U.S. patent application number 15/529550 was filed with the patent office on 2017-11-09 for method and reagent for detecting ovarian clear cell adenocarcinoma.
This patent application is currently assigned to PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY. The applicant listed for this patent is PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY, TOSOH CORPORATION. Invention is credited to Noriaki ARAKAWA, Hisashi HIRANO, Etsuko MIYAGI, Norihisa OHTAKE.
Application Number | 20170322218 15/529550 |
Document ID | / |
Family ID | 56074462 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170322218 |
Kind Code |
A1 |
ARAKAWA; Noriaki ; et
al. |
November 9, 2017 |
METHOD AND REAGENT FOR DETECTING OVARIAN CLEAR CELL
ADENOCARCINOMA
Abstract
The present invention aims to provide a method for detecting,
with high sensitivity and specificity, ovarian clear cell
adenocarcinoma, which is highly malignant, among benign and
malignant ovarian tumors having various tissue types, and a reagent
that can be used for the method. The present invention provides
NT-TFPI2, which is a novel processed tissue factor pathway
inhibitor 2 polypeptide, as a new detection marker for ovarian
clear cell adenocarcinoma. The detection of ovarian clear cell
adenocarcinoma is carried out by measuring the amount of NT-TFPI2,
or the total amount of NT-TFPI2 and intact TFPI2. The reagent for
detecting ovarian clear cell adenocarcinoma contains an antibody
that specifically recognizes NT-TFPI2 and intact TFPI2.
Inventors: |
ARAKAWA; Noriaki;
(Yokohama-shi, JP) ; HIRANO; Hisashi;
(Yokohama-shi, JP) ; MIYAGI; Etsuko;
(Yokohama-shi, JP) ; OHTAKE; Norihisa; (Ayase-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PUBLIC UNIVERSITY CORPORATION YOKOHAMA CITY UNIVERSITY
TOSOH CORPORATION |
Yokohama-shi, Kanagawa
Shunan-shi, Yamaguchi |
|
JP
JP |
|
|
Assignee: |
PUBLIC UNIVERSITY CORPORATION
YOKOHAMA CITY UNIVERSITY
Yokohama-shi, Kanagawa
JP
TOSOH CORPORATION
Shunan-shi, Yamaguchi
JP
|
Family ID: |
56074462 |
Appl. No.: |
15/529550 |
Filed: |
November 26, 2015 |
PCT Filed: |
November 26, 2015 |
PCT NO: |
PCT/JP2015/083272 |
371 Date: |
May 25, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/47 20130101;
G01N 27/62 20130101; C12N 15/09 20130101; C07K 14/8114 20130101;
G01N 33/57449 20130101; G01N 33/574 20130101; G01N 2333/8114
20130101; C07K 16/38 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; C07K 14/81 20060101 C07K014/81; C07K 16/38 20060101
C07K016/38 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 27, 2014 |
JP |
2014-239433 |
Claims
1. A processed tissue factor pathway inhibitor 2 (TFPI2)
polypeptide having the following properties (i) to (iii): (i) a
polypeptide having the amino acid sequence from the 23th residue
aspartic acid to the 131st residue histidine or to the 130th
residue cysteine in the TFPI2 amino acid sequence of SEQ ID NO:1,
or a sequence having an identity of not less than 80% to this
sequence; (ii) a polypeptide that is fractionated into a molecular
weight of about 16,000 by reducing SDS-PAGE; and (iii) a
polypeptide whose peptide fragment obtained after asparagine-linked
sugar chain cleavage treatment is fractionated into a molecular
weight of about 12,000 by reducing SDS-PAGE.
2. A method for detecting ovarian clear cell adenocarcinoma, said
method comprising measuring the amount of the processed TFPI2
polypeptide according to claim 1 in a sample.
3. The method according to claim 2, further comprising measuring
the amount of intact TFPI2 in said sample.
4. The method according to claim 3, wherein ovarian clear cell
adenocarcinoma is judged to be detected in cases where the total of
said amount of the processed TFPI2 polypeptide and said amount of
intact TFPI2 exceeds a reference value calculated from a
control.
5. The method according to claim 2, wherein said measurement is
carried out by antigen-antibody reaction using an antibody that
binds to an antigenic determinant in the region from the 23th
residue aspartic acid to the 131st residue histidine or to the
130th residue cysteine in the TFPI2 amino acid sequence of SEQ ID
NO:1.
6. The method according to claim 5, wherein said antibody is an
antibody that recognizes Kunitz domain 1 of TFPI2.
7. The method according to claim 2, wherein said measurement is
carried out using mass spectrometry.
8. A reagent for detecting ovarian clear cell adenocarcinoma,
comprising an antibody that binds to an antigenic determinant in
the region from the 23th residue aspartic acid to the 131st residue
histidine or to the 130th residue cysteine in the TFPI2 amino acid
sequence of SEQ ID NO:1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel processed
polypeptide of tissue factor pathway inhibitor 2 (TFPI2) protein
(hereinafter referred to as "NT-TFPI2") to be used for detection of
ovarian clear cell adenocarcinoma, which is highly malignant among
ovarian tumors, and a method for detecting ovarian clear cell
adenocarcinoma based on measurement of NT-TFPI2. More specifically,
the present invention relates to a method for detecting ovarian
clear cell adenocarcinoma using a measurement method in which the
total amount of NT-TFPI2 and intact TFPI2 is calculated, and a
reagent for detecting ovarian clear cell adenocarcinoma.
BACKGROUND ART
[0002] Ovarian cancer is the tumor with the highest mortality rate
among gynecological malignancies. In Japan, its annual incidence is
about 7,000 to 8,000, and its annual mortality is about 4,000.
These numbers are expected to increase year by year. Ovarian
surface epithelial malignant tumors account for about 85% of
ovarian cancers, and are classified based on their tissue types
into the serous type, endometrioid type, mucinous type, clear cell
type, and undifferentiated type. In particular, it has been
reported that, while the incidence rate of clear cell
adenocarcinoma is about 5% in Europeans and Americans, it tends to
be high in Japanese with an incidence rate of 20% to 30%. Ovarian
clear cell adenocarcinoma is characterized in that Stage I cases
account for about half of all cases, often become resistant to
chemotherapy using cisplatin, paclitaxel, or the like, and that its
malignancy is extremely high.
[0003] Conventionally, transvaginal ultrasound, CT, MRI, and the
like have been used as methods for detection of ovarian cancer.
[0004] As methods for detecting ovarian cancer using a blood
component such as whole blood, blood cells, serum, or plasma,
methods in which cancer antigen 125 (CA125) is detected are
generally known. CA125 is an antigen which is recognized by a
monoclonal antibody (OC125) established by Bast et al. in 1981
using a human ovarian cancer cell line (OVCA433) as an immunogen.
In cases where CA125 is detected in a blood component, the presence
of ovarian surface epithelial ovarian cancer is suggested with a
high positive rate. Therefore, the CA125 is widely used for
diagnosis including screening of ovarian cancer, evaluation of
therapeutic effects on ovarian cancer, and follow-up after therapy
(Non-patent Documents 1 and 2).
[0005] However, the positive rate of CA125 in ovarian cancer is
generally about 80%, and false-negative results are obtained in
some cases. Thus, judgment by CA125 is impossible in about 20% of
ovarian cancer. Based on comparison among different tissue types of
malignant ovarian cancer, the CA125 positive rate in serous type
cancer is not less than 90%, while the CA125 positive rate in clear
cell adenocarcinoma is about 65%, which is extremely low
(Non-patent Document 3). CA125 is also utilized as an auxiliary
marker for endometriosis, which is a benign tumor, but CA125 cannot
clearly distinguish between benign ovarian tumors and malignant
ovarian tumors, and identification of the tissue types of malignant
tumors is difficult therewith.
[0006] If ovarian clear cell adenocarcinoma, which is highly
malignant, can be specifically identified among ovarian tumors
having various tissue types by a blood test, the identification may
contribute to improvement of the accuracy of diagnosis in screening
and follow-up of ovarian cancer, as well as to future development
of preoperative treatment methods such as preoperative chemotherapy
specific to clear cell adenocarcinoma. Moreover, a canceration
theory has been proposed in which the origin of development of
ovarian clear cell adenocarcinoma is ovarian endometriosis. Thus,
for use in follow-up of ovarian endometriosis and for elucidation
of the mechanism of development of the cancer, identification of a
specific marker molecule to ovarian clear cell adenocarcinoma and
development of a method for detecting this molecule have been
demanded.
[0007] Tissue factor pathway inhibitor 2 (TFPI2) is the same
protein as placental protein 5 (PPS), and is a placenta-derived
serine protease inhibitor having three Kunitz-type protease
inhibitor domains (Non-patent Document 4). TFPI2 has disulfide
bonds at three positions in each Kunitz domain (KD), and it is
reported that a plurality of types of TFPI2 are fractionated near a
molecular weight of about 30,000 Da to 35,000 Da depending on the
asparagine-linked sugar chain structures added to the Kunitz
domains 2 and 3 (Non-patent Document 5).
[0008] In terms of association of TFPI2 with gynecological
diseases, findings such as an increased blood level of TFPI2 in
preeclampsia relative to intrauterine growth retardation (IUGR) or
normal pregnancy (Non-patent Document 6) and an increased blood
level of TFPI2 in patients with endometriosis (Patent Document 1)
have been reported. In terms of association with cancer, gene-level
studies have been intensively carried out, and it has been reported
that TFPI2 is included in a group that shows more than a certain
level of gene expression in clear cell adenocarcinoma in uterine
cancer and ovarian cancer (Non-patent Document 7), that the gene
expression of TFPI2 increases in gastric cancer (Patent Document
2), and that the gene expression of asTFPI2, which is a splicing
variant of TFPI2, increases in cancer (Non-patent Document 8).
Moreover, since hypermethylation of a CpG island in the TFPI2
promoter region occurs in various cancers, studies on epigenetic
markers have been intensively carried out in recent years (Patent
Document 3, Non-patent Documents 9, 10, 11, 12, and 13).
[0009] On the other hand, the present inventors, Arakawa et al.,
elucidated that TFPI2 is specifically produced from the clear cell
adenocarcinoma cell line of ovarian cancer, and that an increased
gene expression of TFPI2 in an ovarian cancer patient tissue
specifically occurs only in patients with clear cell adenocarcinoma
(Patent Document 4). The inventors also discovered that blood TFPI2
is significantly increased in clear cell adenocarcinoma relative to
healthy individuals and cases of endometriosis (Patent Document 5,
Non-patent Document 14).
[0010] However, the presence of the processed TFPI2 polypeptide has
not been known to date. Furthermore, detection of ovarian clear
cell adenocarcinoma by measurement of the processed polypeptide,
and the effect of the processed polypeptide detection, have of
course been unknown.
PRIOR ART DOCUMENTS
Patent Documents
[0011] Patent Document 1: Japanese Translated PCT Patent
Application Laid-open No. 2007-506965 [0012] Patent Document 2: JP
2008-118915 A [0013] Patent Document 3: WO 2008/084219 [0014]
Patent Document 4: JP 2013-79979 A [0015] Patent Document 5: JP
2013-61321 A
Non-patent Documents
[0015] [0016] Non-patent Document 1: J. Clin. Invest., 68, 1331
(1981) [0017] Non-patent Document 2: Human Reproduction, 4, 1(1989)
[0018] Non-patent Document 3: Molecular Tumor Marker Research, 20,
98 (2005) [0019] Non-patent Document 4: J. Biochem., 116, 939
(1994) [0020] Non-patent Document 5: Int. J. Cancer. May 29; 76(5):
749-56 (1998) [0021] Non-patent Document 6: Placenta, 28, 224
(2007) [0022] Non-patent Document 7: Clin. Cancer Res., 11, 6422
(2005) [0023] Non-patent Document 8: Mol. Cancer. Mar 12; 6: 20.
(2007) [0024] Non-patent Document 9: Cancer Genet. Cytogenet., 197,
16 (2010) [0025] Non-patent Document 10: Anticancer Res., 30, 1205
(2010) [0026] Non-patent Document 11: Gynecol. Oncol., 130(i):
132-9 (2013) [0027] Non-patent Document 12: J. Invest. Dermatol.,
13(5): 1278-85 (2013) [0028] Non-patent Document 13: Dig. Dis.
Sci., 58(4): 1010-5 (2013) [0029] Non-patent Document 14: J.
Proteome Res., 2013, 12 (10), pp. 4340-4350
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0030] An object of the present invention is to provide a method
for detecting ovarian clear cell adenocarcinoma which is highly
malignant, with high sensitivity and specificity, among benign and
malignant ovarian tumors having various tissue types, and a reagent
that can be used for the method,
Means for Solving the Problems
[0031] Therefore, the present inventors intensively generated
antibodies that show high affinity to the recombinant TFPI2 protein
derived from mammalian cells and TFPI2 protein derived from cancer
cells, and analysis of properties of these antibodies was carried
out. As a result, the present inventors discovered that intact
TFPI2 and a novel processed TFPI2 polypeptide (NT-TFP2) are present
in the culture supernatant of clear cell adenocarcinoma cells. The
present inventors also discovered that, when intact TFPI2 and
NT-TFPI2 are measured using an antibody that recognizes Kunitz
domain 1 of TFPI2 in ovarian tumor and uterine tumor, a higher
detection specificity for ovarian clear cell adenocarcinoma can be
obtained compared to cases where intact TFPI2 alone is measured, so
that NT-TFPI2 can be a detection marker for ovarian clear cell
adenocarcinoma, thereby completing the present invention.
