JP6760562B2 - Information provision method for predicting the prognosis of patients with clear cell ovarian cancer - Google Patents

Description

The present invention relates to a method for providing information for predicting the prognosis of a clear cell ovarian cancer patient by using tissue factor pathway inhibitor 2 (TFPI2) as a measurement target.

Both the number of ovarian cancer morbidity and the number of deaths in Japan are on the rise, and the National Cancer Center's 2015 Cancer Statistics Prediction shows that the number of ovarian cancers is 10,400 and the number of deaths is 4,800. It is the disease with the highest number of deaths. Ovarian cancer is classified into four main histological types: serous, intimal, mucinous, and clear cells. Among them, clear cell ovarian cancer is less effective than other histological types in many cases with poor prognosis (Non-Patent Document 1), and is the most frequent histological type among young people under 50 years old. Since it occurs frequently as the origin of the outbreak (Non-Patent Document 2), studies for improvement of clear cell ovarian cancer medical care are underway under the initiative of Japan.

Conventionally, cancer antigen 125 (Cancer Antigen 125, CA125) has been useful as a method for detecting ovarian cancer from blood components such as whole blood, blood cells, serum, and plasma, and screening for ovarian cancer and evaluation of therapeutic effect on ovarian cancer have been made. , It is widely used as an effective test for follow-up after treatment.

However, although CA125 is excellent in detecting ovarian cancer in general, it does not have (1) histological differentiation performance, (2) low positive rate for clear cell carcinoma, and (3) endometriosis, which is a benign tumor. There is a problem that the value becomes high due to the disease and it is not possible to accurately diagnose the onset of clear cell carcinoma from endometriosis. As a result, delays in dealing with clear cell cancer cases, overtreatment of benign diseases due to false positive determination of the disease, and accompanying decrease in the QOL of patients have become urgent problems. Therefore, if a biomarker that can predict not only the diagnosis but also the prognosis is created in order to accurately manage clear cell ovarian cancer, extended surgical procedures such as lymph node dissection and resection with other organs will be performed before surgery. It is expected to contribute to the treatment of ovarian cancer, such as making it possible to prepare the surgical system with this in mind.

Tissue factor pathway inhibitor 2 (TFPI2) is a placenta-derived serpin protease inhibitor that is the same protein as placenta protein 5 (Placental Protein 5; PP5) and contains three Knitz-type protease inhibitor domains. TFPI2 was reported by Miyagi et al. Of the present inventor as a secretory serpin protease inhibitor of the placenta (Non-Patent Document 3), and since it has heparin binding and is upregulated in the placenta, it is involved in the blood coagulation system during the perinatal period. It has been considered (Non-Patent Document 4). Regarding the relationship between TFPI2 and cancer, since the TFPI2 gene promoter has undergone methylation modification in many cancer types, clinical research is being actively promoted in the field of liquid biopsy as a genetic diagnostic target using methylation modification as an index. (Non-Patent Documents 5 to 7).

On the other hand, Arakawa et al. Of the present inventor revealed that TFPI2 is specifically produced from a clear cell cancer cell line in ovarian cancer, and that gene expression in ovarian cancer patient tissues is specifically improved only in clear cell cancer patients. (Patent Document 1), it was found that TFPI2 in blood was significantly improved in clear cell carcinoma as compared with healthy subjects and endometrial cases (Patent Document 2, Non-Patent Document 8). Further, a method for detecting clear cell ovarian cancer by measuring TFPI2 is also disclosed (Patent Document 3).
However, to date, the association between TFPI2 and the prognosis of patients with clear cell ovarian cancer has been unclear.

Patent No. 5224309 Japanese Unexamined Patent Publication No. 2013-61321 International Publication No. 2016/084912 Pamphlet

Cancer, 88 (11), 2584-2589 (2000) Int. J. Gynecol. Cancer, 17 (1), 37-43 (2007) J. Biochem. , 116,939 (1994) Placenta, 23 (2), 145-153 (2002). Cancer Genet. Cytogenet. , 197, 16 (2010) Anticancer Research. , 30, 1205 (2010) Clin. Cancer Res. , 11, 6422 (2005) J. Proteome Res. , 2013, 12 (10), pp 4340-4350

An object of the present invention is to provide a method for providing information for predicting the prognosis of a patient with clear cell ovarian cancer, and a reagent that can be used for the method.

As a result of diligent studies and collation of clinical information including survival time of TFPI2 and clear cell ovarian cancer patients, we found that there is a certain correlation between the two, and TFPI2 determines the prognosis of clear cell ovarian cancer patients. The present invention was completed with the idea that it could provide information for prediction.

