U.S. patent application number 17/740864 was filed with the patent office on 2022-09-08 for cytocidal agent.
The applicant listed for this patent is FUJITA ACADEMY. Invention is credited to Michiko FUKUDA, Naohiro KANAYAMA, Motohiro NONAKA, Yuichiro ONODERA, Toshiaki SHIBATA, Kazuhiro SUGIHARA.
Application Number | 20220280596 17/740864 |
Document ID | / |
Family ID | 1000006348636 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220280596 |
Kind Code |
A1 |
SUGIHARA; Kazuhiro ; et
al. |
September 8, 2022 |
CYTOCIDAL AGENT
Abstract
The present invention is a cytocidal agent including a peptide
consisting of an amino acid sequence represented by SEQ ID NO: 1
and a site selectively binding to a target molecule, in which the
peptide consisting of an amino acid sequence represented by SEQ ID
NO: 1 is a peptide exclusively consisting of L-amino acids, a
peptide in which, in amino acid sequences represented by SEQ ID NO:
1, the first to the 14.sup.th amino acids are D-amino acids, and
the 15.sup.th to 19.sup.th amino acids are L-amino acids, a peptide
in which, in amino acid sequences represented by SEQ ID NO: 1, the
first to 14.sup.th amino acids are L-amino acids, and the 15.sup.th
to the 19.sup.th amino acids are D-amino acids, or a peptide
exclusively consisting of D-amino acids.
Inventors: |
SUGIHARA; Kazuhiro;
(Nagoya-shi, JP) ; KANAYAMA; Naohiro;
(Hamamatsu-shi, JP) ; ONODERA; Yuichiro;
(Hokkaido, JP) ; SHIBATA; Toshiaki;
(Hamamatsu-shi, JP) ; FUKUDA; Michiko; (San Diego,
CA) ; NONAKA; Motohiro; (Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FUJITA ACADEMY |
Aichi |
|
JP |
|
|
Family ID: |
1000006348636 |
Appl. No.: |
17/740864 |
Filed: |
May 10, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16640248 |
Feb 19, 2020 |
11357819 |
|
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PCT/JP2018/031136 |
Aug 23, 2018 |
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17740864 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 47/66 20170801; A61K 38/10 20130101 |
International
Class: |
A61K 38/10 20060101
A61K038/10; A61K 47/66 20060101 A61K047/66; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2017 |
JP |
2017-161556 |
Claims
1-10. (canceled)
11. A method of determining a disease onset possibility of a
disease in which disease-causing cells have expressed CNGB3 and
treating the disease, comprising: measuring CNGB3 in an exosome
collected from a subject animal; comparing the obtained measurement
value with a pre-set reference value to determine a possibility
that the subject animal has developed the disease; and treating the
disease with an effective amount of a compound indicated for
treating the disease, wherein the disease is cancer.
12. The method according to claim 11, wherein determining that the
subject animal has a high possibility of developing the disease if
the measurement value exceeds the reference value.
13. The method according to claim 11, wherein the exosome is
isolated from blood collected from the subject animal.
14. The method according to claim 11, wherein measuring CNGB3 by
using a peptide consisting of an amino acid sequence represented by
SEQ ID NO: 2.
15. The method according to claim 11, wherein the subject animal is
a human.
16. (canceled)
17. A biomarker used for determining presence or absence of disease
onset of endometriosis or cancer, comprising: an amount of CNGB3 in
an exosome.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 16/640,248, filed Feb. 19, 2020, which is a 35 U.S.C.
.sctn. 371 of International Patent Application No.
PCT/JP2018/031136, filed Aug. 23, 2018, which claims priority to
Japanese Patent Application No. 2017-161556, filed Aug. 24, 2017,
the entire disclosures of which are hereby incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a peptide drug obtained by
selectively inducing apoptosis in a target cell.
BACKGROUND ART
[0003] Apoptosis means cell death controlled and adjusted as a
proliferation inhibition mechanism among cell deaths of
multicellular organisms. In multicellular organisms, exclusion of
unnecessary cells or harmful cells caused in a generation or
reproduction process is performed by inducing apoptosis in the
cell. In addition, by inducing apoptosis in a cell which is a cause
of a disease and excluding the cell, it is possible to expect
improvement in condition of the disease. In this way, substances
having activity of inducing apoptosis is used for therapeutic use.
For example, by inducing apoptosis in a cancer cell, it can be
expected to remit or cure cancer.
[0004] In a case where apoptosis induction is used for treatment of
a disease, selectivity with respect to a target cell in which
apoptosis is induced is very important. In a case where target
selectivity is low and apoptosis is induced in a cell other than a
target cell, side effects exceed expected therapeutic effects and
are not appropriate as a therapeutic drug from a viewpoint of
safety. If apoptosis can be induced specifically in a target cell,
an effective therapeutic drug in which side effects are
sufficiently suppressed is obtained.
[0005] Endometriosis is a disease in which endometrial cells
proliferate at a site other than an endometrial cavity. In many
cases, endometrial cells proliferate in a peritoneum or ovary of
pelvis. Representative symptoms include menstrual pain
(dysmenorrhea) or infertility, and in a critical case, a patient
can experience severe pain or be fainted. In addition, with
endometriosis being a matrix, there is a case where cancer occurs.