[0032] That is, the present invention includes the following
embodiments. [0033] (1) A processed tissue factor pathway inhibitor
2 (TFPI2) polypeptide having the following properties (i) to
(iii):
[0034] (i) a polypeptide having the amino acid sequence from the
23th residue aspartic acid to the 131st residue histidine or to the
130th residue cysteine in the TFPI2 amino acid sequence of SEQ ID
NO:1, or a sequence having an identity of not less than 80% to this
sequence;
[0035] (ii) a polypeptide that is fractionated into a molecular
weight of about 16,000 by reducing SDS-PAGE; and
[0036] (iii) a polypeptide whose peptide fragment obtained after
asparagine-linked sugar chain cleavage treatment is fractionated
into a molecular weight of about 12,000 by reducing SDS-PAGE.
[0037] (2) A method for detecting ovarian clear cell
adenocarcinoma, the method comprising measuring the amount of the
processed TFPI2 polypeptide according to (1) in a sample. [0038]
(3) The method according to (2), further comprising measuring the
amount of intact TFPI2 in the sample. [0039] (4) The method
according to (3), wherein ovarian clear cell adenocarcinoma is
judged to be detected in cases where the total of the amount of the
processed TFPI2 polypeptide and the amount of intact TFPI2 exceeds
a reference value calculated from a control. [0040] (5) The method
according to any one of (2) to (4), wherein the measurement is
carried out by antigen-antibody reaction using an antibody that
binds to an antigenic determinant in the region from the 23th
residue aspartic acid to the 131st residue histidine or to the
130th residue cysteine in the TFPI2 amino acid sequence of SEQ ID
NO:1. [0041] (6) The method according to (5), wherein the antibody
is an antibody that recognizes Kunitz domain 1 of TFPI2. [0042] (7)
The method according to any one of (2) to (4), wherein the
measurement is carried out using mass spectrometry. [0043] (8) A
reagent for detecting ovarian clear cell adenocarcinoma, comprising
an antibody that binds to an antigenic determinant in the region
from the 23th residue aspartic acid to the 131st residue histidine
or to the 130th residue cysteine in the TFPI2 amino acid sequence
of SEQ ID NO:1.
Effect of the Invention
[0044] The present invention provides a novel detection marker for
ovarian clear cell adenocarcinoma. The present invention also
provides a method in which ovarian clear cell adenocarcinoma, which
is highly malignant, is detected as being positive with high
sensitivity and specificity among benign ovarian tumors and
malignant ovarian tumors having various tissue types, while benign
ovarian tumors and malignant ovarian tumors other than clear cell
adenocarcinoma are detected as being negative.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a diagram showing the results of FACS analysis of
cells to which a GPI-anchor type TFPI2 expression plasmid was
introduced, wherein the analysis was carried out using (a) an
anti-FLAG antibody or (b) an anti-BNC antibody as a negative
control.
[0046] FIG. 2 is a diagram showing the results of ELISA analysis of
secretory TFPI2. The ordinate represents the absorbance, and the
abscissa represents the amount of each solution added per well.
[0047] FIG. 3 is a diagram showing the results (a photograph) of
Western blotting analysis of secretory TFPI2.
[0048] FIG. 4 is a diagram showing the results of measurement of
the serum antibody titer in each mouse by CELISA analysis using
GPI-anchor type TFPI2. The ordinate represents the absorbance.
[0049] FIG. 5 is a diagram showing the results of measurement of
the serum antibody titer in each mouse by ELISA analysis using
secretory TFPI2. The ordinate represents the absorbance.
[0050] FIG. 6 is a diagram showing the results of analysis of each
monoclonal antibody by CELISA analysis using GPI-anchor type TFPI2.
The ordinate represents the absorbance, and the abscissa represents
the concentration of the antibody added.
[0051] FIG. 7 is a diagram (photographs) showing the results of
analysis of each monoclonal antibody by immunoprecipitation-Western
blotting (IP-WB) analysis using culture supernatants of three kinds
of ovarian cancer cells. The ordinate in each graph represents the
signal intensity per unit area.
[0052] FIG. 8-1 is a diagram (WB images) showing the results of
IP-WB analysis of three kinds of monoclonal antibodies using
culture supernatants of two kinds of ovarian cancer cells.
[0053] FIG. 8-2 is a diagram (Ruby-stained image) (photograph)
showing the results of IP-WB analysis of three kinds of monoclonal
antibodies using culture supernatants of two kinds of ovarian
cancer cells.
[0054] FIG. 8-3 is a diagram showing the results of amino acid
sequence analysis of IP products obtained with three kinds of
monoclonal antibodies using culture supernatants of two kinds of
ovarian cancer cells.
[0055] FIG. 9 is a diagram showing the results of analysis of
culture supernatants of various ovarian cancers using two kinds of
AIA assay reagents. "Rate" in the ordinate represents the amount of
4-methylumbelliferone produced per unit time [nmol/(Ls)].
[0056] FIG. 10-1 is a diagram showing the results of AIA analysis
of culture supernatants of three kinds of ovarian cancer obtained
with or without N-type sugar chain digestion treatment.
[0057] FIG. 10-2 is a diagram showing the recovery rate after
immunoprecipitation of culture supernatants of three kinds of
ovarian cancer with or without N-type sugar chain digestion
treatment.
[0058] FIG. 10-3 is a diagram showing WB images of culture
supernatants of three kinds of ovarian cancer obtained with or
without N-type sugar chain digestion treatment.
[0059] FIG. 11-1 is a diagram showing the results of AIA analysis
of changes in TFPI2 over time in culture supernatants of two kinds
of ovarian cancer.
[0060] FIG. 11-2 is a diagram (photographs) showing the results of
IP-WB analysis on changes in TFPI2 over time in culture
supernatants of two kinds of ovarian cancer.
[0061] FIG. 12-1 is a diagram showing the results of AIA analysis
of TFPI2 in five kinds of ovarian cancer cells.
[0062] FIG. 12-2 is a diagram (photographs) showing the results of
IP-WB analysis of TFPI2 in five kinds of ovarian cancer cells.
[0063] FIG. 13 is a diagram showing the results of AIA analysis on
changes in TFPI2 over time during the gestation period.
[0064] FIG. 14 is a diagram showing the results of AIA analysis of
TFPI2 and CA125 in a panel for various gynecological tumors. Each
horizontal line represents the median in each disease group.
[0065] FIG. 15 is a diagram showing box plots of the measured
values of TFPI2 and CA125 in a panel for various gynecological
tumors.
[0066] FIG. 16 is a diagram showing ROC curves of TFPI2 and CA125
for CCA and non-CCA ovarian tumor samples.
[0067] FIG. 17-1 is a diagram showing the results of analysis of
the C-terminal sequence of the NT-TFPI2 polypeptide. A: A
SYPRO-Ruby-stained image (photograph) of the antibody column-bound
fraction; B: Results of mapping of the sequence information
identified from Bands #1 to #3 on the TFPI2 amino acid
sequence.
[0068] FIG. 17-2 is a diagram showing the results of analysis of
the C-terminal sequence of the NT-TFPI2 polypeptide. C: Mass
spectrum charts of precursor ions of representative TFPI2-derived
peptides detected from Band #3.
MODE FOR CARRYING OUT THE INVENTION
[0069] <1> Processed Tissue Factor Pathway inhibitor 2
(TFPI2) Polypeptide of Present Invention
[0070] The polypeptide of the present invention is a processed
tissue factor pathway inhibitor 2 (TFPI2) polypeptide.
[0071] As shown in the later-described Examples, NT-TFPI2 was not
present in the intracellular fraction of cancer cells, and was
present only in the cell culture supernatant. Thus, NT-TFPI2 is
assumed to be a fragment polypeptide of TFPI2 that appears through
a process in which intact TFPI2 is secreted to the outside of
cancer cells, and the secreted intact TFPI2 is localized in the
extracellular matrix, followed by undergoing certain characteristic
processing.
[0072] NT-TFPI2 is a fragment containing Kunitz domain 1, which is
positioned in the N-terminal side of intact TFPI2. More
specifically, SEQ ID NO:1 is an amino acid sequence based on cDNA
of human TFPI2, and the region from the initiating methionine to
the 22nd residue glycine corresponds to a signal peptide, NT-TFPI2
has at least the sequence from the 23th residue aspartic acid to
the 131st residue histidine or to the 130th residue cysteine, which
is a sequence that follows the above region, or an amino acid
sequence having an identity of not less than 80% to the sequence.
The identity is preferably not less than 90%, more preferably not
less than 95%. The polypeptide of the present invention may be a
polypeptide having a sequence which is the same as the above
sequence except that one or several amino acids are deleted,
substituted, inserted, and/or added. The term "several" means
preferably 2 to 20, more preferably 2 to 10, still more preferably
2 to 5.
[0073] NT-TFPI2 may also have an amino acid sequence located in the
C-terminal side of the region from the 23th residue aspartic acid
to the 131st residue histidine or to the 130th residue cysteine in
the TFPI2 amino acid sequence of SEQ ID NO:1. For example, NT-TFPI2
containing histidine as the 131st amino acid or arginine as the
132nd amino acid is also preferred. NT-TPI2 preferably does not
contain the Kunitz domain 3 portion of TFPI2.
[0074] Although NT-TFPI2 may also have other peptide fragments in
both sides of the above sequence, it preferably does not have an
antigenic determinant for an antibody that recognizes Kunitz domain
3 of TFPI2.
[0075] Intact TFPI2 is a peptide represented by the region from the
23th residue to the 235th residue in the amino acid sequence of SEQ
ID NO:1.
[0076] NT-TFPI2 is fractionated into a molecular weight of about
16,000 by reducing SDS-PAGE. More specifically, for example, when
SDS-PAGE is carried out under reducing conditions according to a
conventional method using polyacrylamide gel with a gradient of 10
to 20% by mass, NT-TFPI2 is detected at a position which is
slightly shifted to the lower molecular weight side from the
position of the band corresponding to a molecular weight of 17,000
as indicated by a molecular weight marker, preferably Full Range
Rainbow Molecular Weight Marker (manufactured by GE
Healthcare).
[0077] In addition, the peptide fragment of NT-TFPI2 obtained after
asparagine-linked sugar chain cleavage treatment is fractionated
into a molecular weight of about 12,000 by reducing SDS-PAGE. The
asparagine-linked sugar chain cleavage treatment can be carried out
using N-glycanase or the like. When the resulting polypeptide, from
which the asparagine-linked sugar chain is released, is subjected
to, for example, SDS-PAGE under reducing conditions according to a
conventional method using polyacrylamide gel with a gradient of 10
to 20% by mass, the band of the polypeptide is detected at the
position corresponding to a molecular weight of 12,000 as indicated
by a molecular weight marker, preferably Full Range Rainbow
Molecular Weight Marker (manufactured by GE Healthcare).
[0078] In NT-TFPI2, an asparagine-linked sugar chain is attached to
the asparagine corresponding to the 116th residue from the
N-terminus of the TFPI2 amino acid sequence of SEQ ID NO:1.
<2> Method for Detecting Ovarian Clear Cell Adenocarcinoma of
Present Invention
[0079] The method for detecting ovarian clear cell adenocarcinoma
of the present invention comprises measuring the amount of NT-TFPI2
in a sample. This method is based on the fact that NT-TFPI2 is
characteristically extracellularly present in ovarian clear cell
adenocarcinoma cells, unlike in other tissue types. As shown by the
later-described Examples, by this method, ovarian clear cell
adenocarcinoma can be specifically detected with higher sensitivity
and specificity compared to cases where a conventionally known
tumor marker (CA125) or intact TFPI2 alone is measured.
[0080] In the detection method of the present invention, the amount
of intact TFPI2 may be measured in addition to the amount of
NT-TFPI2. This is because sufficient sensitivity and specificity
can be obtained also by carrying out judgment of detection of
ovarian clear cell adenocarcinoma based on the total amount of
NT-TFPI2 and intact TFPI2 in the sample. This is also because, as
described later, the total amount based on the measurement of both
of these and the measured amount of intact TFPI2 alone may be used
for indirect measurement of the amount of NT-TFPI2 to detect
ovarian clear cell adenocarcinoma.
[0081] In the detection method of the present invention, the method
for measuring the amount of NT-TFPI2 and/or the amount of intact
TFPI2 is not limited. Examples of the method include methods
utilizing antigen-antibody reaction in which an antibody that
recognizes NT-TFPI2 and/or intact TFPI2 is used, and methods
utilizing mass spectrometry.
[0082] Specific examples of the methods utilizing antigen-antibody
reaction in which an antibody that recognizes NT-TFPI2 and/or
intact TFPI2 is used include the following. [0083] (a) A
competition method using a labeled measuring object and an antibody
that recognizes the measuring object, which method utilizes
competitive binding of the labeled measuring object and the
measuring object contained in the sample to the antibody. [0084]
(b) A method using surface plasmon resonance, wherein the sample is
brought into contact with a chip on which an antibody that
recognizes the measuring object is immobilized, and a signal
dependent on binding of the antibody to the measuring object is
detected. [0085] (c) A fluorescence polarization immunoassay using
an antibody that recognizes a fluorescently labeled measuring
object, which immunoassay utilizes the phenomenon that binding of
the antibody to the measuring object causes an increase in the
degree of fluorescence polarization. [0086] (d) A sandwich method
using two kinds of antibodies (one of which is a labeled antibody)
that recognize the measuring object at different epitopes, wherein
formation of a complex of the three molecules, that is, the two
antibodies and the measuring object, is allowed to occur. [0087]
(e) A method in which pretreatment is carried out by concentrating
the measuring object in the sample using an antibody that
recognizes the measuring object, and the polypeptide in the bound
protein is detected using a mass spectrometer or the like.
[0088] Although the methods (d) and (e) are simple and versatile,
the method (d) is more preferred for processing of a large number
of samples since the technologies related to the reagents and the
devices for this method have been sufficiently established.