That is, the present invention includes the following aspects.
[1] A method for providing information for predicting the prognosis of a patient with clear cell ovarian cancer.
A method comprising measuring the amount of TFPI2 in a sample derived from the patient and associating the measured value of the amount of TFPI2 with the prognosis of the patient.
[2] The method according to [1], wherein in the association, when the measured value of the TFPI2 amount exceeds a preset reference value, the patient is associated with a possibility of poor prognosis.
[3] The method according to [1] or [2], wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2.
[4] An antibody that binds to an antigen-determining group in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 in the measurement of the amount of TFPI2. The method according to any one of [1] to [3], which is carried out by an antigen-antibody reaction using.
[5] The method according to [4], wherein the antibody is an antibody that recognizes Kunitz domain 1 of TFPI2.
[6] The method according to any one of [1] to [3], wherein the measurement is performed using a mass spectrometry method.
[7] A clear ovary cell containing an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 of the amino acid sequence shown in SEQ ID NO: 1. A reagent for predicting the prognosis of cancer patients.

INDUSTRIAL APPLICABILITY The present invention provides information for predicting the prognosis of a patient with clear cell ovarian cancer from the amount of TFPI2 in a sample, and preoperatively operates with an expanded surgical procedure such as lymph node dissection or combined resection of other organs in mind. It is expected that more accurate management of clear cell cancer will be possible, such as the ability to establish a system.

The figure which showed the clinical stage (FIGO classification) of clear cell ovarian cancer, and the box plot (Box Plot) of (a) TFPI2 and (b) CA125. The horizontal axis represents the clinical stage (FIGO classification), and the vertical axis represents the blood volume. The figure which showed the correlation of TFPI2 and CA125. The horizontal axis represents the amount of TFPI2, and the vertical axis represents the amount of CA125. The figure which showed the box plot (Box Plot) of (a) TFPI2 and (b) CA125 measurement value in the surviving group or the dying group of clear cell ovarian cancer. The vertical axis represents the blood volume. The figure which showed the ROC curve of the survival group and the death group of clear cell ovarian cancer by (a) TFPI2 and (b) CA125. The vertical axis represents sensitivity and the horizontal axis represents 100% -specificity. The figure which showed the correlation between the postoperative survival days and (a) TFPI2 or (b) CA125. The vertical axis represents the number of days after surgery, and the horizontal axis represents the blood volume. The figure which showed the survival curve of TFPI2 or CA125. The vertical axis represents the survival rate, and the horizontal axis represents the postoperative survival days.

<1> Method for Providing Information for Predicting Prognosis of Clear Cell Ovarian Cell Cancer Patient of the Present Invention The method for providing information for predicting prognosis for clear cell ovarian cell carcinoma patient of the present invention is a sample derived from the patient. Includes measuring the amount of TFPI2 in and associating the measured value of the TFPI2 amount with the prognosis of the patient.
This is a method based on the correlation between the amount of TFPI2 and the prognosis of patients with clear cell ovarian cancer.
The method of the present invention provides information for more accurately predicting the prognosis of a clear cell ovarian cancer patient as compared with the case of measuring CA125, which is a conventionally known tumor marker, as shown in Examples described later. Will be done. The doctor predicts the prognosis of the clear cell ovarian cancer patient by referring to the provided information and the like. That is, the method itself of the present invention does not include the final judgment act regarding the prediction of prognosis, but includes the stage of providing the judgment material to the doctor.
In the present specification, the term "prognosis" refers to, for example, the future survival period (days) of a patient, the survival rate, and the discrimination of survival / death prospects.

The TFPI2 measured in the present invention is not particularly limited, and may be, for example, intact TFPI2 (hereinafter, also referred to as “I-TFPI2”), TFPI2 processing polypeptide (hereinafter, also referred to as “NT-TFPI2”), or both. There may be.

SEQ ID NO: 1 shows the amino acid sequence based on the cDNA of human TFPI2. In SEQ ID NO: 1, from the starting methionine to the glycine at the 22nd residue are signal peptides.
“Intact TFPI2” refers to a peptide represented by residues 23 to 235 of the amino acid sequence of SEQ ID NO: 1.
Further, "NT-TFPI2" refers to a peptide fragment containing Knitz domain 1 located on the N-terminal side of intact TFPI2, as described in Patent Document 3. More specifically, NT-TFPI2 is a peptide containing at least the sequence from aspartic acid at residue 23 of the amino acid sequence of SEQ ID NO: 1 to histidine at residue 131 or cysteine at residue 130, or the above-mentioned peptide. A peptide containing an amino acid sequence having 80% or more identity with the sequence. The identity is preferably 90% or more, more preferably 95% or more. In addition, this polypeptide may be a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, and / or added in the above sequence. The number is preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 5. Further, although other peptide fragments may be provided on both sides of the sequence, it is preferable that the antibody does not have an antigenic determinant that recognizes the Knitz domain 3 of TFPI2.

