derived biological sample as the test sample untreated or after removing and concentrating the high molecular weight protein using an antibody column or other method. -It is performed by subjecting to polyacrylamide gel electrophoresis or isoelectric focusing, and transferring (blotting) the gel after contact with the plate. A known transfer device can be used. The transfer method itself is known. Preferably electrotransfer is used. The sample developed on the gel after the electrophoresis is transferred to the plate for mass spectrometry by various methods (diffusion, electric force, etc.). As a buffer used for electrotransfer, it is preferable to use a buffer having a pH of 7 to 9 and a low salt concentration. Specific examples include Tris buffer, phosphate buffer, borate buffer, and acetate buffer. Tris / Glycine / Methanol buffer, SDS-Tris-Tricine buffer, etc. as Tris buffer, ACN / NaCl / Isotonic phosphate buffer, Sodium phosphate / ACN, Boric acid, etc. as phosphate buffer Examples of the buffer include sodium borate-hydrochloric acid buffer, tris-borate / EDTA, borate / ACN, and the like. Examples of the acetate buffer include tris-acetate / EDTA. Preferred are tris / glycine / methanol buffer and sodium borate-hydrochloric acid buffer. Examples of the composition of the tris / glycine / methanol buffer include Tris 10-15 μmM, glycine 70-120 μmM, and methanol 7-13%. An example of the composition of the sodium borate-hydrochloric acid buffer is about 5 to 20 mM sodium borate.

Thereby, the molecules present in the test sample including the target molecules are efficiently captured on the surface of the support. After the plate is dried, a reagent called matrix is added to absorb the laser light and promote ionization of analyte molecules through energy transfer, which is advantageous for later mass spectrometry (when using MALDI method) You can also. As the matrix, those known in mass spectrometry can be used. For example, sinapinic acid (SPA (= 3,5-dimethoxy-4-hydoroxycinammic acid)), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic acid (DHB) ), Α-cyano-4-hydroxycinammic acid (CHCA), and the like, but is not limited thereto. Preferably, it is DHB or CHCA.

The presence and amount of the peptide of the present invention, which is a target molecule, can be identified from the information on the molecular weight by mass spectrometry of molecules in the test sample captured on the support surface by the above method.
The mass spectrometer measures the molecular weight of a substance by ionizing a gaseous sample and then putting the molecule or molecular fragment into an electromagnetic field, separating it by mass number / charge number from its movement, and obtaining the spectrum of the substance. -It is a device to detect. Matrix-assisted laser deionization (MALDI), in which a sample and a matrix that absorbs laser light are mixed, dried and crystallized, and ionized analytes are brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation. And MALDI-TOFMS method, which uses time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration. Principles of ionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solutions are electrically sprayed into the atmosphere and individual analyte multivalent ions are unfolded into the gas phase A mass spectrometer based on can be used.
A method for mass spectrometry of molecules on a plate for mass spectrometry is known per se. For example, the method described in WO 2004/031759 can be appropriately modified and used as necessary.

From the result of mass spectrometry, the presence or absence and the amount of the target molecule in the test sample can be identified based on the molecular weight information of the target molecule. In this step, it is also possible to output information from the mass spectrometer as differential information by comparing it with mass spectrometry data in a biological sample derived from a healthy person using an arbitrary program. It will be appreciated that such programs are well known and those skilled in the art can easily construct or modify such programs using known information processing techniques.

In a particularly preferred embodiment, each of the above steps is performed using a blot chip manufactured by Protocera as a plate for mass spectrometry, and the peptides of the present invention are quantitatively compared (differential analysis) using a MALDI mass spectrometer. Furthermore, if necessary, the peptides remaining on the same chip can be identified. Alternatively, up to quantitative comparison (differential analysis) of test samples is performed using a blot chip system manufactured by Protocera, and the peptide is identified by a combination device (LC-MS / LC / MS / MS / MS). MS).

The measurement of the peptide of the present invention in the test method of the present invention can also be performed using an antibody against it. In such a method, if an optimized immunoassay system is constructed and a kit is made, the peptide can be detected with high sensitivity and high accuracy without using a special device such as the mass spectrometer. It is particularly useful in that it can.