[0089] Specific examples of the methods for measuring the amount of
NT-TFPI2 and/or the amount of intact TFPI2 utilizing
antigen-antibody reaction include the following. [0090] (A) A
method using an antibody that recognizes both NT-TFPI2 and intact
TFPI2, wherein the total amount of NT-TFPI2 and intact TFPI2 is
measured (NT+I-TFPI2 assay system). The antibody that recognizes
both NT-TFPI2 and intact TFPI2 is preferably an antibody that binds
to an antigenic determinant in the region from the 23th residue
aspartic acid to the 131st residue histidine or to the 130th
residue cysteine in the TFPI2 amino acid sequence represented by
SEQ ID NO:1. The antibody is more preferably an antibody having an
antigenic determinant in Kunitz domain 1 of TFPI2. In cases where
the above-mentioned sandwich method is used in this method, two
kinds of antibodies for different epitopes are used as the
antibody. [0091] (B) A method using an antibody that does not
recognize NT-TFPI2 but recognizes intact TFPI2, wherein the amount
intact TFPI2 alone is measured (I-TFPI2 assay system). The antibody
that does not recognize NT-TFPI2 but recognizes intact TFPI2 is
preferably an antibody having an antigenic determinant in Kunitz
domain 3 of TFPI2. In cases where the above-mentioned sandwich
method is used in this method, two kinds of antibodies for
different epitopes are used as the antibody. At least one of these
is an antibody that does not recognize NT-TFPI2 but recognizes
intact TFPI2, and the other may be either an antibody that does not
recognize NT-TFPI2 but recognizes intact TFPI2, or an antibody that
recognizes both NT-TFPI2 and intact TFPI2. [0092] (C) A method in
which the amount of intact TFPI2 alone measured in the I-TFPI2
assay system of (B) is subtracted from the total amount of NT-TFPI2
and intact TFPI2 measured in the NT+I-TFPI2 assay system of (A), to
calculate the amount of NT-TFPI2 alone. [0093] (D) A method using
an antibody that does not recognize intact TFPI2 but recognizes
NT-TFPI2, wherein the amount NT-TFPI2 alone is measured. Examples
of the antibody that does not recognize intact TFPI2 but recognizes
NT-TFPI2 include antibodies that specifically recognize a peptide
sequence in the C-terminal portion of NT-TFPI2. For example, in
cases where the above-mentioned sandwich method is used, such an
antibody is used as the solid-phase antibody, and an antibody
having a recognition site in Kunitz domain 1 is used as the
detection antibody.
[0094] In the method for detecting ovarian clear cell
adenocarcinoma of the present invention, the amount of NT-TFPI2
alone measured by the method of (C) or (D) may be used as a
criterion. However, sufficient sensitivity and specificity can be
obtained also by using the total amount of NT-TFPI2 and intact
TFPI2 measured by the method of (A) as a criterion. The latter
method is more preferred from the viewpoint of the fact that the
antibody can be easily obtained, and that the measurement can be
simply carried out by a single step.
[0095] The antibody that recognizes NT-TFPI2 and/or intact TFPI2
can be obtained by immunizing an animal using as an immunogen, for
example, the NT-TFPI2 polypeptide or the intact TFPI2 protein
itself, an oligopeptide composed of a partial region of the
NT-TFPI2 polypeptide or the intact TFPI2 protein, or a
polynucleotide encoding the intact molecule or a partial region of
the NT-TFPI2 polypeptide or of the intact TFPI2 protein.
[0096] The animal to be used for the immunization is not limited as
long as the animal has ability to produce antibodies. The animal
may be a mammal normally used for immunization, such as mouse, rat,
or rabbit, or may be a bird such as chicken.
[0097] In cases where the NT-TFPI2 polypeptide or the intact TFPI2
protein itself, or an oligopeptide composed of a partial region of
the NT-TFPI2 polypeptide or the intact TFPI2 protein is used as an
immunogen, its structure may change during the process of preparing
the protein or the oligopeptide. Therefore, in some cases, the
antibody obtained may not have high specificity or binding capacity
to the desired antigen, so that quantification of the concentration
of TFPI2 contained in the sample may be inaccurate. On the other
hand, in cases where a protein expression vector containing a
polynucleotide encoding the intact molecule or a partial region of
the NT-TFPI2 polypeptide or of the intact TFPI2 protein is used as
an immunogen, the intact molecule or partial region of the NT-TFPI2
polypeptide or of the intact TFPI2 protein introduced is expressed
as it is without undergoing a structural change in the body of the
immunized animal. Therefore, an antibody having high specificity
and binding capacity (that is, high affinity) to the NT-TFPI2
polypeptide or intact TFPI2 in the sample can be obtained, which is
preferred.
[0098] The antibody that recognizes TFP12 may be either a
monoclonal antibody or a polyclonal antibody. The antibody is
preferably a monoclonal antibody.
[0099] The method of establishment of a hybridoma cell that
produces an antibody that recognizes NT-TFPI2 and/or intact TFP12
may be appropriately selected from methods whose techniques have
been established. For example, a hybridoma cell that produces a
monoclonal antibody that recognizes NT-TFPI2 and/or intact TFPI2
can be established by collecting B cells from an animal immunized
by the above method, fusing the B cells with myeloma cells
electrically or in the presence of polyethylene glycol, selecting a
hybridoma cell that produces a desired antibody using HAT medium,
and preparing the selected hybridoma cell into a monoclone by the
limiting dilution method.
[0100] The selection of the antibody that recognizes NT-TFPI2
and/or intact TFPI2, for example, the monoclonal antibody that
recognizes NT-TFP12 and/or intact TFP12, used in the method for
detecting ovarian clear cell adenocarcinoma of the present
invention may be carried out based on affinity to GPI
(glycosylphosphatidylinositol)-anchor type TFPI2 or secretory TFPI2
derived from a host expression system.
[0101] The host is not limited, and may be appropriately selected
from microorganism cells such as E. coli or yeast, insect cells,
and animal cells that are usually used for protein expression by
those skilled in the art. The host is preferably a mammalian cell
since it enables expression of a protein having a structure similar
to that of natural NT-TFPI2 and/or intact TFPI2 by
post-translational modification such as disulfide bonding or
glycosylation. Examples of the mammalian cell include the human
embryonic kidney (HEK)-derived 293T cell line, monkey kidney CCS7
cell line, Chinese hamster ovary (CHO) cells, and cancer cells
isolated from human.
[0102] The method of purification of the antibody to be used in the
method for detecting ovarian clear cell adenocarcinoma of the
present invention may be appropriately selected from methods whose
techniques have been established. For example, after culturing
hybridoma cells which are established by the above method and which
produce an antibody, the culture supernatant may be collected, and
the antibody may be concentrated, if necessary, by ammonium sulfate
precipitation. Thereafter, by, affinity chromatography using a
carrier to which Protein A, Protein C, Protein L, or the like is
immobilized, and/or by ion-exchange chromatography, purification of
the antibody is possible.
[0103] The labeled antibody used for the antigen-antibody reaction
in the sandwich method described above may be prepared by labeling
an antibody purified by the above method with an enzyme such as
peroxidase or alkaline phosphatase. The labeling may also be
carried out using a method whose technique has been sufficiently
established.
[0104] The method for measuring the amount of NT-TFPI2 and/or the
amount of intact TFPI2 utilizing mass spectrometry in the detection
method of the present invention is described below concretely.
[0105] In cases of a blood sample, a pretreatment step is
preferably carried out by removing proteins contained in large
amounts in blood such as albumin, immunoglobulin, and transferrin
using Agilent Human 14 or the like, and performing further
fractionation by ion exchange, gel filtration, reverse-phase HPLC,
and/or the like.
[0106] The measurement can be carried out by tandem mass
spectrometry (MS/MS), liquid chromatography-tandem mass
spectrometry (LC/MS/MS), matrix assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF/MS), surface
enhanced laser desorption ionization mass spectrometry (SELDI-MS),
or the like.
[0107] In the detection method of the present invention, ovarian
clear cell adenocarcinoma is preferably judged to be detected when
the amount of NT-TFPI2 obtained by the measurement is higher than a
reference value (cutoff value) calculated from a control.
Alternatively, ovarian clear cell adenocarcinoma is preferably
judged to be detected when the total of the amount of NT-TFPI2 and
the amount of intact TFPI2 obtained by the measurement is higher
than a reference value (cutoff value) calculated from a
control.
[0108] The amount of NT-TFPI2 and the amount of intact TFPI2 used
for the judgment may be either measured values or converted
concentration values. The converted concentration value means a
value converted from the measured value based on a calibration
curve prepared using TFPI2 as a standard sample.
[0109] The reference value (cutoff value) may be appropriately set
to a measured value which provides optimum sensitivity and
specificity, by carrying out measurement for
non-clear-cell-adenocarcinoma samples such as
non-clear-cell-adenocarcinoma ovarian tumors, uterine tumors,
and/or samples from healthy individuals, as well as for
clear-cell-adenocarcinoma samples, and then carrying out receiver
operating characteristic (ROC) curve analysis. More specifically,
for example, in cases where serum is used as the sample, the
reference value (cutoff value) of the total of the amount of
NT-TFPI2 and the amount of intact TFPI2 may be set to 1.9, which is
a measured value (rate:nmol/(Ls)) calculated based on the
concentration of 4-methylumbelliferone produced by alkaline
phosphatase per unit time.
<3> Reagent for Detecting Ovarian Clear Cell Adenocarcinoma
of Present Invention
[0110] The reagent for detecting ovarian clear cell adenocarcinoma
of the present invention comprises an antibody that binds to an
antigenic determinant in the region from the 23th residue amino
acid to the 131st residue or 130th residue amino acid in the TFPI2
amino acid sequence of SEQ ID NO:1. The antibody is preferably an
antibody that recognizes Kunitz domain 1 of TFPI2. The antibody can
recognize both NT-TFPI2 and intact TFPI2.
[0111] In cases where the reagent of the present invention is used
in the sandwich method described above, the reagent preferably
contains, as the antibody, two kinds of antibodies for different
epitopes.
[0112] The reagent for detecting ovarian clear cell adenocarcinoma
of the present invention may further contain a reagent for
detecting a tumor marker for ovarian cancer containing an antibody
that recognizes the tumor marker for ovarian cancer. Examples of
the tumor marker for ovarian cancer include CA125.
[0113] The antibody contained in the reagent of the present
invention may be an antibody itself, a labeled antibody, or an
antibody immobilized on a solid phase.
[0114] The reagent of the present invention is described below
concretely for cases where it is used for a two-step sandwich
method, which is one mode of the sandwich method. However, the
present invention is not limited thereto.
[0115] The reagent of the present invention can be prepared by the
method described in the following (I) to (III). [0116] (I) First,
Antibody 1, one of the two kinds of antibodies for different
epitopes that recognize NT-TFPI2 and intact TFPI2 (hereinafter
referred to as "Antibody 1" and "Antibody 2"), is bound to a
carrier capable of B/F (Bound/Free) separation such as an
immunoplate or magnetic particles. The binding method may be either
physical binding utilizing hydrophobic boning, or chemical bonding
using a linker reagent capable of cross-linking two substances to
each other. [0117] (II) After the binding of the Antibody 1 to the
carrier, the carrier surface is subjected to blocking treatment
using bovine serum albumin, skim milk, a commercially available
immunoassay blocking agent, or the like for preventing non-specific
binding, to provide a primary reagent. [0118] (III) After labeling
the other antibody, Antibody 2, a solution containing the obtained
labeled antibody is provided as a secondary reagent. Preferred
examples of the substance with which Antibody 2 is labeled include
enzymes such as peroxidase and alkaline phosphatase; substances
detectable with detection devices, such as fluorescent substances,
chemiluminescent substances, and radioisotopes; and substances to
which another molecule specifically binds, such as biotin, to which
avidin specifically binds. Preferred examples of the solution for
the secondary reagent include buffers with which antigen-antibody
reaction can be favorably carried out, such as phosphate buffer and
Tris-HCl buffer.
[0119] The thus prepared reagent of the present invention may be
freeze-dried, if necessary.
[0120] In cases of a one-step sandwich method, binding of Antibody
1 to the carrier and subsequent blocking treatment may be carried
out in the same manner as in (I) and (II) to prepare an
antibody-immobilized carrier, and a buffer containing a labeled
Antibody 2 may be further added to the antibody-immobilized
carrier, to provide a reagent.
[0121] For measurement of NT-TFPI2 and intact TFPI2 by a two-step
sandwich method using reagents obtained by the method described
above, the method described in the following (IV) to (VI) may be
carried out. [0122] (IV) The primary reagent prepared in (II) is
brought into contact with a sample for a predetermined period of
time at a constant temperature. In terms of the reaction
conditions, the reaction may be carried out at a temperature within
the range of 4.degree. C. to 40.degree. C. for 5 minutes to 180
minutes, [0123] (V) Unreacted substances are removed by B/F
separation, and then the secondary reagent prepared in (III) is
brought into contact with the resulting reaction product for a
predetermined period of time at a constant temperature to allow
formation of a sandwich complex. In terms of the reaction
conditions, the reaction may be carried out at a temperature within
the range of 4.degree. C. to 40.degree. C. for 5 minutes to 180
minutes. [0124] (VI) Unreacted substances are removed by B/F
separation, and the labeling substance of the labeled antibody is
quantified. Based on a calibration curve prepared using a TFPI2
solution having a known concentration as a standard sample, the
concentration of human NT-TFPI2 and intact TFPI2 in the sample is
quantified.
[0125] The above description on the TFPI2 detection reagent also
applies to the reagent for detecting a tumor marker for ovarian
cancer. The reagent for detecting a tumor marker for ovarian cancer
may be one prepared in the same manner as the reagent of the
present invention, or may be a commercially available product.
[0126] The amount of each reagent component such as the antibody
contained in the detection agent may be appropriately set depending
on conditions such as the amount of the sample, the type of the
sample, the type of the reagent, and the detection method. More
specifically, for example, in cases where the amount of NT-TFPI2
and the amount of intact TFPI2 are measured as described below by a
sandwich method using 50 .mu.L of 2.5-fold diluted serum or plasma
as a sample, the amount of the antibody to be bound to the carrier
may be 100 ng to 1000 .mu.g, and the amount of the labeled antibody
may be 2 ng to 20 .mu.g per the reaction system in which 50 .mu.L
of the sample is reacted with the antibodies.