Endometrial cells proliferate under the stimulus of female hormones
(estrogen). For this reason, as a therapeutic drug of
endometriosis, hormone drugs for inhibiting estrogen secretion, for
example, a low-dose pill, a selective agonist (inhibitor against
ovarian function and endometrial cell proliferation), and a GnRH
agonist (secretion inhibitor of follicle-stimulating hormone) are
used. Here, since curative therapy has only an operation of
removing the uterus and ovary, it is required to develop a
therapeutic drug with which a cure of endometriosis is
expected.
[0006] For example, Patent Literature 1 discloses a peptide
composition including a fusion peptide between a Z13 peptide and an
endosome escape peptide specifically binding to cyclic
nucleotide-gated channel beta 3 (CNGB3) and a fusion peptide
between a Z13 peptide and an apoptosis-inducing peptide. CNGB3 is a
molecule that is specifically highly expressed on a cell surface of
an endometrial cell (endometrial cell present other than
endometriosis) and is not expressed on a peritoneum surface. By
incorporating both peptides in an endometrial cell by the Z13
peptide moiety, it is possible to selectively induce apoptosis in
an endometrial cell. In one example, a peptide composition
including a fusion peptide between a Z13 peptide and an endosome
escape peptide and a fusion peptide between a Z13 peptide and an
apoptosis-inducing peptide was administered to the peritoneum of a
baboon which has developed endometriosis via laparoscopy, selective
apoptosis was induced only in a cell of a lesion of endometriosis,
and apoptosis was not induced in other adjacent cells (refer to NPL
1).
CITATION LIST
Patent Literature
[0007] [PTL 1] United States patent application, Publication No.
2016/145308
Non-Patent Literature
[0007] [0008] [NPL 1] Sugihara, et al., NATURE COMMUNICATIONS,
2014, Volume 5, Article Number 4478.
DISCLOSURE OF INVENTION
Technical Problem
[0009] An object of the present invention is to provide a peptide
drug that selectively induces apoptosis in a target cell.
Solution to Problem
[0010] The present inventors found that, as a result of intensive
examination to solve the problem, a peptide composition including a
fusion peptide between an endosome escape peptide including a
specific peptide sequence, an apoptosis-inducing peptide including
a specific peptide sequence, and a Z13 peptide has a high
efficiency in terms of higher selectivity and can induce apoptosis
in a endometrial cell which expresses CNGB3, compared to the
peptide composition including a fusion peptide between a Z13
peptide and an endosome escape peptide and a fusion peptide between
a Z13 peptide and an apoptosis-inducing peptide. In addition, the
present inventors found that CNGB3 is expressed not only in the
endometrium and retina but also in some cancer cells, thereby
completing the present invention.
[0011] That is, the present invention provides the following
cytocidal agent, endometriosis model animal, and the like.
[0012] [1] A cytocidal agent having a peptide consisting of an
amino acid sequence represented by SEQ ID NO: 1 and a site
selectively binding to a target molecule.
[0013] [2] The cytocidal agent according to [1], in which the
target molecule is a molecule present on a surface of a cell or a
tissue.
[0014] [3] The cytocidal agent according to [1] or [2], in which
the peptide consisting of an amino acid sequence represented by SEQ
ID NO: 1 is a peptide exclusively consisting of L-amino acids, a
peptide in which, in the amino acid sequence represented by SEQ ID
NO: 1, the first to the 14.sup.th amino acids are D-amino acids,
and the 15.sup.th to 19.sup.th amino acids are L-amino acids, a
peptide in which, in the amino acid sequence represented by SEQ ID
NO: 1, the first to 14.sup.th amino acids are L-amino acids, and
the 15.sup.th to the 19.sup.th amino acids are D-amino acids, or a
peptide exclusively consisting of D-amino acids.
[0015] [4] The cytocidal agent according to any one of [1] to [3],
in which the site selectively binding to the target molecule is a
peptide or a protein, and the peptide consisting of the amino acid
sequence represented by SEQ ID NO: 1 and the site selectively
binding to the target molecule are directly or indirectly linked to
each other.
[0016] [5] The cytocidal agent according to any one of [1] to [4],
in which the target molecule is CNGB3 or annexin I.
[0017] [6] The cytocidal agent according to [1], in which the site
selectively binding to the target molecule is a peptide consisting
of an amino acid sequence represented by SEQ ID NO: 2, and the site
selectively binding to the target molecule is directly or
indirectly linked to a downstream of the peptide consisting of the
amino acid sequence represented by SEQ ID NO: 1.
[0018] [7] The cytocidal agent according to [1], in which the site
selectively binding to the target molecule is a peptide consisting
of an amino acid sequence represented by SEQ ID NO: 3, and the
peptide consisting of the amino acid sequence represented by SEQ ID
NO: 1 is directly or indirectly linked to a downstream of the site
selectively binding to the target molecule.
[0019] [8] The cytocidal agent according to any one of [1] to [7],
which is a therapeutic agent of a disease resulting from abnormal
proliferation of a cell.
[0020] [9] The cytocidal agent according to [8], in which the
disease is endometriosis or cancer.
[0021] [10] An endometriosis model animal, in which a
CNGB3-overexpressing cell into which a gene encoding CNGB3 is
introduced is transplanted in a peritoneal cavity. [11] A method of
determining a disease onset possibility of a disease in which
disease-causing cells have expressed CNGB3, including: measuring
CNGB3 in an exosome collected from a subject animal, and comparing
the obtained measurement value with a pre-set reference value to
determine a possibility that the subject animal has developed the
disease.
[0022] [12] The method of determining a disease onset possibility
according to [11], wherein determining that the subject animal has
a high possibility of developing the disease if the measurement
value exceeds the reference value.