The antibody against the peptide of the present invention can be prepared, for example, by isolating and purifying the peptide of the present invention from a biological sample derived from a patient expressing the peptide and immunizing an animal using the peptide as an antigen. Alternatively, when the amount of the obtained peptide is small, the peptide is partially digested with peptidase or the like, the amino acid sequence of the obtained fragment is determined by the Edman method or the like, and the nucleic acid encoding the peptide based on the sequence A hybridizable oligonucleotide is synthesized and a cDNA library derived from the patient is used as a template to obtain a cDNA encoding a protein containing the peptide, or a cDNA derived from the patient is used as a primer. By performing RT-PCR using RNA as a template, a cDNA fragment encoding the peptide is obtained, the cDNA fragment is incorporated into an appropriate expression vector, introduced into an appropriate host cell, and the resulting transformant is cultured. Thus, the peptide of the present invention can be prepared in large quantities by collecting the recombinant peptide. You can. Alternatively, the peptide of the present invention can be obtained by using a cell-free transcription / translation system using the cDNA obtained as described above as a template. Further, it can be prepared in a large amount by an organic synthesis method.

As described above, the peptides 1, 2 and 3 of the present invention are peptides consisting of the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively. Therefore, the antibody against the peptide of the present invention can be synthesized, for example, by synthesizing all or part of the amino acid sequence using a known peptide synthesis method based on the amino acid sequence information, or by fibrinogen Aα isolated by a conventional method. It is desirable to cleave the chain or apoE protein with an appropriate peptidase or the like to obtain a peptide fragment containing all or part of the sequence of the peptide of the present invention, and prepare this as an immunogen.

The antibody against the marker peptide of the present invention (hereinafter sometimes referred to as “the antibody of the present invention”) may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a known immunological technique. The antibody includes not only a complete antibody molecule but also a fragment thereof, and examples thereof include Fab, F (ab ′) 2, ScFv, and minibody.

For example, the polyclonal antibody is cross-linked to a carrier protein such as bovine serum albumin or KLH (Keyhole Limpet Hemocyanin) prepared by any of the above methods or other methods, or a partial peptide thereof. Can be administered as an antigen together with a commercially available adjuvant (for example, complete or incomplete Freund's adjuvant) subcutaneously or intraperitoneally in animals every 2-3 weeks (partial) The antibody titer of the collected serum is measured by a known antigen-antibody reaction, and the increase is confirmed), and it can be obtained by collecting the whole blood about 3 to about 10 days after the final immunization and purifying the antiserum. . Examples of animals to which the antigen is administered include mammals such as rats, mice, rabbits, goats, guinea pigs, and hamsters.

Monoclonal antibodies can be obtained by cell fusion methods (for example, Takeshi Watanabe, Principles of Cell Fusion Methods and Production of Monoclonal Antibodies, Akira Taniuchi, Toshitada Takahashi, “Monoclonal Antibodies and Cancer: Basic and Clinical”, 2-14). Page, Science Forum Publishing, 1985). For example, the peptide of the present invention or a partial peptide thereof is administered to a mouse subcutaneously or intraperitoneally 2-4 times together with a commercially available adjuvant, and the spleen or lymph node is collected about 3 days after the final administration, and white blood cells are collected. The leukocytes and myeloma cells (for example, NS-1, P3X63Ag8) are cell-fused to obtain a hybridoma that produces a monoclonal antibody against the peptide. The cell fusion may be PEG method [J. Immunol. Methods, 81 (2): 223-228 (1985)] or voltage pulse method [Hybridoma, 7 (6): 627-633 (1988)]. A hybridoma producing a desired monoclonal antibody can be selected by detecting an antibody that specifically binds to an antigen from the culture supernatant using a known EIA or RIA method or the like. The culture of the hybridoma producing the monoclonal antibody can be performed in vitro or in vivo such as mouse or rat, or preferably mouse ascites, and the antibody can be obtained from the culture supernatant of the hybridoma and the ascites of the animal, respectively. .

As the N-terminal fragment of fibrinogen Aα chain, in addition to peptide 1 of the present invention, non-phosphorylated FPA and fragments thereof may also be present in the test sample. These peptide groups do not show significant variation in pancreatic cancer, but any of these may be significantly higher in other diseases. If the antibody against peptide 1 of the present invention has cross-reactivity with one or more other N-terminal fragments of fibrinogen Aα chain, the possibility of misdiagnosing other diseases as pancreatic cancer (false positive) is increased. Therefore, the antibody against peptide 1 of the present invention used in the test method of the present invention is preferably a highly specific antibody that does not cross-react with the N-terminal fragment of fibrinogen Aα chain other than the peptide. Such an antibody can be obtained by reacting a plurality of monoclonal antibodies obtained as described above with the other fragments and selecting an antibody that does not cross-react with them. In particular, since peptide 1 of the present invention is phosphorylated FPA, it is desirable that an antibody against peptide 1 is capable of specifically recognizing the phosphorylated site.