Examples of the patient-derived sample (test sample) in the present invention include blood components such as whole blood, blood cells, serum, and plasma, cell or tissue extract, urine, and cerebrospinal fluid. In addition, the ovarian tissue biopsy may be the test target, but in that case, the culture supernatant of the biopsy sample is used as the sample. It is preferable to use a body fluid such as a blood component or urine as a sample because it can be performed easily and non-invasively. Considering the ease of sample collection and versatility for other test items, the blood component is used as a sample. Is particularly preferable. The dilution ratio of the sample may be appropriately selected from undiluted to 100-fold dilution according to the type and condition of the sample to be used.

Further, the time for collecting the sample in the present invention is not particularly limited. For example, it may be any time from preoperative time when ovarian cancer is suspected by diagnostic imaging etc. and detailed examination is performed to follow-up after definitive diagnosis of clear cell ovarian cancer by postoperative pathological examination. Any sample collected at any stage, such as before and after the start, can be used in the method of the present invention. This is because, as shown in Examples described later, the amount of TFPI2 in blood has a certain correlation with the prognosis of the patient, but no clear relationship with the degree of cancer progression in the patient was observed. Be supported.

The association between the measured TFPI2 amount and the patient's prognosis is, for example, when the intact TFPI2 amount, the NT-TFPI2 amount, or the sum of the intact TFPI2 amount and the NT-TFPI2 amount exceeds a preset reference value. Is done by associating with a possible poor prognosis. The prognostic potential indicated by the association is usually provided to the physician as information that can be used as a basis for predicting the prognosis.
The reference value used for the determination may be either a measured value or a converted concentration value. The converted concentration value refers to a value converted from a measured value based on a calibration curve prepared using TFPI2 as a standard sample.

As the reference value for determining the prognosis, for example, a sample derived from a clear cell ovarian cancer patient who survives without recurrence after the first surgery and a sample derived from a clear cell ovarian cancer patient who died after the first surgery are measured. By performing receiver operating characteristic (ROC) curve analysis, it is possible to appropriately set a value indicating optimum sensitivity and specificity. For example, specifically, the reference value (cutoff value) of the total of the NT-TFPI2 amount and the intact TFPI2 amount may be set to 400 pg / mL, or 430 pg / mL as shown in Examples described later. May be good.

Hereinafter, a method for measuring TFPI2 will be described.
In the present invention, the amount of NT-TFPI2 or the amount of intact TFPI2 in the sample may be measured individually, or the values may be summed to obtain the total amount. Further, the total amount of NT-TFPI2 and intact TFPI2 in the sample may be measured by a measuring system capable of measuring at one time. Alternatively, as will be described later, the NT-TFPI2 amount may be indirectly measured from the total amount measured by both measurements and the measured amount of intact TFPI2 alone.
In the method of the present invention, the method for measuring the amount of NT-TFPI2 and / or the amount of intact TFPI2 is not particularly limited. For example, a method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and / or intact TFPI2, and a method using mass spectrometry can be exemplified.

As a measurement method using an antigen-antibody reaction using an antibody that recognizes NT-TFPI2 and / or intact TFPI2, for example, the method described in Patent Document 3 can be mentioned, and specific examples thereof include the following.
(A) A competitive method using a labeled measurement target and an antibody that recognizes the measurement target, and utilizing the competitive binding of the labeled measurement target and the measurement target contained in the sample to the antibody.
(B) A method using surface plasmon resonance in which a sample is brought into contact with a chip on which an antibody that recognizes a measurement target is immobilized, and a signal that depends on the binding between the antibody and the measurement target is detected.
(C) A fluorescence polarization immunoassay method utilizing an antibody that recognizes a fluorescence-labeled measurement target, and the degree of fluorescence polarization increases as the antibody binds to the measurement target.
(D) A sandwich method in which two types of antibodies recognizing a measurement target (one of which is a labeled antibody) having different epitopes are used to form a complex of the two antibodies and the measurement target.
(E) A method in which a measurement target in a sample is concentrated with an antibody that recognizes the measurement target as a pretreatment, and then the polypeptide of the binding protein is detected by a mass spectrometer or the like.
The methods (d) and (e) are simple and highly versatile, but the method (d) is more preferable in treating a large number of samples because the techniques related to reagents and devices are sufficiently established.