[0127] The reagent for detecting ovarian clear cell adenocarcinoma
of the present invention is applicable to either manual detection
or detection using an automatic immunodiagnostic device. Detection
using an automatic immunodiagnostic device is especially preferred
since it enables the detection without being influenced by
endogenous measurement-inhibiting factors and competing enzymes
contained in the sample, and also enables rapid quantification of
the concentrations of NT-TFPI2 and intact TFPI2, as well as tumor
markers for ovarian cancer,
[0128] Examples of the sample (test sample) to be subjected to the
method for detecting ovarian clear cell adenocarcinoma of the
present invention and the detection reagent of the present
invention include blood components such as whole blood, blood
cells, serum, and plasma; extracts from cells and tissues; urine;
and cerebrospinal fluid. Although an ovarian tissue biopsy sample
may be subjected to the test, a culture supernatant of the biopsy
sample is used as the sample in such cases. A blood component or a
body fluid such as urine is preferably used as the sample since it
allows simple noninvasive detection of ovarian clear cell
adenocarcinoma. From the viewpoint of simplicity of sample
collection and versatility for other test items, use of a blood
component as the sample is especially preferred. The dilution rate
of the sample may be appropriately selected from no dilution to
100-fold dilution depending on the type and the conditions of the
sample used. For example, 50 .mu.L of a 2.5-fold diluted sample may
be used in cases of serum or plasma.
EXAMPLES
[0129] Examples are shown below for concrete description of the
present invention. However, these Examples merely show examples of
the present invention, and the present invention is not limited by
the Examples.
Example 1
Construction of Vector for DNA Immunization
[0130] For efficient induction of humoral immunity by DNA
immunization, it is preferred to localize the subject antigen
protein on the cell surface as a membrane-bound protein. Since
TFPI2 is originally a secretory protein, a plasmid vector that can
express a protein in which a GPI (glycosylphosphatidylinositol)
anchor is attached to the C-terminal side of TFPI2 (hereinafter
referred to as GPI-anchor type TFPI2) was constructed for allowing
localization of TFPI2 on the cell surface. [0131] (1) Using the
following primers (a), a polynucleotide composed of the 73rd to
705th bases of TFPI2 cDNA (GenBank No. NM_006528) was amplified by
RT-PCR according to a conventional method. [0132] (a) Primers for
GPI-anchor Type TFPI2 Expression Plasmid Forward:
[0133] 5'-cgatgacgacaagettgetcaggagccaaca-3' (SEQ ID NO:2; wherein
the 15 bases in the 3'-end side correspond to the base sequence
from position 73 to position 87 in GenBank No. NM_006528)
[0134] Reverse:
[0135] 5'-catcagtggtgaattcaaattgcttcttccg-3' (SEQ ID NO:3; wherein
the 15 bases in the 5'-end side correspond to the base sequence
from position 6.sup.-91 to position 705 in GenBank No. NM_006528)
[0136] (2) Into the HindIII-EcoRI site of pFLAG1 (manufactured by
SIGMA), which contains the coding region for the GPI anchor of
placental alkaline phosphatase and the coding region for a FLAG
tag, the RT-PCR amplification product obtained in (1) was inserted
using in-fusion (manufactured by Clontech) according to its
protocol, to construct a GPI-anchor type TFPI2 expression plasmid
in which the FLAG tag peptide is attached to the N-terminal side,
and the GPI anchor is attached to the C-terminal side. [0137] (3)
In order to confirm that TFPI2 expressed by the polynucleotide
inserted in the expression plasmid constructed in (2) is localized
on the cell surface as assumed, the following test was carried out
using the 293T cell line, which is a transiently expressing cell.
[0138] (3-1) The GPI-anchor type TFPI2 expression plasmid
constructed in (2) was transfected into the 293T cell line
according to a conventional method. [0139] (3-2) The transfected
293T cells were cultured in a 5% CO.sub.2 incubator using D-MFM
medium supplemented with 10% FBS (fetal bovine serum) (manufactured
by Wako Pure Chemical industries, Ltd.) for 24 hours at 37.degree.
C., to allow transient expression of TFPI2 protein. [0140] (3-3) To
the cultured cells obtained in (3-2), a mouse anti-FLAG M2 antibody
manufactured by SIGMA, which specifically binds to the FLAG tag, or
a mouse anti-BNC antibody as a negative control, which does not
bind to the FLAG tag, was added, and the cells were incubated for
30 minutes. BNC is a peptide composed of seven amino acids in the
C-terminal side of BNP (brain natriuretic peptide) (JP 2009-240300
A). [0141] (3-4) Thereafter, a fluorescently labeled anti-mouse IgG
antibody (manufactured by BECKMAN COULTER) was added to the cells,
and the cells were incubated for 30 minutes, followed by performing
FAGS (Fluorescence Activated Cell Sorting) analysis.
[0142] The results of the FAGS analysis are shown in FIG. 1. As a
result of the analysis, in the case of the negative control,
wherein the anti-BNC antibody was added (FIG. 1(b)), no increase in
the signal, that is, no shift, due to fluorescently labeled cells
was found. On the other hand, a shift due to fluorescently labeled
cells was found in the case where the anti-FLAG antibody was added
(FIG. 1(a)). From these results, it was shown that the GPI-anchor
type TFPI2 to which the FLAG tag peptide is attached was localized
on the cell surface after its protein expression,
Example 2
Immunization and Blood Collection
[0143] Immunization of mice was carried out by administering, to
four Balb/c mice, 100 .mu.L of a PBS solution prepared such that
the solution contains 40 .mu.g of the GPI-anchor type TFPI2
expression plasmid constructed in Example 1 (2) in terms of the
amount of DNA. On Day 7, Day 14, Day 21, Day 28, and Day 35 after
the first immunization, additional administration was carried out.
On Day 42 after the first immunization, blood was collected to
prepare antiserum to provide antisera A-1 to A-4.
Example 3
Preparation of GPI-Anchor Type TFPI2 Constantly Expressing
Cells
[0144] For evaluation of the antisera, a
Chinese-hamster-ovary-derived cell line CHO-K1 that can constantly
express the GPI-anchor type TFPI2 was prepared by the following
method. [0145] (1) The TFPI2 expression plasmid constructed in
Example 1 [0146] (2) was introduced into the CHO-K1 cell line by
gene transfer according to a conventional method. Thereafter, the
cells were cultured in a 5% CO.sub.2 incubator using Hams F12
medium supplemented with 10% FBS (manufactured by Wako Pure
Chemical Industries, Ltd.) for 24 hours at 37.degree. C. [0147] (2)
Thereafter, a solution of the antibiotic Geneticin (manufactured by
Invitrogen) was added to the culture at 250 .mu.g/mL, and culture
was carried out for additional three weeks. [0148] (3) CHO-K1 cells
that constantly express the GPI-anchor type TFPI2 were obtained
with an anti FLAG antibody using a cell sorter.
Example 4
Construction of Secretory TFPI2 Expression Plasmid
[0149] In the TFPI2 expression plasmid constructed in Example 1
(2), an oligonucleotide encoding a BNC peptide composed of seven
amino acids the C-terminal side of BNP (brain natriuretic peptide)
(Patent Document 5) was further inserted into the position between
the inserted TFPI2 gene and the GPI anchor-coding region present in
its 3'-end side. By this, a plasmid capable of expressing a
secretory TFPI2 having the FLAG peptide in the N-terminal side and
the BNC peptide in the C-terminal side, but having no GPI anchor
was prepared. The preparation method is described below more
concretely. [0150] (1) Using the following primers (b), a
polynucleotide in which an oligonucleotide encoding the BNC peptide
is added to the 3'-end side of intact TFPI2 cDNA excluding the
start codon and the stop codon (the region from position 148 to
position 780 in GenBank No. NM_016528) was amplified by RT-PCR
according to a conventional method. [0151] (b) Primers for
Secretory TFPI2 Expression Plasmid Forward:
[0152] 5'-cgatgacgacaagottgctcaggagccaaca-3' (SEQ ID NO:4; wherein
the 15 bases in the 3'-end side correspond to the base sequence
from position 73 to position 87 in GenBank No. NM_006528) [0153]
Reverse:
[0154]
5'-agcatcagtggtgaattctcattagtggcgacgcagaactttgcaaaattgcttcttccg-3'
(SEQ ID NO:5; wherein the 15 bases in the 5'-end side correspond to
the base sequence from position 691 to position 705 in GenBank No.
NM_006528) [0155] (2) Into the HindIII-EcoRI site of pFLAG1
(manufactured by SIGMA), which is a plasmid containing the GPI
anchor region of placental alkaline phosphatase, the RT-PCR
amplification product of (1) was inserted using in-fusion
(manufactured by Clontech) according to its protocol, to construct
a secretory TFPI2 expression plasmid. [0156] (3) In order to
confirm that the secretory TFPI2 expressed from the polynucleotide
inserted in the pFLAG1 has the FLAG tag in the N-terminal side and
the BNC tag in the C-terminal side, the following test was carried
out using the 293T cell line, which is a transiently expressing
cell. [0157] (3-1) In the same manner as described in Example 1,
the secretory TFPI2 expression plasmid constructed in (2) was
transfected into the 293T cell line, and secretory TFPI2 protein
was transiently expressed. The culture liquid after 72 hours of
culture was centrifuged, and the resulting supernatant was
collected as a secretory TFPI2 protein solution. [0158] (3-2) Using
the secretory TFPI2 protein solution as a sample, (A) an enzyme
immunoassay (ELISA) and (B) Western blotting (WB) were carried
out.
(A) ELISA
[0158] [0159] (A-1) A rabbit anti-FLAG polyclonal antibody
(manufactured by ROCKLAND) was diluted with carbonate buffer (pH
9.8) such that its amount became 100 ng/well, and then immobilized
on a MaxiSorp 96-well plate (manufactured by NUNC). [0160] (A-2)
After allowing the reaction to proceed at 4.degree. C. overnight,
the ate was washed three times with TBS (Tris-Buffered Saline), and
TBS solution supplemented with 3% bovine serum albumin (BSA) was
added to each well at 250 .mu.L/well. The plate was incubated at
room temperature for 2 hours. [0161] (A-3) The plate was then
washed three times with TBS. A secretory TFPI2 protein solution
and, as a negative control, a culture supernatant of the 293T cell
line which was not transfected TFPI2 expression plasmid were added
thereto at 50 .mu.L/well. The plate was incubated at room
temperature for 1 hour. [0162] (A-4) After washing the plate three
times with TBS supplemented with 0.5% Tween 20 (TBS-T), a mouse
anti-BNC monoclonal antibody solution diluted to 1 .mu.g/mL with
TBS-T supplemented with 1% ESA (1% BSA/TBS-T) was added to the
plate at 50 .mu.L/well. The plate was incubated at room temperature
for 1 hour. [0163] (A-5) After washing the plate three times with
TBS-T, a horseradish peroxidase (HRP)-labeled anti-mouse
immunoglobulin G-Fc antibody (manufactured by SIGMA) solution
10,000-fold diluted with 1% BSA/TES-T was added to the plate at 50
.mu.L/well, and the plate was incubated at room temperature for 1
hour. [0164] (A-6) After washing the plate four times with TBS-T,
TMB Microwell Peroxidase Substrate (manufactured by KPL) was added
to the plate, and the reaction was stopped with 1 mol/L phosphoric
acid solution, followed by measuring the absorbance at 450 nm using
an absorbance measurement plate reader.
(B) Western Blotting
[0164] [0165] (B-1) The secretory TFPI2 protein solution obtained
in (B-1), and, as a negative control, a culture supernatant of the
293T cell line which was not transfected TFPI2 expression plasmid,
were separated by SDS-PAGE, and transferred to a PVDF membrane
(manufactured by GE Healthcare). [0166] (B-2) The membrane was
blocked by reaction with TBS-T supplemented with 5% skim milk
(blocking solution) at room temperature for 2 hours, and an
alkaline phosphatase-labeled anti-BNC antibody was added to the
blocking solution at 1 .mu.g/sheet, incubated at 4.degree. C.
overnight. [0167] (B-3) The membrane was washed with TBS-T, and the
obtained chemiluminescence was detected with a photosensitive film
using Western Lightning CDP-Star (manufactured by Perkin-Elmer
Corp.).
[0168] The analysis results obtained by ELISA are shown in FIG. 2.
In the case of the secretory TFPI2 protein solution (culture
supernatant of secretory TFPI2), clear signals could be observed in
a manner dependent on the amount of the sample added, unlike the
case of the negative control (culture supernatant of non
transfected 293T). Thus, production of the secretory TFPI2 protein
into the culture supernatant was shown.
[0169] The analysis results obtained by Western blotting are shown
in FIG. 3. In the case of the secretory TFPI2 protein solution
(culture supernatant of secretory TFPI2), a clear band was detected
near a molecular weight of about 35 kDa. Thus, production of
secretory TFPI2 protein having the FLAG tag in the N-terminus and
the BNC tag in the C-terminus into the culture supernatant was
shown.
Example 5
Evaluation of Mouse Antisera
[0170] The mouse antisera collected in Example 2 were analyzed by
cell enzyme immunoassay (CELISA) using the GPI-anchor type TFPI2
expression CHO-K1 cells prepared in Example 3, and by ELISA using
the secretory TFPI2 protein solution obtained in Example 4, For
studying specificity, expression plasmids that express a non-TFPI2
protein as a GPI-anchor type (having a FLAG tag in the N-terminal
side and a GPI anchor in the C-terminal side) or as a secretory
type (having a FLAG tag in the N-terminal side and a BNC tag in the
C-terminal side) were constructed based on a known gene sequence in
the same manner as the TFPI2 expression plasmid described above,
and the constructed expression plasmids were transfected into the
293T cell line or CHO-K1 cells. The non-TFPI2 protein is
hereinafter also referred to as control protein.