[0023] [13] The method of determining a disease onset possibility
according to [11] or [12], wherein the exosome is isolated from
blood collected from the subject animal.
[0024] [14] The method of determining a disease onset possibility
according to any one of [11] to [13], wherein measuring CNGB3 by
using a peptide consisting of an amino acid sequence represented by
SEQ ID NO: 2.
[0025] [15] The method of determining a disease onset possibility
according to any one of [11] to [14], in which the subject animal
is a human.
[0026] [16] The method of determining a disease onset possibility
according to any one of [11] to [15], in which the disease is
endometriosis or cancer. [17] A biomarker which includes an amount
of CNGB3 in an exosome and is used for determining presence or
absence of disease onset of endometriosis or cancer.
Advantageous Effects of Invention
[0027] The cytocidal agent according to the present invention can
very efficiently induce apoptosis in a target cell. For this
reason, the cytocidal agent is particularly effective as a
therapeutic agent of a disease resulting from abnormal
proliferation of a cell of endometriosis or cancer. In addition,
the endometriosis model animal according to the present invention
is useful for drug efficacy tests on therapeutic agent candidate
substances of endometriosis. In addition, by the disease onset
evaluation method and the biomarker according to the present
invention, it is possible to conveniently and efficiently evaluate
a disease onset possibility of a disease that specifically
expresses CNGB3 such as endometriosis and cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0028] FIG. 1 is a view illustrating a measurement result of
emission intensity (RLU) of a reaction solution treated with each
peptide in Reference Example 1.
[0029] FIG. 2 is a view illustrating a measurement result of
emission intensity (RLU) of a reaction solution treated with each
peptide in Example 1.
[0030] FIG. 3 is a view illustrating a measurement result of
emission intensity (RLU) of a reaction solution treated with each
peptide of a peptide 8 and peptides A1 to A6 in Example 2.
[0031] FIG. 4 is a view illustrating a measurement result of
emission intensity (RLU) of a reaction solution treated with each
peptide of the peptide 8 and peptides B1 to B7 in Example 2.
[0032] FIG. 5 is a view illustrating a measurement result of
emission intensity (RLU) of a reaction solution treated with each
peptide in Example 3.
[0033] FIG. 6 is a view illustrating a measurement result of the
number of alive cells of A431-CNGB3-myc cells treated with a
peptide A2 in Example 4.
[0034] FIG. 7 is a staining image in which Apop-Tag assay and
nuclear staining were performed on the A431-CNGB3-myc cells treated
with the peptide A2 in Example 4.
[0035] FIG. 8 is a view illustrating a measurement result of
emission intensity (RLU) of peritoneum of an endometriosis model
mouse in which the peptide A2 was administered in the peritoneal
cavity one time in Example 5.
[0036] FIG. 9 is a view illustrating a measurement result of
emission intensity (RLU) of peritoneum of an endometriosis model
mouse in which the peptide A2 was administered in the peritoneal
cavity multiple times in Example 5.
[0037] FIG. 10 is a view illustrating a measurement result of a
relative ATP amount (%) of the A431-CNGB3-myc cells treated with
each peptide in Example 6.
[0038] FIG. 11 is a view illustrating a measurement result of a
relative ATP amount (%) of Ishikawa cells treated with each peptide
in Example 6.
[0039] FIG. 12 is a view illustrating a measurement result of a
relative ATP amount (%) of SNG-II cells treated with each peptide
in Example 6.
[0040] FIG. 13 is a view illustrating a measurement result of a
relative ATP amount (%) of Hec-1A cells treated with each peptide
in Example 6.
[0041] FIG. 14 is a view illustrating a measurement result of a
relative ATP amount (%) of RL95-2 cells treated with each peptide
in Example 6.
[0042] FIG. 15 is a view illustrating a result of time-dependently
measuring an increase rate (%) of the number of photons of a
cancer-carrying mouse to which an IF7 (RR)-(K+H) peptide is
administered in Example 8.
[0043] FIG. 16 is a view illustrating a result of time-dependently
measuring an increase rate (%) of a tumor volume of the
cancer-carrying mouse to which the IF7 (RR)-(K+H) peptide is
administered in Example 8.
BEST MODE FOR CARRYING OUT THE INVENTION
[0044] <Cytocidal Agent>
[0045] The cytocidal agent according to the present invention has a
peptide consisting of an amino acid sequence (KLAKLAKKLAKLAKHLAHL)
represented by SEQ ID NO: 1 (hereinafter, referred to as "effector
peptide") and a site selectively binding to a target molecule. The
effector peptide is a peptide in which a peptide having an
apoptosis-inducing activity and a peptide having an endosome escape
activity are linked in tandem. The cytocidal agent according to the
present invention binds to a target molecule through a site
selectively binding to the target molecule and is taken into the
target cell by endocytosis. Subsequently, as a result of
destructing an endosome membrane by the action of the peptide site
having endosome escape activity, the cytocidal agent included in
endosome taken into the target cell is released in cytoplasm of the
target cell. The cytocidal agent released in cytoplasm induces
apoptosis of the target cell by interfering with mitochondrial
membrane by the action of the peptide site having
apoptosis-inducing activity.
[0046] The cytocidal agent according to the present invention
includes all of a peptide moiety having apoptosis-inducing
activity, a peptide moiety having endosome escape activity, and a
site selectively binding to a target molecule in a molecule. For
this reason, it is possible to very efficiently induce apoptosis in
a target cell, compared to a peptide composition each independently
including a peptide in which a peptide selectively binding to a
target molecule is linked to a peptide having apoptosis-inducing
activity and a peptide in which a peptide selectively binding to a
target molecule is linked to a peptide having endosome escape
activity.