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

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

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

In the present invention, the amount of NT-TFPI2 alone measured by the methods (C) and (D) described above may be used as a criterion for determination, but NT-TFPI2 and intact TFPI2 measured by the method (A). The latter is more preferable because sufficient sensitivity and specificity can be obtained even if the total amount of the above is used as a criterion for determination, and the antibody can be easily obtained and measured in one step.

Antibodies that recognize NT-TFPI2 and / or intact TFPI2 are NT-TFPI.
2 Polypeptide or intact TFPI2 protein itself, oligopeptide consisting of a partial region of NT-TFPI2 polypeptide or intact TFPI2 protein, NT-TFPI2 polypeptide or a polynucleotide encoding the intact or partial region of intact TFPI2 protein as an immunogen , Can be obtained by immunizing animals.
The animal used for immunity is not particularly limited as long as it has an antibody-producing ability, and may be a mammal normally used for immunity such as a mouse, a rat or a rabbit, or a bird such as a chicken.

As the immunogen, an NT-TFPI2 polypeptide or intact TFPI2 protein itself, or an oligopeptide consisting of a partial region of the NT-TFPI2 polypeptide or intact TFPI2 protein can be used. However, the protein or oligopeptide does not reflect the three-dimensional structure of TFPI2 in vivo, or its structure may change during the preparation process. Therefore, the obtained antibody may not have high specificity or binding force to the desired in vivo TFPI2, and even if a measurement system is constructed using this antibody, it is contained in the sample as a result. It may not be possible to accurately quantify the TFPI2 concentration. On the other hand, when an expression vector containing an intact or partial region of the NT-TFPI2 polypeptide or intact TFPI2 protein is used as an immunogen, the NT as introduced does not undergo structural changes in the body of the immunized animal. -Because the intact or partial region of the TFPI2 polypeptide or intact TFPI2 protein is expressed, an antibody having high specificity and binding force (that is, high affinity) to the NT-TFPI2 polypeptide or intact TFPI2 in the sample It is preferable because it can be obtained.

In addition, TFPI1 known as a homologue of TFPI2 is also present in the blood. Therefore, it is desirable to use an antibody that specifically recognizes only TFPI2 without crossing with TFPI1.

The antibody that recognizes TFPI2 may be a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is preferable.
The establishment of hybridoma cells that produce an antibody that recognizes NT-TFPI2 and / or intact TFPI2 may be appropriately selected from the methods for which techniques have been established. As an example, selection of hybridoma cells in which B cells are collected from an animal immunized by the method described above, the B cells and myeloma cells are fused electrically or in the presence of polyethylene glycol, and a desired antibody is produced by HAT medium. By monocloning the selected hybridoma cells by the limiting dilution method, hybridoma cells that produce a monoclonal antibody that recognizes NT-TFPI2 and / or intact TFPI2 can be established.

The selection of the antibody that recognizes NT-TFPI2 and / or intact TFPI2 used in the present invention, for example, the monoclonal antibody that recognizes NT-TFPI2 and / or intact TFPI2, is derived from the host expression system and is a GPI (glycosylphosphatidy secretion) anchor type. It may be done based on the affinity for TFPI2 or secretory TFPI2.
The host is not particularly limited and may be appropriately selected from microbial cells such as Escherichia coli and yeast, insect cells, and animal cells that are usually used by those skilled in the art for protein expression, but disulfide bonds or sugar chains are added. It is preferable to use a mammalian cell as a host, which can express a protein having a structure similar to that of natural NT-TFPI2 and / or intact TFPI2 by post-translational modification such as. Examples of mammalian cells include conventionally used human fetal kidney-derived cell (HEK) 293T cell line, monkey kidney cell COS7 strain, Chinese hamster ovary (CHO) cell, and cancer cells isolated from humans. Be done.

Purification of the antibody used in the present invention may be carried out by appropriately selecting from the methods for which the technique has been established. As an example, after culturing the antibody-producing hybridoma cells established by the method described above, the culture supernatant is collected, and if necessary, the antibody is concentrated by ammonium sulfate precipitation, and then protein A, protein G, protein L, etc. are fixed. The antibody can be purified by affinity chromatography and / or ion exchange chromatography using the proteinated carrier.