The test method of the present invention using the antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the test sample is determined by chemical or physical means. Any measurement method may be used as long as it is a measurement method that is detected and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used.

As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.

For insolubilization of the antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize and immobilize proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.

In the sandwich method, a test sample is reacted with an insolubilized antibody of the present invention (primary reaction), and another labeled antibody of the present invention is reacted (secondary reaction), followed by a labeling agent on an insolubilized carrier. By measuring the amount (activity), the amount of the peptide of the present invention in the test sample can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times.

The monoclonal antibody against the polypeptide of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competition method, the antigen in the test sample and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated ( B / F separation), measure the amount of labeled B or F, and quantify the amount of antigen in the test sample. In this reaction method, a soluble antibody is used as an antibody, a B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or The first antibody is soluble, and the second antibody is a solid phase method using a solid phase antibody.
In the immunometric method, the antigen of the test sample and the immobilized antigen are competitively reacted with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen and excess in the test sample are separated. Then, the solid phase antigen is added, and then the solid phase antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test sample.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test sample is small and only a small amount of precipitate is obtained.

In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the peptide of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (2nd edition) (Medical School, published in 1982), "Enzyme Immunoassay" edited by Eiji Ishikawa et al. (3rd edition) (Medical School, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid. Vol. 74 (Immunochemical Techniques (Part C)), ibid. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (Above, published by Academic Press) It is possible to irradiation.

Since the peptides of the present invention are composed of proteolytic products, various molecules such as undegraded proteins and similar peptides with a common cleavage site may affect the measured value in the usual “sandwich ELISA system”. Therefore, in the first step, a biological sample is immunoaffinity purified with an antibody, the fraction bound to the antibody is subjected to mass spectrometry in the second step, and so-called immunomass spectrometry, in which identification and quantification are performed based on a precise molecular weight. (For example, see Rapid Commun. Mass Spectrom. 2007, 21: 352-358). For example, when a blood sample is used as a biological sample, a biomarker peak is not observed even if the sample is directly measured with a MALDI mass spectrometer. However, according to immunomass spectrometry, undegraded protein is also a similar peptide. Is completely separated by a mass spectrometer, and can be quantified with high specificity and sensitivity based on the exact molecular weight of the biomarker.

Alternatively, as another test method of the present invention using the antibody of the present invention, the antibody is immobilized on the surface of a probe that can be adapted to a mass spectrometer as described above, and a test sample is applied to the antibody on the probe. Examples include a method of detecting a peak corresponding to the molecular weight of a marker peptide recognized by the antibody by subjecting the biological sample component captured by the antibody to mass spectrometry.

When the level of peptide 1 in a sample derived from a subject measured by any of the above methods is about 2 times or more, preferably about 5 times or more than the level of peptide 1 in a control sample from a healthy person, The subject can be diagnosed as having a high possibility of having pancreatic cancer. Furthermore, for example, when a sample from a cancer patient in which peptide 1 such as lung cancer shows a relatively high value can be used as a control, the value is statistically significantly higher than the peptide level in the control sample. In addition, it can be diagnosed that the subject is likely to have pancreatic cancer.
In addition, when the level of peptide 1 in a sample derived from a subject is statistically significantly higher than the level of peptide 1 in a control sample derived from a healthy subject, Can be diagnosed as having a high possibility of having lung cancer, hematopoietic tumor or gallbladder / bile duct cancer in addition to pancreatic cancer. Furthermore, if the level of peptide 1 in a sample from a subject is statistically significantly lower than the level of peptide 1 in a control sample from a healthy person, the subject is prostate cancer, It can be diagnosed that there is a high possibility of having breast cancer, endometrial cancer, colorectal cancer, esophageal cancer, cervical cancer or stomach cancer.