[0047] In the present invention and the present specification, the
"target cell" is a subject cell in which apoptosis is induced. The
"target molecule" is a molecule present on a surface of a subject
cell in which apoptosis is induced or a tissue surface on which the
cell is present, and is a molecule to which the cytocidal agent
according to the present invention selectively binds.
[0048] As disclosed in Patent Literature 1, a peptide (hereinafter,
referred to as "KLAK peptide") consisting of an amino acid sequence
(hereinafter, referred to as "KLAK sequence") consisting of 4 amino
acid repeats of KLAK has an action (apoptosis-inducing activity) of
inducing apoptosis by interfering with mitochondrial membrane. In
addition, a peptide (hereinafter, referred to as "HLAH peptide")
consisting of an amino acid sequence (hereinafter, referred to as
"HLAH sequence") consisting of 4 amino acid repeats of HLAH has an
action (endosome escape activity) of destructing the endosome
membrane. The peptide in which the KLAK peptide is linked to the
HLAH peptide has endosome escape activity and apoptosis-inducing
activity. However, the intensity of the respective activities is
affected by the length of amino acids in each peptide and the order
of the linkage. Both of the peptide in which the HLAH peptide is
linked to a downstream (C-terminal) of the KLAK peptide and the
peptide in which the KLAK peptide is linked to a downstream
(C-terminal) of the HLAH peptide has endosome escape activity and
apoptosis-inducing activity, but the peptide in which the HLAH
peptide is linked to a downstream (C-terminal) of the KLAK peptide
has high apoptosis-inducing activity.
[0049] The effector peptide consisting of an amino acid sequence
represented by SEQ ID NO: 1 is a peptide in which an HLAH sequence
consisting of 5 amino acids is linked to a downstream of the KLAK
sequence consisting of 14 amino acids. That is, in the amino acid
sequence of SEQ ID NO: 1, the first to the 14.sup.th amino acids
are sites having apoptosis-inducing activity and the 15.sup.th to
the 19.sup.th amino acids are sites having endosome escape
activity. In order to obtain the highest apoptosis-inducing
activity in a case of being taken into the target cell, the
effector peptide is a peptide in which the length of the KLAK
sequence, the length of the HLAH sequence, and the order of linkage
of the KLAK sequence and the HLAH sequence are optimized. Since the
cytocidal agent has the effector peptide, the cytocidal agent
according to the present invention has very high apoptosis-inducing
activity.
[0050] The effector peptide included in the cytocidal agent
according to the present invention is not particularly limited as
long the peptide is a peptide consisting of an amino acid sequence
represented by SEQ ID NO: 1, may be a peptide consisting of a
L-amino acid, may be a peptide consisting of a D-amino acid, or may
be a peptide consisting of a L-amino acid and a D-amino acid. Since
stability is high in the endosome and higher apoptosis-inducing
activity is obtained, the effector peptide is preferably a peptide
consisting of at least some D-amino acids, at least one of the KLAK
sequence (in the amino acid sequence represented by SEQ ID NO: 1,
the first to the 14.sup.th amino acids) and the HLAH sequence (in
the amino acid sequence represented by SEQ ID NO: 1, the 15.sup.th
to the 19.sup.th amino acids) is more preferably a peptide
consisting of a D-amino acid, and all thereof is particularly
preferably a peptide consisting of a D-amino acid.
[0051] In the cytocidal agent according to the present invention,
the effector peptide including a site selectively binding to a
target molecule and an amino acid sequence represented by SEQ ID
NO: 1 may directly bind, or may indirectly bind via a linker. The
linker is not particularly limited, and examples thereof include a
peptide of 1 to 20 amino acids, a sugar chain, polyethylene glycol,
polyolefin, and the like. Since synthesis is relatively easy, as
the cytocidal agent according to the present invention, those in
which a site selectively binding to a target molecule is a peptide
or protein, and which binds to the effector peptide directly or via
a peptide of 1 to 20 amino acids are preferable. In addition, in
the cytocidal agent according to the present invention, the site
selectively binding to a target molecule may be linked to an
N-terminal side of the effector peptide consisting of an amino acid
sequence represented by SEQ ID NO: 1, or may be linked to a
C-terminal side as long as binding properties to the target
molecule is not hindered.
[0052] The site selectively binding to a target molecule in the
cytocidal agent according to the present invention is not
particularly limited, and is determined according to the target
molecule. The site may be a peptide or protein, may be an oligo
nucleotide or nucleic acid, may be a sugar chain, may be a lipid,
or may be a low-molecular compound.
[0053] The target molecule to which the cytocidal agent according
to the present invention selectively binds is not particularly
limited as long as the target molecule is a molecule present on a
surface of a target cell or tissue in which apoptosis is induced,
and may be a protein, may be a sugar chain, or may be a lipid.
[0054] Selectivity of the cytocidal agent according to the present
invention becomes high, and thus side effects are decreased in a
case of being used as a therapeutic agent of various diseases. From
this, as the target molecule to which the cytocidal agent according
to the present invention selectively binds, a molecule, in which an
expression amount on a surface of a target cell or a tissue surface
including the target cell is prominently large compared to the
expression amount in many other cells or tissues, is preferable,
and a molecule, which is specifically expressed on the surface of
the target cell or the tissue surface including the target cell, is
more preferable.