As for the labeled antibody used when carrying out the antigen-antibody reaction by the above-mentioned sandwich method, the antibody purified by the above-mentioned method may be labeled with an enzyme such as peroxidase or alkaline phosphatase, and the technique for labeling has been sufficiently established. It may be done by using a method.

In the method of the present invention, a method of measuring the amount of NT-TFPI2 and / or the amount of intact TFPI2 by using mass spectrometry will be specifically described below.
When the sample is blood, as a pretreatment step, major proteins such as albumin, immunoglobulin, transferrin, etc., which are abundant in blood, are removed by Agent Human 14, etc., and then further separated by ion exchange, gel filtration, reverse phase HPLC, etc. It is preferable to draw. Alternatively, it is also possible to specifically recover only TFPI2 by an immunological method using an anti-TFPI2 antibody.

Measurements include tandem mass spectrometry (MS / MS), liquid chromatography and tandem mass spectrometry (LC / MS / MS), matrix-assisted laser desorption / ionization flight time mass spectrometry (matrix assisted laser resolution ionization time-of-fly mass spectrometry). , MALDI-TOF / MS), surface enhanced laser ionization mass spectrometry, SELDI-MS, and the like.

<2> Reagent for Predicting Prognosis of Clear Cell Cancer Patient of the Present Invention The reagent for predicting the prognosis of the clear cell cancer patient of the present invention is 23 residues of the amino acid sequence TFPI2 represented by SEQ ID NO: 1. It contains an antibody that binds to an antigenic determinant in the region from the amino acid of the base to the amino acid of the 131st or 130th residue. The antibody is preferably an antibody that recognizes Kunitz domain 1 of TFPI2. These antibodies can recognize both NT-TFPI2 and intact TFPI2.

When the reagent of the present invention is used in the sandwich method described above, it is preferable that the antibody contains two types of antibodies having different epitopes.
The antibody contained in the reagent of the present invention may be the antibody itself, may be labeled, or may be immobilized on a solid phase.

Among the reagents of the present invention, the case of using the reagent in the two-step sandwich method, which is one aspect of the sandwich method described above, will be specifically described below. However, the present invention is not limited to this.
First, the reagent of the present invention can be prepared by the methods shown in (I) to (III) below.
(I) First, of the two types of antibodies having different epitopes (hereinafter referred to as "antibody 1" and "antibody 2") that recognize NT-TFPI2 and intact TFPI2 used in the sandwich method, antibody 1 is immunoplate. It is bound to a B / F (Bound / Free) separable carrier such as magnetic particles or magnetic particles. The bonding method may be a physical bond using a hydrophobic bond, or a chemical bond using a linker reagent capable of cross-linking between two substances.
(II) After binding the antibody 1 to the carrier, the surface of the carrier is blocked with bovine serum albumin, skim milk, a commercially available blocking agent for immunoassay, or the like to prepare a primary reagent in order to avoid non-specific binding.
(III) Label the other antibody 2 and prepare a solution containing the obtained labeled antibody as a secondary reagent. Substances labeled with antibody 2 include enzymes such as peroxidase and alkaline phosphatase, fluorescent substances, chemically luminescent substances, substances that can be detected by a detector such as radioisotope, or substances that have a specific binding partner such as avidin to biotin. Etc. are preferable. Further, as the solution of the secondary reagent, a buffer solution capable of performing an antigen-antibody reaction well, for example, a phosphate buffer solution, a Tris-HCl buffer solution, or the like is preferable. The reagent of the present invention produced in this manner may be freeze-dried if necessary.

In the case of the one-step sandwich method, the antibody 1 was bound to the carrier in the same manner as in (I) to (II) described above to carry out a blocking treatment, and the labeled antibody 2 was applied to the antibody-immobilized carrier. The reagent may be prepared by further adding the containing buffer solution.
Next, in order to detect and measure NT-TFPI2 and intact TFPI2 by the two-step sandwich method using the reagents obtained by the above-mentioned method, the methods shown in (IV) to (VI) below may be used. ..
(IV) The primary reagent prepared in (II) and the sample are brought into contact with each other for a certain period of time at a certain temperature. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 to 180 minutes.
(V) The unreacted substance is removed by B / F separation, and then the secondary reagent prepared in (III) is brought into contact with the secondary reagent for a certain period of time at a certain temperature to form a sandwich complex. The reaction conditions may be a temperature range of 4 ° C. to 40 ° C. for 5 to 180 minutes.
(VI) Unreacted substances are removed by B / F separation, labeled substances of labeled antibodies are quantified, and human NT-TFPI2 and intact TFPI2 in the sample are quantified by a calibration curve prepared using a known concentration of TFPI2 solution as a standard. To do.