(1) CELISA Analysis
[0171] (1-1) To a 96-well plate, the GPI-anchor type TFPI2
expression CHO-K1 cells prepared in Example 3, and, as negative
control cells, CHO-K1 cells that express the control protein as a
GPI-anchor type, were added at 5.times.10.sup.4 cell/well, and the
cells were cultured in a 5% CO.sub.2 incubator using Hams F12
medium supplemented with 10% FES (manufactured by Wako Pure
Chemical Industries, Ltd.) for 24 hours at 37.degree. C., [0172]
(1-2) To the GPI-anchor type TFPI2-expressing cells and the
negative control cells, each of 2000-fold diluted antisera (A1 to
A4) or a mouse anti-FLAG M2 antibody (manufactured by SIGMA) was
added as a primary antibody, and the reaction was incubated at room
temperature for 1 hour. [0173] (1-3) After the reaction, the plate
was washed, and a horseradish peroxidase (HRP)-labeled anti-mouse
immunoglobulin G-Fc antibody (manufactured by SIGMA) was added to
the plate as a secondary antibody, incubated at room temperature
for 1 hour. [0174] (1-4) After the incubation, the plate was
washed, and TMB Microwell Peroxidase Substrate (manufactured by
KPL) was added to the plate. The reaction was then stopped with 1
mol/L phosphoric acid solution, and the absorbance at 450 nm was
measured using an absorbance measurement plate reader.
(2) ELISA Analysis
[0175] Each mouse antiserum was evaluated by the same method as in
Example 4(A) except that a culture supernatant of the 293T cell
line that expresses the control protein as a secretory type
(hereinafter also referred to as secretory-type control protein
solution) was used as the negative control.
[0176] The results of the CELISA analysis are shown in FIG. 4. In
the negative control cells, signals could be hardly found for any
of the antisera. In contrast, the TFPI2-expressing cells showed
clear signals. From these results, it was shown that anti-TFPI2
antisera having high specificity could be obtained by the DNA
immunization carried out in Example 2.
[0177] The results of the ELISA analysis are shown in FIG. 5.
Similarly to the results of CELISA, when the antisera (A1 to A4)
were used, clear signals were found only for TFPI2. Thus, this
method also showed that antisera having high specificity could be
obtained by the DNA immunization carried out in Example 2.
[0178] The results shown in FIG. 4 and FIG. 5 together indicate
that the mouse antisera obtained in Example 2 are antisera having
high specificity to both the GPI anchor type TFPI2 and the
secretory TFPI2.
Example 6
Establishment of Hybridomas
[0179] Hybridomas capable of producing antibodies against TFPI2
were established by the following method. [0180] (1) From mice in
which an increase in the antibody titer due to the DNA immunization
was found in Example 2, spleen cells were collected, and splenic
cells were obtained therefrom. [0181] (2) Cell fusion was carried
out with the splenic cells collected and the mouse myeloma cell
line SP2/0 in the presence of polyethylene glycol according to a
conventional method. [0182] (3) By culturing the resulting cells
with HAT (manufactured by Sigma-Aldrich)/GIT medium (manufactured
by Wako Pure Chemical Industries, Ltd.) for about 10 days,
selection of antibody-producing cell hybridomas was carried out.
[0183] (4) Culture supernatants of the selected antibody-producing
cell hybridomas were subjected to the CELISA described in Example 5
(1) and the ELISA described in Example 4 (A) to perform screening
of hybridomas capable of producing antibodies against TFPI2. [0184]
(5) From the cells in the wells selected by the screening,
monoclones were obtained by the limiting dilution method, and the
monoclones were subjected to acclimation culture from HT
(manufactured by Sigma-Aldrich)/GIT medium to GIT medium. Finally,
10 kinds of hybridomas (TS-TF01 to TS-TF10) were established.
Example 7
Identification of Antigenic Determinants
[0185] The antigenic determinant of each antibody was identified
using cells expressing variants of KD1, KD2, and KD3, which are
Kunitz domains of TFPI2. The preparation method for plasmids that
express the variants is described below concretely. [0186] (1)
Using the primers described in the following (c), (d), and (e),
polynucleotides corresponding to the KD1 region, the KD2 region, or
the region from KD3 to the C-terminus, of TFPI2 were amplified by
RT-PCR according to a conventional method. [0187] (c) Primers for
GPI-Anchor Type TFPI2-KD1
[0188] Forward:
[0189] 5'-cgatgacgacaagettgetcaggagccaaca-3' (SEQ ID NO:6; wherein
the 15 bases in the 3'-end side correspond to the base sequence
from position 73 to position 87 in GenBank No. NM_006528)
[0190] Reverse:
[0191] 5'-catcagtggtgaattotttttctatcctcca-3' (SEQ ID NO:7; wherein
the 15 bases in the 5'-end side correspond to the base sequence
from position 259 to position 273 in GenBank No. NM_006528) [0192]
(d) Primers for GPI-Anchor Type TFPI2-KD2
[0193] Forward:
[0194] 5'-cgatgacgacaagettgttcccaaagtttgc-3' (SEQ ID NO:8; wherein
the 15 bases in the 3'-end side correspond to the base sequence
from position 274 to position 288 in GenBank No. NM_006528)
[0195] Reverse:
[0196] 5'-catcagtggtgaattctttctttggtgcgca-3' (SEQ ID NO:9; wherein
the 15 bases in the 5'-end side correspond to the base sequence
from position 445 to position 459 in GenBank No. NM_006528) [0197]
(e) Primers for GPI-Anchor Type TFPI2-KD3
[0198] Forward:
[0199] 5'-cgatgacgacaagcttattccatcattttqc-3' (SEQ ID NO:10; wherein
the 15 bases in the 3'-end side correspond to the base sequence
from position 460 to position 474 in GenBank No. NM_006528)
[0200] Reverse:
[0201] 5'-catcagtggtgaattcaaattgcttcttccg-3' (SEQ ID NO:11; wherein
the 15 bases in the 5'-end side correspond to the base sequence
from position 691 to position 705 in GenBank No. NM_006528) [0202]
(2) Three kinds of GPI-type TFPI2 expression plasmids were
constructed by the method described in Example 1 (2). [0203] (3)
Transiently expressing cells for the three kinds of polypeptides
were prepared by the method described in Example 3, and the
antigenic determinants of the 10 kinds of monoclonal antibodies
described in Example 6 were identified.
[0204] Table 1 shows the antigenic determinant of each antibody
revealed from the result of FACS analysis.
TABLE-US-00001 TABLE 1 Antigen determinant KD1 KD3 1 TS-TF01
TS-TF05 2 TS-TF02 TS-TF06 3 TS-TF03 TS-TF07 4 TS-TF04 TS-TF08 5
TS-TF09 6 TS-TF10
Example 8
Preparation of Monoclonal Antibodies and CELISA Analysis Using
Purified Antibodies
[0205] From the 10 kinds of hybridomas established in Example 6,
monoclonal antibodies against TFPI2 (anti-TFPI2 monoclonal
antibodies) were prepared by the following method, and CELISA
analysis was carried out. [0206] (1) With 50% ammonium sulfate, 300
mL of the culture supernatant of each hybridoma established in
Example 6 was fractionated, and the obtained fraction was dialyzed
against TBS (Tris-Buffered Saline; 10 mM Tris-HCl+150 mM NaCl (pH
7.4)), followed by purification of a monoclonal antibody by the
following method using HiTrap Protein G HP (manufactured by GE
Healthcare). [0207] (1-1) The column was preliminarily subjected to
buffer replacement with PBS (Phosphate Buffer Saline; 10 mM
phosphoric acid 150 mM NaCl (pH 7.4)), and the culture supernatant
of the hybridoma was passed through the column at a flow rate of 10
mL/min. [0208] (1-2) The column was sufficiently washed with 5
column volumes of PBS to remove unbound protein. In this process,
by confirming that the absorbance at OD280 of the buffer that had
passed through the column was not more than 0.01, unbound protein
was judged not to be remaining. [0209] (1-3) After washing the
column, the bound antibody was eluted with an eluent (100 mM
glycine (pH 2.5)). The eluted antibody was immediately neutralized
by adding 1/10 volume of 1 M Tris (pH 8.0), and immediately
subjected to dialysis with TBS, followed by quantification of the
protein concentration of the purified antibody using an absorption
spectrometer. [0210] (2) From a mouse anti-FLAG M2 antibody
(manufactured by SIGMA) as a positive control, an anti-TFPI2 P-2
antibody (manufactured by Santa Cruz) as a comparative control, and
the above 10 kinds of anti-TFPI2 antibodies, antibody dilutions
were prepared using 1% FES/PBS solution such that the amount of
each antibody added was 200 ng/well to 2.5 ng/well. [0211] (2-1)
The antibody dilutions were subjected to CELISA analysis by the
method described in Example 5 (1). Only in the cases where the
antibody was added at. 200 ng/well, analysis was carried out for
both the TFPI2-expressing cells and the control cells. In the cases
where the antibody dilutions at not more than 66.6 ng/well were
used, analysis was carried out only for TFPI2-expressing cells. The
results of the CELISA analysis are shown in FIG. 6. The anti-FLAG
antibody, which is a positive control, showed clear signals in both
the TFPI2-expressing cells and the negative control cells. The
anti-TFPI2 P-2 antibody, which is a comparative control, showed no
signal in the TFPI2-expressing cells. Since this antibody is for
use in WB, that is, for use in detection of denatured protein, it
is very natural that no reactivity can be found in this analysis
system, wherein TFPI2 having a naturally occurring higher-order
structure is used. On the other hand, the above 10 kinds of
anti-TFPI2 antibodies generally showed specific and
concentration-dependent signals in the TFPI2-expressing cells
although the signal varied among the antibodies.
Example 9
Evaluation of Immunoprecipitability of Magnetic Beads on which
Monoclonal Antibodies are Immobilized
[0212] Magnetic particles on which the 10 kinds of anti-TFPI2
monoclonal antibodies prepared in Example 8 are immobilized were
prepared, and proteins in the culture supernatant of ovarian clear
cell adenocarcinoma cells that specifically bind to the antibodies
were identified by the following method. As the ovarian clear cell
adenocarcinoma cells, three kinds of cells OVISE, OVMANA, and
OVSAYO were used. [0213] (1) Part of the purified monoclonal
antibodies obtained in Example 8 were immobilized on Dynabeads
M-280 Tosylactivated magnetic particles (manufactured by
Invitrogen), and blocking was carried out with PBS supplemented
with 0.5% BSA, to prepare antibody-immobilized magnetic particles.
[0214] (2) Immunoprecipitation-Western :Blotting Method (IP-WB
Method) [0215] (2-1) The three kinds of cancer cells were cultured
in RPMI 1640 medium (manufactured by Wako Pure Chemical Industries,
Ltd.) supplemented with 10% fetal bovine serum at 100% confluence
for three days. [0216] (2-2) After centrifugation of the culture
supernatant, each of the ten kinds of antibody-immobilized magnetic
particles prepared by the method described in Example 8 were added
to 0.1 mL of the supernatant, and the resulting mixture was stirred
at room temperature for 1 hour to allow the reaction to proceed.
[0217] (2-3) The antibody-immobilized magnetic particles were
washed twice with PBST-NP40 (0.1% Tween 20, 1% NP40), and then
three times with PBS that does not contain a detergent. [0218]
(2-4) Proteins bound to each kind of antibody-immobilized magnetic
particles were analyzed by the Western blotting described in
Example 4 (E). As molecular weight markers, Full-Range Rainbow
Molecular Weight Markers (manufactured by GE) were used. As the gel
for the SDS-PAGE, a 10-20% gradient gel (manufactured by Marysol)
was used. For the Western blotting, a detection antibody was
prepared by labeling an anti-TFPI2 peptide antibody that recognizes
the N-terminus of intact TFPI2 (see Non-patent Document 6) using an
alkaline phosphatase labeling kit (manufactured by Dojindo
Laboratories), which antibody was established by the department of
gynecology, Yokohama City University. The detection was carried out
by the method described in Example 4. The WB data was analyzed by
Labo 1D software attached to Chemi-Stage (manufactured by Kurabo
Industries Ltd.), wherein the signal intensities per unit area in A
and B, where strong signals of the antibodies were found, were
digitized. This study was carried out three times. The results of
the areal Labo 1D software analysis were obtained by calculating
the signal average and the standard error for the results of the
three times of tests.
[0219] Images obtained by the analysis by the IP-WB method and the
results of analysis of WB signals by the Labo 1D software are shown
in FIG. 7. From the culture supernatant, clear signals were
detected near a molecular weight of about 28,000 and near a
molecular weight of about 16,000. Since an antibody that recognizes
the N-terminus of TFPI2 is used in the WB analysis, the signal (A)
near a molecular weight of about 28,000 was assumed to correspond
to intact TFPI2, and the signal (B) near a molecular weight of
about 16,000 was assumed to be a TFPI2 fragment polypeptide
produced by reduction of the molecular weight for some reason. Its
signal per unit area varied among the antibodies. The TS-TF01
antibody and the TS-TF04 antibody, which have antigenic
determinants in Kunitz domain 1, tended to show high signals. This
TFPI2 fragment polypeptide, which contains Kunitz domain 1, was
provided as NT-TFPI2.