[0055] For example, CNGB3 is a membrane protein highly expressed
only in the endometrium and retina in normal tissues. Local
existence of CNGB3 in normal tissues is very biased, and it is only
shown that moderate expression is confirmed in pineal bodies and
weak expression is confirmed in bone marrow, choroid plexus,
oviduct, eyes, ovaries, and testes. For this reason, the cytocidal
agent having CNGB3 as a target molecule in the cytocidal agent
according to the present invention is a cytocidal agent capable of
specifically inducing apoptosis in the endometrium or retina, and
is useful as a therapeutic agent for diseases of the endometrium or
retina. In particular, since apoptosis is not induced in normal
cells in the peritoneal cavity or pelvis, and apoptosis can be
selectively induced in endometrial cells, it is very appropriate as
a therapeutic agent for endometriosis.
[0056] In addition, CNGB3 is relatively strongly expressed in
various cancer cells as well. For this reason, the cytocidal agent
having CNGB3 as a target molecule in the cytocidal agent according
to the present invention has high selectivity with respect to a
cancer cell that expresses CNGB3 and is useful as an anti-cancer
drug in which side effects are suppressed. Examples of cancers that
expresses CNGB3 include uterine cancer, cervical cancer, pelvic
cavity cancer, ovarian cancer, breast cancer, peritoneal wall
cancer, omentum majus tumor, esophagus cancer, stomach cancer,
small intestinal cancer, colon cancer, rectal cancer, appendix
cancer, gall bladder cancer, pancreatic cancer, liver cancer,
splenic cancer, kidney cancer, tongue cancer, pharynx cancer, nasal
cancer, parotid gland cancer, thyroid cancer, malignant lymphoma,
bone tumor, skin cancer, lung cancer, mediastinal cancer, testis
cancer, prostate cancer, bladder cancer, brain tumor, and the like.
Even cancer cells derived from the same kinds of tissues include
both cancer cells which express CNGB3 and cancer cells which do not
express CNGB3. For this reason, in a case where the cytocidal agent
having CNGB3 as a target molecule in the cytocidal agent according
to the present invention is used as an anti-cancer drug, it is
preferable to check in advance by biopsy and the like that a target
cancer cell is a cell which expresses CNGB3.
[0057] In a case where CNGB3 is used as a target molecule, examples
of the site selectively binding to the target molecule include a
peptide consisting of an amino acid sequence represented by SEQ ID
NO: 2 (VRRAXNXPG; X represents an optional amino acid present
naturally), a peptide consisting of an amino acid sequence obtained
by partially modifying the amino acid sequence represented by SEQ
ID NO: 2 (hereinafter, the peptide is referred to as "CNGB3-binding
peptide"), and the like (Patent Literature 1). Examples of the
peptide consisting of an amino acid sequence obtained by partially
modifying the amino acid sequence represented by SEQ ID NO: 2
include a peptide in which 1, 2, or 3 amino acids of the amino acid
sequence represented by SEQ ID NO: 2 are deleted, replaced, or
added, and maintains binding properties to CNGB3; a peptide which
has at least 75% or more, preferably 85% or more, and more
preferably 90% or more sequence identity to the amino acid sequence
represented by SEQ ID NO: 2 and maintains binding properties to
CNGB3; and the like. Specific examples of the CNGB3-binding peptide
include a peptide consisting of an amino acid sequence represented
by SEQ ID NO: 3 (VRRADNRPG) (hereinafter, referred to as "Z13
peptide"), a peptide consisting of an amino acid sequence
represented by SEQ ID NO: 4 (VRRAENRPG), a peptide consisting of an
amino acid sequence represented by SEQ ID NO: 5 (VRRANNLPG), a
peptide consisting of an amino acid sequence represented by SEQ ID
NO: 6 (VRRANNRPG), and the like.
[0058] From a viewpoint of stronger apoptosis-inducing activity in
a cell which expresses CNGB3, as the cytocidal agent having CNGB3
as a target molecule, a peptide in which the Z13 peptide and the
effector peptide are linked directly or indirectly via a linker is
preferable, a peptide in which the Z13 peptide and the effector
peptide are linked directly or via a peptide consisting of 1 to 20
amino acids is more preferable, a peptide in which the Z13 peptide
is linked to the C-terminal of the effector peptide directly or via
1 to 20 amino acids are further more preferable, a peptide in which
the C-terminal of the effector peptide is directly linked to the
N-terminal of the Z13 peptide (SEQ ID NO: 26:
KLAKLAKKLAKLAKHLAHLVRRADNRPG) is even further more preferable, and
a peptide in which the C-terminal of the effector peptide
exclusively consisting of D-amino acids is directly linked to the
N-terminal of the Z13 peptide exclusively consisting of all L-amino
acids is particularly preferable.