When the level of peptide 2 in a sample derived from a subject measured by any of the above methods is about 2 times or more, preferably about 5 times or more than the level of peptide 2 in a control sample from a healthy person, The subject can be diagnosed as having a high possibility of having pancreatic cancer or gallbladder / bile duct cancer. Furthermore, for example, when a sample from a cancer patient in which peptide 2 such as ovarian cancer shows a relatively high value can be used as a control, the value was statistically significantly higher than the peptide level in the control sample. Sometimes it can be diagnosed that the subject is likely to have pancreatic cancer or gallbladder / bile duct cancer. Further, pancreatic cancer can be differentiated from gallbladder / bile duct cancer by combining peptide 1 and / or peptide 3 as a marker.
On the other hand, when the level of peptide 2 in the sample derived from the subject is statistically significantly higher than the level of peptide 2 in the control sample derived from the healthy subject, Can be diagnosed as having a high possibility of having ovarian cancer, esophageal cancer or hematopoietic tumor in addition to pancreatic cancer and gallbladder / bile duct cancer.

When the level of peptide 3 in a sample derived from a subject measured by any of the above methods is statistically significantly higher than the level of peptide 3 in a control sample derived from a healthy subject, The subject can be diagnosed as having a high probability of having pancreatic cancer.
On the other hand, when the level of peptide 3 in the sample derived from the subject is statistically significantly lower than the level of peptide 3 in the control sample derived from the healthy subject, the subject is diagnosed with cervical cancer. It can be diagnosed that there is a high possibility of suffering from colon / rectal cancer, ovarian cancer, esophageal cancer, gallbladder / bile duct cancer, prostate cancer, endometrial cancer, stomach cancer, breast cancer or lung cancer.

The test method of the present invention is preferably carried out by collecting biological samples from patients in time series and examining the time course of the expression of the peptide of the present invention in each sample. The collection interval of the biological sample is not particularly limited, but it is desirable to sample as frequently as possible within a range that does not impair the patient's QOL. For example, when plasma or serum is used as a sample, blood is collected at intervals of about 1 minute to about 12 hours It is preferable to carry out. Among the peptides of the present invention, peptides 1 and 3 tend to increase in serum level as the stage of pancreatic cancer progresses, at least in stage 0-II of the disease classification. On the other hand, Peptide 2 shows a markedly high value at stage 0, and maintains a significantly high value as compared with healthy individuals even when the stage progresses. Therefore, when the level of the peptide of the present invention decreases with time, it can be determined that there is a high possibility that the pathological condition of pancreatic cancer in the patient is improved.

Furthermore, the method for examining pancreatic cancer based on the above time-series sampling is such that when a treatment measure for the disease is taken for a patient who is a subject between the previous sampling and the current sampling, treatment by the measure is performed. It can be used to evaluate the effect. That is, for a sample sampled before and after treatment, when it is determined that the condition after treatment is improved compared to the condition before treatment, it can be evaluated that the treatment is effective. On the other hand, when it is determined that the condition after treatment is not improved or further deteriorated as compared with the condition before treatment, it can be evaluated that the treatment has no effect.

Furthermore, the peptide of the present invention can also provide an active drug discovery target for pancreatic cancer in addition to diagnosis. That is, when the peptide itself has a physiological function in the direction of treatment (remission) of the disease (referred to as “therapeutic peptide”), by administering to the patient a substance that increases the amount or activity of the peptide, When the peptide itself has physiological functions in the direction of exacerbation of the disease (referred to as “exacerbation peptide”), the disease can be treated by administering a substance that reduces the amount or activity of the peptide.