Example 10
identification of Monoclonal Antibody-Bound Proteins by Mass
Spectrometry
[0220] Further study by IP-WB was carried out using magnetic
particles on which a total of three kinds of antibodies (the
TS-TF01 antibody and the TS-TF04 antibody, which were found to have
high affinity to NT-TFPI2 in Example 9, and the TS-TF05 antibody as
a control) are immobilized. The NT-TFPI2 polypeptide, which binds
to the TS-TF01 antibody and the TS-TF04 antibody, was analyzed by
mass spectrometry. Using the culture supernatants of two kinds of
ovarian cancer cells OVISE and OVMANA prepared in Example 9, IP-WB
was carried out for samples prepared by the method described in
Example 9. Only for the OVMANA sample, sample preparation for mass
spectrometry was carried out as follows. [0221] (1) The OVMANA
sample solution was concentrated using an evaporator, and separated
by SDS-PAGE was carried out. The separated proteins were stained by
Ruby staining (manufactured by Invitrogen). [0222] (2) Three kinds
of stained fragments near a molecular weight of about 16,000 and
one kind of unstained fragment in the vicinity (TS-TF01: fragments
shown in A and B in Ruby staining in FIG. 8; TS-TF04: fragments
shown in C and D in Ruby staining in FIG. 8), that is, a total of
four fragments, were cut out, and subjected to reduction-alkylation
using dithiothreitol and iodoacetamide, followed by carrying out
in-gel digestion with trypsin. [0223] (3) The peptide fragments
produced by the trypsin digestion were separated by ID nano-LC
system (Ultimate 3000, manufactured by Thermo Fisher Scientific
Inc.) using a C18 reverse-phase column, and MS/MS measurement was
carried out using an LTQ Orbitrap mass spectrometer (manufactured
by Thermo Fisher Scientific Inc.). The obtained data were analyzed
using Protein Discoverer 1.3 (manufactured by Thermo Fisher
Scientific Inc.), and proteins were identified by performing search
against amino acid sequences in the Swiss-Prot database.
[0224] The proteins identified from the WB images and the
Ruby-stained image, and from the IP products near a molecular
weight of about 16,000, obtained from OVMANA, are shown in FIG. 8.
In the WB images, clear signals that were assumed to be obtained
from NT-TFPI2 were found near a molecular weight of about 16,000
for both TS-TF01 antibody and TS-TF04 antibody as well as for both
OVISE and OVMANA. In the Ruby-stained image, the molecular weight
of the clear band that was assumed to correspond to a TFPI2
fragment polypeptide having a decreased molecular weight seems to
be slightly increased. This is, however, assumed to be due to
disturbance of the electrophoresis due to influence of an increase
in the salt concentration caused by sample concentration. From the
mass spectrometry data of the OVMANA IP products, it was
demonstrated that the total of three kinds of proteins A, B, and C
near a molecular weight of about 16,000, whose staining could be
clearly observed, are NT-TFPI2. From information on the C-terminal
peptide identified (FFSGGCH; SEQ ID NO:12) and the results obtained
with the N-terminus-recognizing antibody, the NT-TFPI2 polypeptide
was found to be a polypeptide containing at least the region from
the 23rd aspartic acid to the 131st residue histidine in the amino
acid sequence of the TFPI2 protein represented by SEQ ID NO:1.
[0225] In the present Example, the in-gel digestion was carried out
using trypsin, which characteristically digests arginine and lysine
residues. It can therefore be naturally thought that the
polypeptide may contain a sequence that follows the 131st residue
histidine in the C-terminal side of TFPI2 protein. That is, the
present Example does not limit the sequence in the C-terminus of
the NT-TFPI2 polypeptide.
Example 11
Preparation of TFPI2 Assay Reagents
[0226] Since both intact TFPI2 and NT-TFPI2 were found to be
present in the clear cell adenocarcinoma culture supernatants in
Example 9 and Example 10, an "NT+I-TFPI2 assay system" assay
reagent that comprehensively measures intact TFPI2 and NT-TFPI2 was
prepared as follows using the TS-TF04 antibody, which has an
antigenic determinant in Kunitz domain 1, in the solid-phase side,
and the TS-TF01 antibody, which has an antigenic determinant in
Kunitz domain 1, in the detection side. In addition, an "I-TFPI2
assay system" assay reagent that measures only intact TFPI2 was
prepared as follows using the TS-TF04 antibody, which has an
antigenic determinant in Kunitz domain 1, in the solid-phase side,
and the TS-TF05 antibody, which has an antigenic determinant in
Kunitz domain 3, in the detection side. [0227] (1) Physical
adsorption of an anti-TFPI2 monoclonal antibody (TS-TF04) to
water-insoluble ferrite carriers was allowed at room temperature
for one day and night such that the adsorption occurred at 100
ng/carrier, and blocking was then carried out with 100 mM Tris
buffer (pH 8.0) supplemented with 1% ESA at 40.degree. C. for 4
hours, to prepare anti-TFPI2 antibody-immobilized carriers. [0228]
(2) Two kinds of anti-TFPI2 labeled antibodies were prepared with
anti-TFPI2 monoclonal antibodies (TS-TF01 and TS-TF05) using an
alkaline phosphatase labeling kit (manufactured by Dojindo
Laboratories). [0229] (3) In each of magnetic force-permeable
containers (volume, 1.2 mL), 12 antibody-immobilized carriers
prepared in (1) were placed, and 50 .mu.L of a buffer (Tris buffer
supplemented with 3% ESA, pH 8.0) supplemented. with 0.5 .mu.g/ml,
of a labeled antibody prepared in (2) was added thereto, followed
by carrying out freeze-drying, to prepare two kinds of TFPI2 assay
reagents Assay Reagent A (NT+I-TFPI2 assay reagent using the
TS-TF04 antibody/the TS-TF01 antibody) and Assay Reagent B (I-TFPI2
assay reagent using the TS-TF04 antibody/thy TS-TF05 antibody). The
TFPI2 assay reagents prepared were tightly closed and sealed under
nitrogen gas, and stored at 4.degree. C. until the assay.
Example 12
Evaluation of Performances of TFPI2 Assay Reagents
[0230] Each 10-fold dilutions of the recombinant TFP12 supernatant
prepared in Example 4 and OVISE and OVMANA prepared in Example 9 in
FBS were provided as samples containing TFPI2, and FBS alone was
provided as a sample containing no TFPI2, to provide a total of
four pseudosamples. The pseudosamples were used for evaluation of
the two kinds of TFPI2 assay reagents prepared in Example 11, by
five-point measurement.
[0231] As an evaluation device, a fully automatic enzyme
immunoassay device AIA-1800 (manufactured by Tosoh Corporation;
manufacturing/marketing notification number, 13E3X90002000002) was
used. Measurement using the fully automatic enzyme immunoassay
device AIA-1800 was carried out by: [0232] (1) automatically
dispensing 20 .mu.L of a diluted sample and 80 .mu.L of a diluent
containing a surfactant to a container storing a TFPI2 assay
reagent prepared in Example 11; [0233] (2) carrying out
antigen-antibody reaction at a constant temperature of 37.degree.
C. for 10 minutes; [0234] (3) carrying out eight times of washing
using a buffer containing a surfactant; and [0235] (4) adding
4-methylumbelliferyl phosphate to the container.
[0236] The concentration of 4-methylumbelliferone produced by
alkaline phosphatase per unit time was provided as the measured
value (nmol/(Ls)).
[0237] The measured values for the pseudosamples are shown in Table
2. Since any of the pseudosamples excluding FBS showed a
coefficient of variation of not more than 3% in the five-point
measurement, it was demonstrated that results obtained with the
TFPI2 assay reagents prepared in Example 11 are reliable.
TABLE-US-00002 TABLE 2 Measured value [nmol/(L s)] FBS Recombinant
TFPI2 OVISE OVMANA A) I + NT-TFPI2 1 0.09 230.25 33.09 112.22 2
0.08 233.26 34.59 112.34 3 0.09 236.27 33.35 111.87 4 0.10 229.63
34.44 113.27 5 0.10 224.61 35.09 114.93 Average 0.09 230.80 34.11
112.93 SD 0.01 4.36 0.85 1.23 CV [%] 7.28 1.89 2.50 1.09 B) I-TFPI2
1 0.18 178.52 20.12 80.48 2 0.19 176.78 20.43 79.95 3 0.20 183.75
20.52 80.45 4 0.19 181.32 20.37 78.91 5 0.18 186.21 21.36 80.30
Average 0.19 181.32 20.56 80.02 SD 0.01 3.82 0.47 0.65 CV [%] 4.31
2.11 2.30 0.82
Example 13
Study of TFPI2 Assay Reagents Using Culture Supernatant Panel for
Various Ovarian Cancer Cells
[0238] The present inventors. Arakawa et al., used a panel of
cultured cells of various ovarian cancers including the clear cell
type, serous type, and mucinous type, to carry out mass
spectrometry of culture supernatants and real-time PCR analysis of
gene expression in the cells. As a result, the present inventors
identified TFPI2 as a molecule that is characteristically produced
in the clear cell type (Patent Document 4). Therefore, if high
measured values of TFPI2 are similarly observed only in culture
supernatants of clear cell adenocarcinoma when the TFPI2 assay
reagents described in Example 11 are used, the target of
measurement by these reagents can be judged to be TFPI2. The
culture supernatant panel for various ovarian cancer cells used by
Arakawa et al. was analyzed with the TFPI2 assay reagents. [0239]
1) The following groups of cells were cultured in RPMI 1640 medium
(manufactured by Wako Pure Chemical Industries, Ltd.) supplemented
with 10% fetal bovine serum at 100% confluence for three days.
[0240] Clear cell type: OVISE, OVTOKO, OVMANA, OVSAYO [0241] Serous
type: OVKATE, OVSAHO [0242] Mucinous type: RMUG-S, MCAS [0243] 2)
By the method described in Example 12, a total of 8 kinds of
culture supernatants were analyzed with the two kinds of TFPI2
assay reagents.
[0244] The analysis results are shown in FIG. 9. In the cases of
the clear cell type, all kinds of cells except OVTOKO showed high
signals. On the other hand, the cells of the serous type and the
mucinous type hardly showed signals. Very strong correlation was
shown between the assay reagents. From these results, it was shown
that TFPI2 is the target of measurement by the TFPI2 assay
reagents.
Example 14
Study of Molecular Weight of NT-TFPI2 Polypeptide by N-Glycanase
Treatment
[0245] From common databases such as UniProt, and known information
from literatures and the like, TFPI2 is known to have
asparagine-linked sugar chains attached to the asparagine residues
at two positions 116 and 170 in the amino acid sequence of SEQ ID
NO:1. Thus, from the results of Example 10, the NT-TFPI2
polypeptide was assumed to be a glycoprotein containing an
asparagine-linked (N-type) sugar chain at the 116th residue. In
view of this, changes in the molecular weight of the NT-TFPI2
polypeptide by sugar chain digestion treatment using N-glycanase
were studied by carrying out IP-WB using the three kinds of
antibodies described in Example 10, and the supernatants of OVISE,
OVMANA, and OVSAYO described in Example 9 in the
N-glycanase-treated group and the untreated group. At the same
time, the IP efficiency of TFPI2 in each supernatant was analyzed
by IP-AIA, wherein the calculation was carried out using the assay
reagents prepared in Example 11. [0246] (1) Two test groups each of
which uses 400 .mu.L each of supernatants of OVISE, OVMANA, and
OVSAYO were provided. One of the test groups was used as an
Ar-glycanase-treated group, wherein 1000 U PNGase F (manufactured
by NEB) was added, and the other test group was used as an
untreated group. In both test groups, incubated at 37.degree. C.
for 16 hours. [0247] (2) For each antibody group, 100 .mu.L of the
supernatant treated in (1) was used. A control group in which
magnetic particles alone were added and groups in which the three
kinds of antibody-immobilized magnetic particles were added, that
is, a total of four test groups, were provided. IP was carried out
by the method described in Example 9. The solution after the
reaction was separated into the supernatant fraction and the
magnetic particle fraction using a magnet. [0248] (3) The
supernatant fraction was subjected to measurement using the two
kinds of assay reagents described in Example 10. The influence of
the N-glycanase treatment on the measured values of TFPI2, and the
IP efficiencies of the three kinds of antibodies, were calculated.
The calculation of the IP efficiency (recovery (%)) was carried out
as follows: 100-(measured value for the supernatant described in
(2)/measured value for the reaction solution obtained with magnetic
particles to which no antibody was bound). [0249] (4) WB was
carried out for the magnetic particle fraction by the method
described in Example 9, and its luminescence signal was
detected.
[0250] The influence of the N-glycanase treatment on the measured
value of TFPI2, the IP recovery, and the WB images are shown in
FIG. 10.
[0251] Whether or not the N-glycanase treatment was carried out did
not cause changes in the measured value of TFPI2 and the IP
efficiency. No inhibition of the reaction system by the N-glycanase
treatment was found. In terms of the IP efficiency, the TS-TF04 and
TS-TF01 IP groups showed recoveries of as high as not less than 97%
in any of the results obtained using the three kinds of
supernatants and the two kinds of assay systems. Thus, almost
complete recovery of TFPI2 molecules was demonstrated. On the other
hand, in the cases of TS-TF05 IP, a significant difference was
found between A) the recovery by the NT+I-TFPI2 assay reagent,
which was 74.3% in terms of the average calculated for four points,
and B) the recovery by the I-TFPI2 reagent, which was 91.3% in
terms of the average calculated for four points. It was suggested
that region in TFPI2 recognized by TS-TF05 is different from those
recognized by TS-TF04 and TS-TF01.
[0252] In terms of the WB results, in the cases where the
N-glycanase treatment was not carried out, clear signals that were
assumed to be obtained from the NT-TFPI2 polypeptide were found
near a molecular weight of about 16,000 for both TS-TF01 antibody
and TS-TF04 antibody as well as for all of the three kinds of
culture supernatants. On the other hand, in the group in which the
N-glycanase treatment was carried out, clear signals that were
assumed to be obtained from the NT-TFPI2 polypeptide were found
near a molecular weight of about 12,000 for all of the three kinds
of culture supernatants. Since the signals that were assumed to be
obtained from the NT-TFPI2 polypeptide, which has high affinity to
the TS-TF01 antibody and the TS-TF04 antibody, clearly showed a
decreased molecular weight due to the sugar chain digestion, it
became clear that the NT-TFPI2 polypeptide has an N-type sugar
chain modification.