The amount of reagent components such as antibodies contained in the reagent of the present invention may be appropriately set according to various conditions such as the amount of sample, the type of sample, the type of reagent, and the measurement method. Specifically, for example, when 20 μL of serum or plasma is used as a sample and the amount of NT-TFPI2 and the amount of intact TFPI2 are measured by the sandwich method as described later, the reaction system in which 20 μL of the sample is reacted with the antibody is applied. The amount of antibody bound to the carrier may be 100 ng to 1000 μg, and the amount of labeled antibody may be 2 ng to 20 μg.

The reagent of the present invention can also be used for measurement by a conventional method, and can also be used for measurement using an automatic immunodiagnosis device. In particular, measurement using an automatic immunodiagnosis device can be performed without being affected by intrinsic measurement obstructing factors and competing enzymes contained in the sample, and NT-TFPI2 and intact TFPI2 in the sample can be measured in a short time. It is preferable because it can be quantified.

Examples are shown below in order to specifically explain the present invention, but these examples show an example of the present invention, and the present invention is not limited to the examples.
<Example 1> Construction of TFPI2 expression plasmid for DNA immunization A TFPI2 expression plasmid for DNA immunization was constructed according to the method of Patent Document 2.
(1) Using the primers of (a) below, a polynucleotide consisting of 73 to 705 bases of TFPI2 cDNA (GenBank No. NM_006528) was amplified by the RT-PCR method according to a conventional method.
(A) Primer for GPI-anchored TFPI2 expression plasmid Forward:
5'-cgatagagacaagctttggtagccaca-3'(SEQ ID NOS: 2, 3'terminal 15 bases correspond to the 73rd to 87th base sequences of GenBank No. NM_006528)
Reverse:
5'-catctaggtgaattcaattgtttctccg-3'(SEQ ID NOS: 3, 5'terminal 15 bases correspond to the 691 to 705th base sequences of GenBank No. NM_006528)

(2) The HindIII-EcoRI site of plasmid pFLAG1 (manufactured by SIGMA) containing the coding region of the GPI anchor and the coding region of the FLAG tag of Placental Alkaline phosphatase was subjected to In-fusion (manufactured by C-terminus) according to the protocol. The RT-PCR amplification product obtained in 1) was inserted to construct a GPI-anchored TFPI2 expression plasmid in which a FLAG tag peptide was added to the N-terminal side and a GPI anchor was added to the C-terminal side.

<Example 2> Evaluation of antiserum Mice and rats were immunized with the TFPI2 expression plasmid according to the method of Patent Document 2. A TFPI2-specific antibody titer increase was confirmed by a cell enzyme immunoassay (CELISA) using GPI-anchored TFPI2-expressing CHO-K1 cells and an ELISA using a secretory TFPI2 solution.

<Example 3> Selection of hybridoma and identification of antigenic determinant A hybridoma that produces an antibody against TFPI2 was established according to the method of Patent Document 2. The antigenic determinants of each antibody were identified by variant-expressing cells of each Knitz domain of TFPI2, KD1, KD2 and KD3. The specific preparation method of each variant expression plasmid is shown below.
(1) Using the primers described in (c), (d) and (e) below, polynucleotides corresponding to the KD1 region, KD2 region and KD3 to C-terminal region of TFPI2 are prepared by the RT-PCR method according to a conventional method. Amplified.
(C) Primer for GPI anchor type TFPI2-KD1 Forward:
5'-cgatagagacaagctttggtaggaccaca-3'(SEQ ID NOS: 4, 3'terminal 15 bases correspond to the 73rd to 87th base sequences of GenBank No. NM_006528)
Reverse:
5'-catctaggtgaattctttttctcatccca-3'(SEQ ID NO: 5, 5'terminal 15 bases correspond to the 259th to 273rd base sequences of GenBank No. NM_006528)
(D) Primer for GPI anchor type TFPI2-KD2 Forward:
5'-cgatagagacaagctgtgtcccaagttgtgt-3'(SEQ ID NOS: 6, 3'terminal 15 bases correspond to 274 to 288 base sequences of GenBank No. NM_006528)
Reverse:
5'-catctaggtgaattctttctttgtgtggccgca-3'(SEQ ID NO: 7, 5'terminal 15 bases correspond to the 445-459th base sequence of GenBank No. NM_006528)
(E) Primer for GPI anchor type TFPI2-KD3 Forward:
5'-cgatagagacaagctattatctcatttgtgt-3'(SEQ ID NO: 8, 3'terminal 15 bases correspond to the 460th to 474th base sequence of GenBank No. NM_006528)
Reverse:
5'-catctaggtgaattcaattgtttctccg-3'(SEQ ID NO: 9, 5'terminal 15 bases correspond to the 691 to 705th base sequences of GenBank No. NM_006528)