The invention also increases the amount or activity of the peptide when the peptide of the invention acts as a therapeutic peptide, and / or the amount or amount of the peptide when the peptide of the invention acts as an exacerbation peptide. Methods of treating pancreatic cancer by reducing activity are provided. The therapeutic method specifically comprises an effective amount of a substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide, Administration to patients with pancreatic cancer. Accordingly, the present invention also includes a substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and / or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. Provide a therapeutic agent.
Specifically, examples of the substance that increases the activity of the peptide of the present invention as a therapeutic peptide include the peptide itself or a molecule having an agonistic action similar thereto. Alternatively, as a substance that increases the activity of the peptide of the present invention as a therapeutic peptide, a non-neutralizing antibody, preferably an agonist antibody, of the peptide can also be mentioned. On the other hand, examples of the substance that reduces the activity of the peptide of the present invention as an exacerbation peptide include a molecule having an antagonistic action of the peptide, or a neutralizing antibody against the peptide.
The substance that increases the production of the peptide of the present invention as a therapeutic peptide includes a degrading enzyme that releases the peptide from a parent protein (fibrinogen, apoE) existing in the living body, the N-terminal side of the peptide and / or C Further comprising an amino acid sequence that is recognized and cleaved by the decomposing enzyme on the terminal side, a substrate or a substrate analog molecule of the decomposing enzyme, a molecule (including an analogous compound) that promotes the production of the decomposing enzyme, Examples include molecules that promote, molecules that suppress the production of inhibitors of the degrading enzyme, and the like. The presence of a degrading enzyme that liberates the peptide is suggested from the amino acid sequence at the N-terminal side and / or C-terminal side of the peptide, and degradation using the amino acid sequence at the N-terminal side and / or C-terminal side of the peptide as a probe Enzyme search and identification becomes possible. A substrate or substrate analog molecule of a decomposing enzyme thus identified, that is, a peptide molecule further comprising an amino acid sequence recognized and cleaved by the decomposing enzyme on the N-terminal side and / or C-terminal side of the peptide, Since it is cleaved by the degrading enzyme in the body to release the peptide of the present invention or its analog molecule as a therapeutic peptide, the same therapeutic effect can be obtained. On the other hand, substances that promote the production and / or activity of the identified degrading enzymes can also indirectly increase the production of the peptides of the invention as therapeutic peptides. If the target degrading enzyme is identified, these substances can be screened or molecularly designed by a method known per se.
On the other hand, the substance that reduces the production of the peptide of the present invention as an exacerbation peptide includes a molecule that suppresses the production of a degrading enzyme that liberates the peptide from a protein present in the living body, an inhibitor of the decomposing enzyme, and the production of the inhibitor And molecules that promote The degrading enzyme that liberates the peptide of the present invention as an exacerbation peptide can be searched and identified by the same method as the peptide of the present invention as the therapeutic peptide. By using a degrading enzyme thus identified, a substance that suppresses (inhibits) the production or activity of the degrading enzyme directly or indirectly can be screened or molecularly designed by a method known per se.

Substances that increase the amount or activity of the peptides of the invention as therapeutic peptides and substances that reduce the amount or activity of the peptides of the invention as exacerbation peptides can be formulated according to conventional means.
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a method known per se, and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above compound or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-mentioned compound or a salt thereof with an ordinary suppository base.

The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, particularly 5 to 100 mg for injections, Other dosage forms preferably contain 10 to 250 mg of the above compound.
Each of the above-described compositions may be mixed with a substance that increases the amount or activity of the peptide of the present invention as the therapeutic peptide or a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide. Other active ingredients may be contained as long as the action is not caused.

Since the preparation thus obtained is safe and has low toxicity, it can be administered, for example, orally or parenterally to humans.
A substance that increases the amount or activity of the peptide of the present invention as a therapeutic peptide and a substance that decreases the amount or activity of the peptide of the present invention as an exacerbation peptide are determined by its action, administration route, patient severity, Although there are differences depending on age, body weight, drug acceptability, etc., for example, the amount of active ingredient per day for an adult is about 0.0008 to about 25 mg / kg, preferably about 0.008 to about 2 mg / kg, This can be administered once or in several divided doses.

Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the present invention is not limited thereto.

Example 1 Profiling analysis using BlotChip 1.5 μL of serum of various cancer patients and healthy subject serum were mixed with 4.5 μL of sample treatment solution for electrophoresis (NuPAGE (registered trademark) LDS Sample Buffer 4x; Invitrogen) for 10 minutes at 70 ° C. After heat treatment, it was applied to a 4-12% gradient polyacrylamide gel (Invitorogen) and subjected to electrophoresis. After the completion of electrophoresis, the gel was cut out, layered on BLOTCHIP (registered trademark) (Protosera, Inc.), and transferred in an electric transfer buffer (BLOTBuffer ^™ ; Protosera, Inc.) at 90 mA for 120 minutes. After the transfer, rinse the surface of the chip with ultrapure water, apply matrix (α-Cyano-4-hydroxy cinamic acid) to the entire chip, and then apply matrix-assisted laser desorptionionization time-of-flight (MALDI-TOF) mass Mass spectrometry was performed using a spectrometer (Ultra-FlexII manufactured by Bruker Daltonics). The measurement parameters were Detector voltage 1685V, Supression 1000, Laser Intensity 28 to 35 Fuzzy mode, 415 points per chip, 500 times laser irradiation per point, total 207,500 times laser irradiation. Each peak intensity in the obtained spectrum was integrated for each M / z and converted into one integrated spectrum. Differential profiling analysis was performed between patient serum and healthy subject serum using ClinProTools (Bruker Daltonik GmbH). Furthermore, the analysis results thus obtained were collated with the peaks in the actual integrated spectrum.
As a result, in the pancreatic cancer patient group, three types of peptides having molecular weights of about 1616, about 2410, and about 2930 (named as peptides 1 to 3, respectively) whose peak intensities are significantly higher than those in the healthy subjects group. Detected (Tables 1-3).