Example 15
Study of Temporal Changes in NT-TFPI2 Polypeptide
[0253] Aiming at elucidation of the mechanism of production of the
NT-TFPI2 polypeptide, temporal changes of TFPI2 dynamics in a
culture supernatant was analyzed by the IP-WB described in Example
9 or the IP-AIA method described in Example 14. [0254] (1) OVISE,
OVMANA, and OVSAYO cells were precultured in 15-cm dishes to
confluence. [0255] (2) To each dish, 20 mL of fresh medium was
added, and the culture supernatant was collected after Hours 24,
48, 72, and 144 during the culture. [0256] (3) The four
supernatants collected in (2) were subjected to IP using 100 .mu.L
of each supernatant per antibody by the method described in Example
9. Each of the three kinds of solutions containing
antibody-immobilized magnetic particles was separated into the
supernatant fraction and the magnetic particle fraction using a
magnet. [0257] (4) The supernatant fraction was subjected to
measurement using the two kinds of assay reagents described in
Example 11, to measure changes in the TFPI2 level in the culture
supernatant over time. [0258] (5) Since TFPI2 increases over time
during the culture period, the TFPI2 levels in the four kinds of
supernatants collected at different times need to be averaged
before carrying out WB. Thus, based on the measured value of TFPI2
in the culture supernatant at Hour 24 in (4), the three magnetic
particle fractions collected thereafter were appropriately diluted
to adjust the amount of TFPI2 to be added to each lane in the
SDS-PAGE in order to carry out the WB described in Example 9. The
measured values of TFPI2 in the culture supernatants collected over
time and the WB images are shown in FIG. 11.
[0259] TFPI2 in the culture supernatant showed a tendency to
increase over time in both the NT+I-TFPI2 assay system and the
I-TFPI2 assay system. In the WB results, clear signals that were
assumed to be obtained from NT-TFPI2 were found near a molecular
weight of about 16,000 in the supernatants collected after 144
hours of the culture for both TS-TF01 antibody and TS-TF04 antibody
as well as for the culture supernatants of both OVISE and OVMANA.
It became clear that these signals were increased sequentially both
OVISE and OVMANA, they are present after Hour 24 in OVMANA.
Example 16
Study of Intracellular TFPI2 Molecules in Cancer Cells
[0260] Production of intact TFPI2 and the NT-TFPI2 polypeptide
secreted to the culture supernatant was shown by Examples 13 and
14. It is, of course, thought that the molecules are also localized
in the cells. In view of this, intracellular TFPI2 molecules in the
cells were analyzed by the IP-WB method. [0261] 1) According to the
method described in Example 13, OVISE, OVMANA, and OVSAYO were
cultured as the clear cell type, and OVKATE and OVSAHO were
cultured as the serous type. [0262] 2) As a cell lysis buffer, a
solution of 2 M thiourea, 7 M urea, 3% CHAPS, and 1% Triton X-100
was prepared. The medium was removed from the cell culture plate,
and the culture plate was then washed three times with PBS,
followed by adding the cell lysis buffer to the culture plate.
[0263] 3) The cells were dissociated using a cell scraper from the
culture plate, and centrifugation was carried out at 15,000 rpm for
20 minutes. The resulting supernatant fraction was provided as a
cell lysate. [0264] 4) Using the cell lysate, measurement by the
two kinds of TFPI2 assay reagents described in Example 11 and IP-WB
using the three kinds of antibodies described in Example 9 were
carried out.
[0265] The analysis results obtained with the TFPI2 assay reagents
and the WB images are shown in FIG. 12. Clear-cell-specific
expression of TFPI2 was found in the cells as well as in the
culture supernatant. In terms of the molecules in the cells, two
clear signals were found between molecular weights of 24,000 and
31,000. Based on their molecular weights, the signals were assumed
to be obtained from intact TFPI2 having a sugar chain, and intact
TFPI2 having no sugar chain. On the other hand, the signal near a
molecular weight of 16,000, which corresponds to NT-TFPI2, was
lower than the detection limit. From these results, it was
suggested that, while a large amount of intact TFPI2 molecules are
present in the cells, whereas NT-TFPI2 may be present in the cells
only in a small amount, or may be absent.
[0266] From Examples 14, 15, and 16, the NT-TFPI2 polypeptide was
shown to be a glycoprotein produced by clear cell adenocarcinoma,
to have an N-type sugar chain, and to be fractionated near a
molecular weight of about 16,000. It was also shown that the
NT-TFPI2 polypeptide is secreted to the culture supernatant and
continuously accumulates therein, and that the NT-TFPI2 polypeptide
is expressed only in a very small amount relative to intact TFPI2
in cancer cells, or is not expressed therein. In terms of the
production mechanism, NT-TFPI2 is thought to be produced by
undergoing processing for some reason after secretion.
Example 17
Measurement of TFPI2 in Pregnancy Serum Samples
[0267] Sixty two pregnancy serum samples (samples purchased from
ProMedDx) were used in the present Example. They were samples
obtained during the period from the 5th week of pregnancy to the
40th week of pregnancy including the first trimester to the third
trimester of pregnancy. All serum samples were obtained from
Europeans and Americans with informed consent, according to
description of the samples.
[0268] Using a fully automatic enzyme immunoassay device AIA-1800
(manufactured by Tosoh Corporation) as a device for the evaluation,
measurement was carried out using the two kinds of assay reagents
prepared in Example 11: the A) NT+I-TFPI2 assay reagent and the B)
I-TFPI2 assay reagent.
[0269] A box plot of the measured values of TFPI2 is shown in FIG.
13. The minimum value, 25 percentile, median, 75 percentile,
maximum value, and concentration range in the 95% confidence
interval in each of the first, second, and third trimesters for
each assay reagent are shown in Table 3. Since the measured values
of TFPI2 showed strong correlation with the number of weeks of
pregnancy and the blood level, the target of measurement by the two
kinds of assay reagents prepared in Example 11 was detected TFPI2
in the serum samples.
TABLE-US-00003 TABLE 3 TFPI2 1st trimester 2nd trimester 3rd
trimester A)I + NT-TFPI2 B)I-TFPI2 A)I + NT-TFPI2 B)I-TFPI2 A)I +
NT-TFPI2 B)I-TFPI2 Minimum value 2.2 1.7 78.3 76.1 129.2 93.0 25
Percentile 3.8 3.7 78.8 86.9 245.6 177.0 Median 12.3 9.6 115.1
121.2 337.7 249.1 75 Percentile 40.2 30.2 180.7 218.5 409.4 348.0
Maximum value 44.0 58.4 184.6 252.2 700.0 667.0 95% Confidence
interval 1.3-33.6 -1.2-36.8 74.2-176.2 70.6-218.9 304.4-387.9
235.7-311.1
Example 18
Measurement of TFPI2 in Ovarian Cancer Samples
[0270] The sample panel (123 cases) used in the present Example is
shown in Table 4. The samples are serum samples collected by the
same protocol in the department of gynecology, Yokohama City
University. The collection was carried out with informed consent
and approval by the ethical committee of Yokohama City
University.
TABLE-US-00004 TABLE 4 <Sample panel content> Number of cases
Ovarian tumor Benign Mucinous 15 Teratoma 15 Serous 5 Fibrothecoma
4 Endometriosis 13 Other benign 4 Borderline Serous, mucinos 7
malignant Malignant Clear 11 Serous 10 Endometrioid 7 Mucinius 5
Other cancer 9 Uterine tumor Benign Uterine fibroids 5 Malignant
Cervical cancer 8 Endometrial cancer 5 Total 123
[0271] Using a fully automatic enzyme immunoassay device AIA-1800
(manufactured by Tosoh Corporation) as a device for the evaluation,
measurement was carried out using the two kinds of TFPI2 assay
reagents prepared in Example 11 and a CA125 assay reagent
(manufactured by Tosoh Corporation; approval number,
20700AMZ00504000).
[0272] The measured values of TFPI2 by the NT+I-TFPI2 assay system
and the I-TFPI2 assay system, and the measured values of CA125 are
shown in FIG. 14. CA125 tended to be at a high level generally in
ovarian malignancies. The measured values by the NT+I-TFPI2 assay
system and the I-TFPI2 assay system tended to be high in clear cell
adenocarcinoma.
[0273] The results of analysis of the panel were classified into
those for five groups (benign ovarian tumor, endometriosis,
borderline malignancy, clear cell adenocarcinoma, and other
malignant ovarian tumors) as shown in FIG. 15. The minimum value,
25 percentile, median, 75 percentile, maximum value, and
concentration range in the 95% confidence interval in each group
for the measured values of the two kinds of TFPI2 and the measured
values of CA125 are shown in Table 5. CA125 tended to be at a
higher level in endometriosis than in benign ovarian tumor, and
also tended to be clearly at a high level in malignant tumors in
general. Since CA125 is an auxiliary marker for endometriosis, it
is natural that it showed a high value in endometriosis among
benign tumors. Since almost the same medians were obtained between
clear cell adenocarcinoma and other malignant ovarian tumors, it
can be said that CA125 cannot distinguish clear cell
adenocarcinoma. On the other hand, the NT+I-TFPI2 assay system and
the I-TFPI2 assay system tended to show a high measured value only
in clear cell adenocarcinoma. It was also shown that the measured
value by the NT+I-TFPI2 assay system tends to be higher.
TABLE-US-00005 TABLE 5-1 Ovarian benign Endometriosis A)I +
NT-TFPI2 B)I-TFPI2 CA125 A)I + NT-TFPI2 B)I-TFPI2 CA125 Minimum
value 0.25 0.17 6.00 0.39 0.31 10.00 25 Percentile 0.53 0.27 11.00
0.65 0.35 17.00 Median 0.69 0.33 18.00 0.73 0.39 50.00 75
Percentile 0.89 0.47 31.00 0.86 0.41 147.50 Maximum value 5.84 2.75
161.00 1.08 0.58 809.00 95% Confidence interval 0.59-1.10 0.32-0.56
18.11-35.51 0.64-0.85 0.35-0.44 2.44-267.10
TABLE-US-00006 TABLE 5-2 Borderline malignant CCA Ovarian cancer
A)I + NT-TFPI2 B)I-TFPI2 CA125 A)I + NT-TFPI2 B)I-TFPI2 CA125 A)I +
NT-TFPI2 B)I-TFPI2 CA125 Minimum 0.41 0.28 16.00 0.59 0.26 15.00
0.43 0.16 16.00 value 25 Percentile 0.72 0.28 23.00 1.99 0.54 72.00
0.63 0.30 33.00 Median 0.75 0.45 32.00 3.37 0.73 137.00 0.75 0.33
122.00 75 Percentile 0.82 0.48 40.00 6.68 2.80 409.00 0.99 0.47
482.00 Maximum 1.11 0.61 162.00 34.30 8.06 1691.00 3.97 1.63
5761.00 value 95% 0.57-0.95 0.32-0.54 1.82-95.61 0.44-13.73
0.32-3.45 4.30-697.00 0.70-1.19 0.32-0.50 67.67-1264.00 Confidence
interval
[0274] The results of the receiver operating characteristic (ROC)
curve analysis of the data obtained by the NT+I-TFPI2 assay system.
I-TFPI2 assay system, and CA125 measurement between the ovarian
clear cell adenocarcinoma group and the other-ovarian-tumors group
are shown in FIG. 16, and AUC (Area Under the Curve; area under the
ROC curve) and the P-value in the significance test are shown in
Table 6. The measured values of the two kinds of TFPI2 were
significantly different between the ovarian clear cell
adenocarcinoma group and the other-ovarian-tumors group with
p<0.0002. Based on the statistical significance observed, it was
shown that the two kinds of TFPI2 assay reagents are more useful
for detection of ovarian clear cell adenocarcinoma than CA125. It
was also shown that the NT+I-TFPI2 assay system, which
comprehensively measures intact TFPI2 and NT-TFPI2, shows a better
P-value and a better AUC than the I-TFPI2 assay system, which
measures only intact TFPI2.
TABLE-US-00007 TABLE 6 A) I + NT-TFPI2 B) I-TFPI2 CA125 Area under
the 0.9101 0.8511 0.7234 curve (AUC) Standard error 0.06454 0.0779
0.07529 95% Confidence 0.7835 to 1.037 0.6983 to 1.004 0.5758 to
0.8710 interval P-value <0.0001 0.0001474 0.01568
[0275] Table 7 shows the sensitivity and the specificity between
the ovarian clear cell adenocarcinoma group and the
other-ovarian-tumors group as calculated by using the value
obtained by the ROC analysis as the TFPI2 reference value (cutoff
value) and using a value of 36 U/mL, which is close to a common
reference value, as the CA125 reference value. Although the
conditions were disadvantageous for CA125, usefulness of TFPI2
became clear at least from the viewpoint of specific diagnosis of
clear cell adenocarcinoma. It was also shown that the A) NT+I-TFPI2
assay reagent has higher specificity than the B) I-TFPI2 assay
reagent.
TABLE-US-00008 TABLE 7 A) I + NT-TFPI2 B) I-TFPI2 CA125 Reference
value 1.91 0.54 36.0 U/mL Sensitivity [%] 81.82 81.82 81.82
Specificity [%] 97.87 84.04 58.51
[0276] Table 8 shows the positive rates for all clinical samples
described in. Example 17 as calculated by using the above reference
value for TFP12 and using 35 U/mL, which is a common reference
value, for CA125. In this panel, the A) NT+I-TFPI2 assay reagent
showed an extremely low false positivity, and allowed
identification of clear cell adenocarcinoma with a high
probability. Thus, the reagent was shown to have a sufficient
performance as a diagnostic marker for clear cell
adenocarcinoma.