[0059] In addition, annexin I is a membrane protein which is
expressed in cytoplasm in normal vascular endothelial cells, but is
specifically expressed on the blood stream side of the vascular
endothelial cells of new blood vessels (new tumor blood vessel) in
malignant tumor tissues. A molecule selectively binding to the
annexin I administered into a living body is taken into the
vascular endothelial cells of new tumor blood vessels via a bond to
annexin I expressed on the blood stream side of the new tumor blood
vessels in the malignant tumor tissues. The molecule taken into the
vascular endothelial cells are transported from the apical side to
the basal side by vesicle transport and discharged to interstitial
cells. The discharged molecule is diffused in interstitial cells
and taken into tumor cells. For this reason, a cytocidal agent
having annexin I as a target molecule in the cytocidal agent
according to the present invention is a cytocidal agent capable of
inducing apoptosis in a cancer cell, and is useful as an
anti-cancer drug. The cytocidal agent having annexin I as a target
molecule is specifically taken into the vascular endothelial cells
of new tumor blood vessels, and in the vesicle transport process in
the vascular endothelial cells or in the vesicle transport process
in the taken tumor cells, a membrane of endosome is destructed by a
function of HLAH peptide moiety and released into cytoplasm. After
that, by a function of KLAK peptide moiety, cells into which the
cytocidal agent is taken are killed. In a case where a cytocidal
agent having annexin I as a target molecule is used as an
anti-cancer drug, cancers which are therapeutic subjects are not
particularly limited, and can be used for the same cancer as those
exemplified described above.
[0060] In a case of having annexin I as a target molecule, examples
of a site selectively binding to a target molecule include a
peptide consisting of an amino acid sequence represented by SEQ ID
NO: 7 (IFLLWQR) (hereinafter, referred to as "IF7 peptide") and a
peptide consisting of an amino acid sequence in which a moiety of
the amino acid sequence represented by SEQ ID NO: 7 is modified
(hereinafter, the peptide is referred to as "annexin I-binding
peptide"). Examples of the peptide consisting of an amino acid
sequence in which a moiety of the amino acid sequence represented
by SEQ ID NO: 7 is modified include a peptide in which one, two, or
three amino acids of the amino acid sequence represented by SEQ ID
NO: 7 are deleted, substituted, or added and which maintains
binding performance to annexin I or a peptide which has at least
70% or more, and preferably 85% or more sequence identity with the
amino acid sequence represented by SEQ ID NO: 7 and maintains
binding performance to annexin I.
[0061] From a viewpoint of having stronger apoptosis-inducing
activity to cancer cells, the cytocidal agent having annexin I as a
target molecule is preferably a cytocidal agent in which an IF7
peptide and the effector peptide are linked directly or indirectly
via a linker, more preferably a peptide in which the IF7 peptide
and the effector peptide are linked directly or via a peptide
consisting of 1 to 20 amino acids, even more preferably a peptide
in which the effector peptide is linked to a C-terminal of the IF7
peptide directly or via a peptide consisting of 1 to 20 amino
acids, still even more preferably a peptide in which the effector
peptide is linked to a C-terminal of the IF7 peptide via a peptide
consisting of 1 to 5 amino acids, and particularly preferably a
peptide in which a C-terminal of the IF7 peptide exclusively
consisting of L-amino acids is linked to a N-terminal of the
effector peptide exclusively consisting of D-amino acids via a
peptide consisting of 1 to 5 L-amino acids. Examples of the peptide
in which the C-terminal of the IF7 peptide is linked to the
N-terminal of the effector peptide via a peptide consisting of 1 to
5 amino acids include an amino acid sequence
(IFLLWQRRRKLAKLAKKLAKLAKHLAHL) represented by SEQ ID NO: 40.
[0062] In a case where the cytocidal agent according to the present
invention is used as a therapeutic drug, the administration path is
not particularly limited, and appropriately determined depending on
the target cell and tissues including thereof. Examples of the
administration path of the cytocidal agent according to the present
invention include oral administration, intravenous administration,
intraperitoneal administration, enema administration, and the
like.
[0063] The cytocidal agent according to the present invention can
be formulated as an oral solid agent such as a powder, a granule, a
capsule, a tablet, and a chewable agent, an oral liquid agent such
as a solution agent and a syrup agent, an injection, an enema
agent, a spray agent, a patch, and an ointment by a general
method.
[0064] The cytocidal agent according to the present invention is
formulated by being mixed with an excipient, a binding agent, a
lubricant, a disintegrating agent, a fluidizing agent, a solvent, a
solubilizing agent, a buffer, a suspending agent, an emulsifier, an
isotonizing agent, a stabilizer, an antiseptic agent, an
anti-oxidant, a flavoring agent, a coloring agent, and the like,
depending on the formulation necessity.
[0065] Examples of the excipient include saccharides such as
lactose, glucose, and D-mannitol, celluloses such as starch and
crystalline cellulose, sugar alcohols such as erythritol, sorbitol,
and xylitol, dicalcium phosphate, calcium carbonate, and kaoline.
Examples of the binding agent include pregelatinized starch,
gelatin, Arabic rubber, methyl cellulose, carboxy methyl cellulose,
sodium carboxy methyl cellulose, crystalline cellulose, D-mannitol,
trehalose, hydroxy propyl cellulose, hydroxy propyl methyl
cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, and the like.
Examples of the lubricant include stearic acid, calcium stearate,
talc, sucrose fatty acid ester, polyethylene glycol, and the like.
Examples of the disintegrating agent include crosslinking polyvinyl
pyrrolidone (crospovidone), low substitution degree hydroxy propyl
cellulose, starch, alginic acid, sodium alginate, and the like.
Examples of the fluidizing agent include silicic acid, silicic
anhydride, aluminum silicate, calcium silicate, a magnesium
metasilicate aluminate compound, an aluminum oxide, an aluminum
hydroxide, a magnesium oxide, a magnesium hydroxide, and the like.