Next, as a result of measuring and comparing the peak intensity of the peptide for each tissue type for pancreatic cancer patients, the cancer in situ (Tis), which is positioned at stage 0, which is the earliest of all 6 stages of disease classification (UICC) However, the peptide was clearly detected (Table 4). This is an early stage of so-called “invisible cancer”, in which it is impossible to diagnose histopathological microinvasion by imaging (MRI, CT, etc.) even in stage II of disease classification (2 cm or more, with metastasis). It means that a diagnosis can be possible.

Example 2 Identification of peptides by de novo MS / MS analysis on BlotChip For identification, matrix-assisted laser desorption ionization   Using time-of-flight (MALDI-TOF) mass spectrometer (Ultra-FlexII by Bruker Daltonics), Bradykinin, AngiotensinII, AngiotensinI, SubstanceP, Bombesin, Renin Substrate, ACTH Clip {1-17}, ACTH Clip {18 -39}, mass calibration was performed using Somatostatin. After that, profiling is performed in reflectron measurement mode, and selected peptide peaks and their fragment ions are identified by MS / MS analysis through the MASCOT search engine built into Biotools (Bruker Daltonik GmbH) with NCBInr and SwissProt databases. Went.
As a result, peptides 1 to 3 were identified as peptides each having the amino acid sequence shown in SEQ ID NOs: 1 to 3, respectively. As a result of the homology search, peptide 1 was phosphorylated from FPA which is a thrombin degradation product of fibrinogen, peptide 2 was a fragment corresponding to positions 170-192 of apoE protein, peptide 3 was 571 of fibrinogen A α chain precursor polypeptide -It became clear that it was a fragment corresponding to position 601.

The clinical test method using the novel diagnostic marker for pancreatic cancer according to the present invention is useful in that pancreatic cancer can be determined quickly and accurately, so that early detection and early treatment of the disease are possible. In addition, since the peptide of the present invention as a measurement target in the present invention can itself be a drug discovery target in these diseases, it can be used for screening for novel therapeutic agents for pancreatic cancer and for treating the diseases using them. This is extremely useful.
This application is based on Japanese Patent Application No. 2008-000776 filed in Japan, the contents of which are incorporated in full herein.

Claims (11)

1. Pancreatic cancer in the subject, characterized in that the amount of one or more peptides selected from the peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2 and 3 in a biological sample collected from the subject is measured. Inspection method for diagnosis.
2. The method according to claim 1, wherein the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is phosphorylated.
3. The method according to claim 1 or 2, wherein the biological sample is a body fluid.
4. The method according to claim 3, wherein the body fluid is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, pleural effusion, ascites, joint fluid, tear fluid, aqueous humor, vitreous humor and lymph fluid. .
5. The method according to any one of claims 1 to 4, comprising subjecting the biological sample to mass spectrometry.
6. 5. One or more antibodies selected from the group of antibodies specifically recognizing each peptide consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 are used. the method of.
7. A biological sample is collected from a patient in time series, and the change over time in the amount of one or more peptides selected from the peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 in the sample is examined. The method according to any one of claims 1 to 6.
8. A method for evaluating a therapeutic effect in a patient with pancreatic cancer, which is selected from a peptide group consisting of each amino acid sequence shown in SEQ ID NOs: 1, 2, and 3 in a biological sample collected from the patient before and after the treatment. A method characterized by examining the change in the amount of the above peptide.
9. The method according to claim 8, wherein the peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated.
10. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2.
11. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 3.