(2) A TFPI2 expression plasmid was constructed by the method described in Example 1 (2).
(3) In order to confirm that TFPI2 expressed by the polynucleotide inserted in the three types of GPI-anchored TFPI2 expression plasmids constructed in (2) is localized on the cell surface as expected, one A 293T cell line, which is a hyperexpressing cell, was used and verified by the following method.
(3-1) The GPI-type TFPI2 expression plasmid constructed in (2) was introduced into a 293T cell line according to a conventional method.
(3-2) The 293T cell line into which the expression plasmid was introduced was introduced into a 5% CO ₂ incubator using D-MEM medium (manufactured by Wako Junyaku Co., Ltd.) supplemented with 10% FBS (Fetal Bovine Serum) 24. The cells were cultured at 37 ° C. for hours to transiently express TFPI2. (3-3) A mouse anti-FLAG M2 antibody manufactured by SIGMA that specifically binds to the cultured cells obtained in (3-2), or a mouse anti-BNC antibody that does not bind to the FLAG tag as a negative control. Was added and allowed to stand for 30 minutes. BNC is a peptide consisting of 7 amino acids on the C-terminal side of BNP (brain natriuretic peptide) (Japanese Patent Laid-Open No. 2009-240300).
(3-4) After standing, a fluorescently labeled anti-mouse IgG antibody (manufactured by BECKMAN COULTER) was added, and after standing for another 30 minutes, FACS (Flow cytometry Activated Cell Sorting) analysis was performed to obtain 10 types of monoclonal antibodies. The antigenic determinant was identified.
Table 1 shows the antigenic determinants of each antibody found from the results of FACS analysis.

<Example 4> Preparation of TFPI2 measuring reagent Intact TFPI2 using TS-TF04 antibody having an antigenic determinant in Knitz domain 1 on the solid phase side and TS-TF01 antibody having an antigenic determinant in Knitz domain 1 on the detection side. And the measurement reagents of the "NT + I-TFPI2 measurement system" that comprehensively targets NT-TFPI2 were prepared as follows.
(1) An anti-TFPI2 monoclonal antibody (TS-TF04) was physically adsorbed on a water-insoluble ferrite carrier at room temperature for a whole day and night so as to reach 100 ng / carrier, and then 100 mM Tris buffer (pH 8.0) containing 1% BSA. An anti-TFPI2 antibody-immobilized carrier was prepared by blocking at 53 ° C. for 4 hours.
(2) An alkaline phosphatase-labeled anti-TFPI2 antibody was prepared using an alkaline phosphatase labeling kit (manufactured by Dojin Chemical Co., Ltd.) using an anti-TFPI2 monoclonal antibody (TS-TF01).

(3) A buffer containing 1 μg / mL of the alkaline phosphatase-labeled antibody prepared in (2) after placing the 12 antibody-immobilized carriers prepared in (1) in a magnetically permeable container (volume 1.2 mL). A TFPI2 measurement reagent was prepared by adding 100 μL of a solution (Tris buffer solution containing 3% BSA, pH 8.0) and performing freeze-drying. The prepared TFPI2 measurement reagent was sealed and sealed under nitrogen filling, and stored at 4 ° C. until measurement.

<Example 5> Evaluation of clinical specimens The 18 cases of clear cell ovarian cancer used in this example were serum specimens collected by the same protocol at Yokohama City University Gynecology from 2003 to 2014, and informed consent. And the approval of the Yokohama City University Ethics Committee.

As the evaluation device, a fully automatic enzyme immunoassay device AIA-2000 (manufactured by Tosoh Corporation: Manufacturing and Sales Notification No. 13B3X90002000009) was used. The measurement of TFPI2 by the fully automatic enzyme immunoassay device AIA-2000 was performed by the following procedure.
(1) 20 μL of the sample and 80 μL of the diluted solution containing the surfactant were automatically dispensed into the container containing the TFPI2 measurement reagent prepared in Example 4.
(2) Perform an antigen-antibody reaction for 10 minutes at a constant temperature of 37 ° C.
(3) After B / F separation, wash 8 times with a buffer solution containing a surfactant.
(4) Add 4-methylimidazole phosphate and add
The concentration of 4-methylumbelliferone produced by alkaline phosphatase per unit time was used as the measured value (nmol / (L · s)), and a calibration curve was prepared using a commercially available TFPI2 recombinant protein (R & D) as a standard product in the sample. The TFPI2 concentration was calculated.
The CA125 was measured using the E-test TOSOHII CA125 measuring reagent (manufactured by Tosoh Corporation, approval number 20700AMZ00504000).