Examples of the solvent include purified water, a physiological
saline solution, and the like. Examples of the solubilizing agent
include dextran, polyvinyl pyrrolidone, sodium benzoate, ethylene
diamine, salicylate amide, nicotinic acid amide, a polyoxy ethylene
hydrogenated castor oil derivative, and the like. Examples of the
buffer include sodium citrate hydrate, sodium acetate hydrate,
sodium hydrogen carbonate, trometamol, boric acid, borax, dibasic
sodium phosphate hydrate, sodium dihydrogen phosphate, and the
like. Examples of the suspending agent or the emulsifier include
sodium lauryl sulfate, Arabic rubber, gelatin, lecithin, glyceryl
monostearate, polyvinyl alcohol, polyvinyl pyrrolidone, celluloses
such as sodium carboxy methyl cellulose, polyoxy ethylene
hydrogenated castor oil, and the like. Examples of the isotonizing
agent include saccharides such as lactose, glucose, and D-mannitol,
sodium chloride, potassium chloride, glycerin, propylene glycol,
polyethylene glycol, urea, and the like. Examples of the stabilizer
include polyethylene glycol, sodium dextran sulfate, sodium
sulfite, and the like. Examples of the antiseptic agent include
para-hydroxybenzoic esters, chlorobutanol, benzyl alcohol,
phenethyl alcohol, chlorocresol, dehydroacetic acid, sorbic acid,
and the like. Examples of the anti-oxidant include sulfite,
ascorbic acid, and the like. Examples of the flavoring agent
include sweeteners generally used in the field of the therapeutic
drug and the food, aromatic chemical, and the like. Examples of the
coloring agent include coloring materials generally used in the
field of the therapeutic drug and the food.
[0066] The cytocidal agent according to the present invention may
be used as it is, and can be also used as a therapeutic composition
including other components. Examples of the other components
included in the therapeutic composition include the excipient, the
binding agent, the lubricant, the disintegrating agent, the
fluidizing agent, the solvent, the solubilizing agent, the buffer,
the suspending agent, the emulsifier, the isotonizing agent, the
stabilizer, the antiseptic agent, the anti-oxidant, the flavoring
agent, the coloring agent, and the like. In addition, the
therapeutic composition may contain other effective components than
the cytocidal agent according to the present invention.
[0067] The cytocidal agent according to the present invention is
preferably administered to mammals, more preferably administered to
humans or livestock such as mouse, rat, rabbit, guinea pig,
hamster, monkey, sheep, horse, cattle, pig, donkey, dog, and cat,
and further more preferably administered to humans.
[0068] <Endometriosis Model Animal>
[0069] Endometriosis cells express CNGB3. In addition, in many
endometriosis, the endometriosis cells proliferate in the
peritoneal cavity. For this reason, it is possible to set the
animal in which CNGB3-overexpressing cells are transplanted in the
peritoneal cavity as an endometriosis model animal.
[0070] Overexpressed CNGB3 is not particularly limited as long as
the CNGB3 is a protein exhibiting the same function as that of a
wild type human CNGB3 (Gene ID: 54714 of NCBI) expressed in the
endometriosis cells. For example, the CNGB3 may be a human CNGB3,
may be a homolog protein of human CNGB3 derived from an animal
other than a human, may be a modified body of a human CNGB3 or the
homolog, or may be a protein having the same function as that of a
human CNGB3.
[0071] Examples of the modified body include a protein consisting
of an amino acid sequence in which one or a plurality of amino
acids of an amino acid sequence of the human CNGB3 or the homolog
are deleted, substituted, or added and having the function as
CNGB3. The number of amino acids to be deleted and the like is
preferably 1 to 50, more preferably 1 to 30, further more
preferably 1 to 20, and even more preferably 1 to 10. Examples of
amino acids added to the N-terminal or C-terminal of the human
CNGB3 or the homolog include a tag peptide such as His tag, Myc
tag, and Flag tag. In addition, examples of the modified body
include a protein that includes an amino acid sequence having 70%
or more sequence identity, preferably 80% or more sequence
identity, further more preferably 85% or more sequence identity,
even more preferably 90% or more sequence identity, and even
further more preferably 95% or more sequence identity with the
amino acid sequence of the human CNGB3 or the homolog and has a
function as CNGB3.
[0072] In the present invention and the specification of the
present application, "amino acids are deleted in protein" means
that some amino acids constituting protein are lost (removed).
[0073] In the present invention and the specification of the
present application, "amino acids in protein are substituted" means
that amino acids constituting protein are changed into other amino
acids.
[0074] In the present invention and the specification of the
present application, "amino acids in protein are added" means that
new amino acids are inserted in protein.
[0075] In order to prepare CNGB3-overexpressing cells, a cell (host
cell) into which genes (CNGB3 genes) encoding CNGB3 are introduced
may not be particularly limited, may be a uterine endothelial cell,
may be a primary culture cell of a cell other than the uterine
endothelial cell collected from animal tissues, or may be a culture
cell strain. In addition, the host cell into which CNGB3 genes are
introduced may be a human-derived cell, or may be a cell derived
from an animal other than a human.
[0076] The CNGB3-overexpressing cell is obtained by introducing
CNGB3 genes in a host cell in an expressible state. Specifically,
an expression vector into which an expression cassette including a
combination of DNA required for expressing CNGB3 is incorporated is
introduced into the host cell. The expression cassette includes a
gene encoding the expressed protein and a promoter controlling
expression of the gene. The expression cassette may further contain
any one or more of terminators, a 5'-untranslated region, and a
3'-untranslated region. A preferable expression cassette is an
expression cassette including all of a gene sequence encoding a
protein, a promoter, a terminator, a 5'-untranslated region, and a
3'-untranslated region. The CNGB3 gene introduced into the host
cell may be any gene as long as the gene include a base sequence
encoding CNGB3 consisting of a target amino acid sequence, or may
be modified depending on codon frequency of the host cell.