TABLE-US-00009 TABLE 8 NT + I-TFPI2 I-TFPI2 CA125 Number Number of
Positive Number of Positive rate Number of Positive rate <Sample
panel content> of cases positive cases rate (%) positive cases
(%) positive cases (%) Ovarian Benign Mucinous 15 0 0.0 2 13.3 2
13.3 tumor Teratoma 15 0 0.0 5 33.3 4 26.7 Serous 5 0 0.0 0 0.0 0
0.0 Fibrothecoma 4 1 25.0 1 25.0 3 75.0 Endometriosis 13 0 0.0 1
7.7 7 53.8 Other benign 4 0 0.0 0 0.0 0 0.0 Borderline Serous, 7 0
0.0 1 14.3 3 42.9 malignant mucinos Malignant Clear 11 9 81.8 9
81.8 9 81.8 Serous 10 0 0.0 1 10.0 9 90.0 Endometrioid 7 1 14.3 1
14.3 5 71.4 Mucinius 5 0 0.0 0 0.0 3 60.0 Other cancer 9 0 0.0 3
33.3 6 66.7 Uterine Benign Uterine fibroids 5 0 0.0 0 0.0 0 0.0
tumor Malignant Cervical cancer 8 0 0.0 1 12.5 1 12.5 Endometrial 5
2 40.0 2 40.0 1 20.0 cancer Total 123
Example 19
Sequence Analysis of C-Terminus of NT-TFPI2 Polypeptide by Mass
Spectrometry
[0277] The C-terminal sequence of the NT-TFPI2 polypeptide was
analyzed by mass spectrometry using the heavy-oxygen water
method.
[0278] Using the OVMANA prepared in Example 9 as an ovarian cancer
cell culture supernatant, a sample for the mass spectrometry was
prepared as follows using a TS-TF01 antibody column. [0279] (1)
Using a HiTrap NHS column (manufactured by GE Healthcare) and the
TS-TF01 antibody, a TS-TF01 antibody-bound column was prepared
according to a conventional method. [0280] (2) Using a syringe, 10
mL of the OVMANA culture supernatant was passed through the
antibody column three times, and washing was carried out with TBS
three times. [0281] (3) Through the antibody column, 1 mL of 0.1 M
glycine (pH 2.0) solution was passed three times to collect an
antibody column-bound fraction solution. [0282] (4) The antibody
column-bound fraction solution was concentrated with
trichloroacetic acid, and developed by SDS-PAGE, followed by
staining by SYPRO-Ruby staining (manufactured by Invitrogen).
[0283] (5) A total of three gel sections that were found to be
stained (the sections #1 to #3 shown in the SYPRO-Ruby-stained
image in FIG. 17A) were excised, and dehydrated with acetonitrile,
followed by performing in-gel digestion using V8 protease
(manufactured by SIGMA). In this step, in order to judge whether
the obtained C-terminal sequence is produced by the in-gel
digestion or by the action of processing by the cells based on the
mass information, the in-gel digestion was carried out in 10 mM
aqueous ammonium bicarbonate solution in which normal ultrapure
water (H.sub.2O.sup.16) and heavy-oxygen water (H.sub.2O.sup.18)
are mixed together at a ratio of 1:1. [0284] (6) The resulting
peptide digest was analyzed with a Triple TOF 5600 mass
spectrometer (manufactured by AB Sciex) connected to a C18
reverse-phase nano-LC system. The measurement data were subjected
to analysis using the Protein Pilot software (manufactured by AB
Sciex), wherein the amino acid sequence was identified by
performing search against amino acid sequences in the Swiss-Prot
database.
[0285] FIG. 17A shows a SYPRO-Ruby-stained image of the antibody
column-bound fraction; FIG. 17B shows the results of mapping of the
sequence information identified from the bands #1 to #3 on the
TFPI2 amino acid sequence; FIG. 17C shows mass spectra of precursor
ions of representative TFPI2-derived peptides detected from the
band #3; and Table 9 shows identification information of the
corresponding ions. All of the bands of 28 kDa (#1), 24 kDa (#2),
and 17.5 kDa (#3), which showed strong signals in the
SYPRO-Ruby-stained image of the antibody column-bound fraction,
were shown to correspond to the TFPI2 peptide. Among the peptides
having a confidence value of not less than 99 and having a glutamic
acid (E) or aspartic acid (D) residue immediately before the
N-terminus (excluding .sub.23DAAQEPTGNNAE.sub.34 (SEQ ID NO:13)
immediately after the secretory signal sequence),
.sub.12KFFSGGCH.sub.131 (SEQ ID NO: 15) and .sub.124KFFSGGC.sub.130
(SEQ ID NO:16) were detected only from #3.
TABLE-US-00010 TABLE 9 Precursor ion Precursor Theoretical
molecular ion Confidence molecular weight m/z Valency Sequence
Modification value weight 1215.50 608.76 2 DAAQEPTGNNAE No Label:
180(1)@ C-term 99 1215.50 1217.51 609.76 2 DAAQEPTGNNAE Label:
180(1)@ C-term 99 1217.50 1296.52 649.26 2 ACDDACWRIE
Carbamidomethyl(C)@ 2, Carbamidomethyl(C)@ 6, 99 1296.51 Label:
18O(1)@ C-term 1294.51 648.26 2 ACDDACWRIE Carbamidomethyl(C)@ 2,
Carbamidomethyl(C)@ 6, 99 1294.51 No Label: 18O(1)@ C-term 938.40
470.21 2 KFFSGGCH Carbamidomethyl(C)@ 7, 99 938.41 No Label:
18O(1)@ C-term 801.35 401.68 2 KFFSGGC Carbamidomethyl(C)@ 7, 99
801.35 No Label: 18O(1)@ C-term 1319.68 440.90 3 DCKRACAKALK
Carbamidomethyl(C)@ 2, Carbamidomethyl(C)@ 6, 99 1319.68 No Label:
18O(1)@ C-term
[0286] In the present Example, which aims at identification of the
C-terminal sequence, an aqueous solution in which ultrapure water
(H.sub.2O.sup.16) and heavy-oxygen water (H.sub.2O.sup.18) are
mixed together at a ratio of 1:1 was used in the experiment system
for the in-gel digestion using V8 protease. The peptides produced
by the protease digestion in the gel are labeled with O.sup.16 or
O.sup.18 almost to the same extent at their C-termini. In contrast,
C-termini produced by the action of processing during the cell
culture are not labeled with O.sup.18.
[0287] For .sub.23DAAQEPTGNNAE.sub.34 (SEQ ID NO:13) and
.sub.81ACDDACWRIE.sub.90 (SEQ ID NO:14), which were assumed to be
internal peptides based on the representative examples of mass
spectra of precursor ions of the peptides detected in the present
Example (FIG. 17C) and the identification information of the
corresponding ions (Table 9), peptides produced by labeling with
O.sup.18 at their C-termini and peptides produced normally with
O.sup.16 were simultaneously detected and identified. For example,
as shown in FIG. 17C, monoisotopic ions of divalent ions
corresponding to m/z 608.76 and m/z 609.76 labeled with O.sup.16
and O.sup.18 were detected for .sub.23DAAQEPTGNNAE.sub.34 (SEQ ID
NO: 13), and precursor ions were detected as a mixture of their
corresponding isotopomers. A similar result was obtained for
.sub.81ACDDACWRIE.sub.90 (SEQ ID NO:14). The above data indicate
that these peptides are not positioned at the C-terminus, and
demonstrate correctness of the present analysis system, which is
intended for identification of the C-terminal sequence.
[0288] On the other hand, for .sub.124KFFSGGCH-.sub.131 (SEQ ID
NO:15), .sub.124KFFSGGC.sub.130 (SEQ ID NO:16), and
.sub.203DCKRACAKALK.sub.212 (SEQ ID NO:17), peptides produced with
the O.sup.16 label were detected and identified, but peptides
produced with the O.sup.18 label were not detected. It is thus
strongly suggested that the C-terminal sequence of the NT-TFPI2
polypeptide is His131 or Cys130.
[0289] Taking into account the total results of the Examples of the
present description, it is thought that the full-length TFPI2
detected in Western blotting correspond to Band #1 and Band #2; the
N-terminus corresponds to Asp23; the C-terminus corresponds to
Lys212; and the sequence after Lys212 is cleaved off for some
reason. The difference in the mobility observed between Band #1 and
Band #2 in SDS-PAGE may be due to a difference in the sugar chain
structure of TFPI2. On the other hand, it became clear that the
NT-TFPI2 polypeptide of 17.5 kDa detected by WB corresponds to Band
#3, and that, although its N-terminus corresponds to Asp23
similarly to full-length TFPI2, its C-terminus corresponds to His
131 or Cys130.
[0290] Since .sub.203DCKRACAKALK.sub.212 (SEQ ID NO:17), which is
positioned in the C-terminal side in full-length TFPI2, was also
detected from Band #3, it is suggested that Band #3 includes not
only NT-TFPI2, but also a polypeptide having a sequence positioned
closer to the C-terminus relative to Arg132, that is, CT-TFP12.
However, since the C-terminal sequence of the NT-TFPI2 polypeptide
was defined from mass information obtained by the heavy-oxygen
water method, its validity is not deteriorated.
INDUSTRIAL APPLICABILITY
[0291] The present invention provides a novel detection marker for
ovarian clear cell adenocarcinoma. The present invention also
provides a method for detecting, with high sensitivity and
specificity, only ovarian clear cell adenocarcinoma among benign
ovarian tumors and malignant ovarian tumors having various tissue
types. These are industrially very useful since they can be
favorably applied to uses such as screening and postoperative
follow-up of ovarian clear cell adenocarcinoma, and follow-up of
endometriosis.
Sequence CWU 1
1
171235PRTHomo Sapiens 1Met Asp Pro Ala Arg Pro Leu Gly Leu Ser Ile
Leu Leu Leu Phe Leu 1 5 10 15 Thr Glu Ala Ala Leu Gly Asp Ala Ala
Gln Glu Pro Thr Gly Asn Asn 20 25 30 Ala Glu Ile Cys Leu Leu Pro
Leu Asp Tyr Gly Pro Cys Arg Ala Leu 35 40 45 Leu Leu Arg Tyr Tyr
Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln Phe 50 55 60 Leu Tyr Gly
Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr Thr Trp Glu 65 70 75 80 Ala
Cys Asp Asp Ala Cys Trp Arg Ile Glu Lys Val Pro Lys Val Cys 85 90
95 Arg Leu Gln Val Ser Val Asp Asp Gln Cys Glu Gly Ser Thr Glu Lys
100 105 110 Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu Lys Phe Phe
Ser Gly 115 120 125 Gly Cys His Arg Asn Arg Ile Glu Asn Arg Phe Pro
Asp Glu Ala Thr 130 135 140 Cys Met Gly Phe Cys Ala Pro Lys Lys Ile
Pro Ser Phe Cys Tyr Ser 145 150 155 160 Pro Lys Asp Glu Gly Leu Cys
Ser Ala Asn Val Thr Arg Tyr Tyr Phe 165 170 175 Asn Pro Arg Tyr Arg
Thr Cys Asp Ala Phe Thr Tyr Thr Gly Cys Gly 180 185 190 Gly Asn Asp
Asn Asn Phe Val Ser Arg Glu Asp Cys Lys Arg Ala Cys 195 200 205 Ala
Lys Ala Leu Lys Lys Lys Lys Lys Met Pro Lys Leu Arg Phe Ala 210 215
220 Ser Arg Ile Arg Lys Ile Arg Lys Lys Gln Phe 225 230 235
231DNAArtificial SequenceSynthetic Forward primer for GPI-anchor
Type TFPI2 Expression Plasmid 2cgatgacgac aagcttgctc aggagccaac a
31331DNAArtificial SequenceSynthetic Reverse primer for GPI-anchor
Type TFPI2 Expression Plasmid 3catcagtggt gaattcaaat tgcttcttcc g
31431DNAArtificial SequenceSynthetic forward primer for Secretory
TFPI2 Expression Plasmid 4cgatgacgac aagcttgctc aggagccaac a
31560DNAArtificial SequenceSynthetic reverse primer for Secretory
TFPI2 Expression Plasmid 5agcatcagtg gtgaattctc attagtggcg
acgcagaact ttgcaaaatt gcttcttccg 60631DNAArtificial
SequenceSynthetic forward primer for GPI-anchor Type TFPI2-KD1
6cgatgacgac aagcttgctc aggagccaac a 31731DNAArtificial
SequenceSynthetic reverse primer for GPI-anchor Type TFPI2-KD1
7catcagtggt gaattctttt tctatcctcc a 31831DNAArtificial
SequenceForward primer for GPI-anchor Type TFPI2-KD2 8cgatgacgac
aagcttgttc ccaaagtttg c 31931DNAArtificial SequenceReverse primer
for GPI-anchor Type TFPI2-KD2 9catcagtggt gaattctttc tttggtgcgc a
311031DNAArtificial SequenceForward primer for GPI-anchor Type
TFPI2-KD3 10cgatgacgac aagcttattc catcattttg c 311131DNAArtificial
SequenceReverse primer for GPI-anchor Type TFPI2-KD3 11catcagtggt
gaattcaaat tgcttcttcc g 31127PRTHomo sapiens 12Phe Phe Ser Gly Gly
Cys His 1 5 1312PRTHomo sapiens 13Asp Ala Ala Gln Glu Pro Thr Gly
Asn Asn Ala Glu 1 5 10 1410PRTHomo sapiens 14Ala Cys Asp Asp Ala
Cys Trp Arg Ile Glu 1 5 10 158PRTHomo sapiens 15Lys Phe Phe Ser Gly
Gly Cys His 1 5 167PRTHomo sapiens 16Lys Phe Phe Ser Gly Gly Cys 1
5 1711PRTHomo sapiens 17Asp Cys Lys Arg Ala Cys Ala Lys Ala Leu Lys
1 5 10
* * * * *