<Example 6> Relationship with clinical stage (FIGO classification) TFPI2 and CA125 in clinical stage (FIGO classification) of clear cell ovarian cancer specimens (18 cases collected from 2003 to 2014) are shown in FIG. Shown. Neither the median TFPI2 nor CA125 showed a clear correlation with the clinical stage (FIGO classification), and there was no tendency for the blood concentration to increase with the progression of cancer. The correlation analysis result of TFPI2 and CA125 is shown in FIG. In this analysis, no correlation was found between TFPI2 and CA125, suggesting that the blood kinetics of TFPI2 and CA125 are not correlated.

<Example 7> Comparison between surviving group and dead group Surviving group (n =) based on the results of a research study in December 2014 for clear cell ovarian cancer specimens (11 cases collected from 2003 to 2010). 4) and the dead group (n = 7) are divided, and the blood TFPI2 value and the blood CA125 measured value in the twin groups are shown in FIG. The results of ROC analysis in the comparison between the surviving group and the dead group are shown in FIG. TFPI2 was found to be significantly higher in the deceased group than in the surviving group (Mann-Whitney U test, p = 0).
.. 0424). Furthermore, as a result of ROC analysis (Fig. 4), when the AUC (area under the curve) value is 0.8929 and the cutoff value (reference value) is 437 pg / mL, the sensitivity is 100% and the specificity is 75%. It became clear that TFPI2 can clearly distinguish between the surviving group and the dead group. On the other hand, CA125 did not show a statistically significant difference between the surviving group and the deceased group (p = 0.4848), and no differential performance like TFPI2 was observed in the ROC analysis.

<Example 8> Relationship with postoperative survival days The relationship between postoperative survival days of clear-cell ovarian cell carcinoma specimens (11 cases collected from 2003 to 2010), blood TFPI2 value, and blood CA125 measurement value It is shown in FIG. Although blood TFPI2 value was negatively correlated highly with respect to post-operative survival days (Spearman rank correlation coefficient, r _s = -
0.76), there was no correlation in the CA125 value (r _s = -0.236).

<Example 9> Relationship with survival curve In clear cell ovarian cancer specimens (11 cases collected from 2003 to 2010), blood TFPI2 was less than 430 pg / mL (4 cases), and the above group. Divided into (7 cases), the survival curve using the Kaplan-Meier method is shown in FIG. A marked decrease in survival was observed in the group of patients with blood TFPI2 of 430 pg / mL or more, which was the group of patients with a blood CA125 value of 35 U / mL (standard value used in clinical practice) or more (10 cases). Was lower than.

The present invention provides information for predicting the prognosis of patients with clear cell ovarian cancer by blood tests. This is industrially expected to contribute to the treatment of ovarian cancer, such as making it possible to prepare the surgical system with expanded surgical procedures such as lymph node dissection and resection of other organs in mind before surgery. Very useful.

Claims (7)

A method of providing information for predicting the prognosis of patients with clear cell ovarian cancer.
A method comprising measuring the amount of TFPI2 in a sample derived from the patient and associating the measured value of the amount of TFPI2 with the prognosis of the patient. The method according to claim 1, wherein in the association, when the measured value of the TFPI2 amount exceeds a preset reference value, the patient is associated with a possibility of poor prognosis. The method according to claim 1 or 2, wherein the amount of TFPI2 is the sum of the amount of TFPI2 processing polypeptide and the amount of intact TFPI2. The TFPI2 amount was measured using an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 of the amino acid sequence of SEQ ID NO: 1 to histidine at residue 131 or cysteine at residue 130. The method according to any one of claims 1 to 3, which is carried out by an antigen-antibody reaction. The method according to claim 4, wherein the antibody is an antibody that recognizes the Knitz domain 1 of TFPI2. The method according to any one of claims 1 to 3, wherein the measurement is performed using a mass spectrometry method. A patient with clear cell carcinoma of the ovary containing an antibody that binds to an antigenic determinant in the region from aspartic acid at residue 23 to histidine at residue 131 or cysteine at residue 130 of the amino acid sequence shown in SEQ ID NO: 1. Reagents for predicting prognosis.

Images