[0077] The promoter and the terminator may be any ones as long as
the promoter and the terminator function in the host cell. The
promoter and the terminator that function in the host cell may be
originally included in the host cell, or may not be originally
included in the host cell.
[0078] As a vector for preparing an expression vector into which an
expression cassette of a CNGB3 gene is incorporated, it is possible
to use an optional vector generally used in introduction to the
host cell. The vector may be a circular plasmid vector, may be a
straight-chain vector, or may be a virus vector such as adeno
virus. It is possible to prepare an expression vector also by
incorporating a CNGB3 gene into a cloning site of a commercially
available mammal cell expression vector.
[0079] The CNGB3-overexpressing cell may be a transformant in which
the expression cassette of the CNGB3 gene is held as an
extrachromosomal gene in the cell of the host cell or a
transformant incorporated in the chromosome of the host cell. In
addition, as a transformation method of introducing an expression
vector into a mammal cell serving as a host, it is possible to
appropriately select a method among known transformation methods
such as lipofection method, a calcium phosphate deposition method,
a lithium acetate method, and an electroporation method and perform
thereof. The obtained CNGB3-overexpressing cell can be cultured
under the same condition as that of the mammal cell serving as a
host.
[0080] The kind of the organism of non-human animals in which
CNGB3-overexpressing cell is transplanted in a peritoneal cavity is
not particularly limited. As the endometriosis model animal
according to the present invention, livestock such as mouse, rat,
rabbit, guinea pig, hamster, monkey, sheep, horse, cattle, pig,
donkey, dog, and cat or test animals are preferable, for example.
Transplantation of the CNGB3-overexpressing cell into a peritoneal
cavity can be generally performed.
[0081] <Biomarker>
[0082] Exosome includes biomolecules expressed in cell membranes of
original cells from which the exosome was released. As described
above, the large amount of CNGB3 is expressed in the cell membranes
of the endometrial cell, and the exosome released from the
endometrial cell includes CNGB3. Similarly, CNGB3 is also expressed
in various cancer cells, and the exosome released from cancer cells
includes CNGB3. For this reason, the amount of CNGB3 in the exosome
is useful as a biomarker of endometriosis or cancer.
[0083] <Method of Determining Disease Onset Possibility>
[0084] A method of determining a disease onset possibility
according to the present invention (hereinafter, referred to as
"evaluation method according to present invention") is a method of
determining a disease onset possibility of a disease in which CNGB3
is expressed, using the CNGB3 in exosomes as a biomarker. In an
animal body in which disease-causing cells have expressed CNGB3 to
develop a disease (hereinafter, referred to as "CNGB3 high
expression disease"), a large amount of exosomes including a large
amount of CNGB3 is secreted from the disease-causing cells. On the
other hand, since CNGB3 is expressed only in limited tissues in
normal cells, exosomes including a large amount of CNGB3 are very
few in the animal body not developing CNGB3 high expression
disease. For this reason, based on the amount of CNGB3 in the
exosome, it is possible to identify the patient group and the
non-patient group of the CNGB3 high expression disease. In the
evaluation method according to the present invention, a possibility
that the subject animal has developed CNGB3 high expression
diseases is evaluated by measuring CNGB3 in the exosome collected
from the subject animal, and comparing the obtained measurement
value with a pre-set reference value.
[0085] As the CNGB3 high expression diseases, endometriosis or
cancer in which CNGB3 is expressed is exemplified. In addition, the
subject animal which is a subject evaluated by the evaluation
method according to the present invention may be a human, or may be
a non-human animal. The kind of organism of the non-human animals
is not particularly limited, and examples thereof include livestock
such as mouse, rat, rabbit, guinea pig, hamster, monkey, sheep,
horse, cattle, pig, donkey, dog, and cat or test animals. As the
subject animal which is a subject evaluated by the evaluation
method according to the present invention, humans, livestock, and
test animals are preferable, and humans are more preferable.
[0086] Specifically, in a case where the measurement value of CNGB3
in the exosome is more than the pre-set reference value, it is
evaluated that the subject animal from which the exosome is
collected has a high possibility of developing a CNGB3 high
expression disease. The reference value is a reference value for
identifying patients and non-patients of the CNGB3 high expression
disease.
[0087] The reference value can be experimentally obtained as a
threshold capable of measuring the amount of CNGB3 in the exosome
of the patient group and the non-patient group of a CNGB3 high
expression disease and distinguishing the both groups. A method of
determining a reference value of the amount of CNGB3 in the exosome
in the present invention is not particularly limited, and, for
example, is obtained by a general statistical method.
[0088] As an example of a method of obtaining a reference value,
for example, an exosome of a patient diagnosed as a patient with a
target CNGB3 high expression disease is collected by other methods
such as a generally performed pathological examination, and the
amount of the CNGB3 is measured. After performing measurement on a
plurality of patients, it is possible to calculate the amount of
CNGB3 in the exosome thereof by the average value or median value
and to set a numerical value including thereof as a reference
value.
[0089] In addition, it is possible to perform measurement on the
amount of CNGB3 in the exosome with respect to a plurality of CNGB3
high expression patients and a plurality of CNGB3 high expression
non-patients, to calculate both of the amount of CNGB3 in the
exosome of the CNGB3 high expression patient group and the CNGB3
high expression non-patient group and variation by an average value
or median value, to obtain a threshold in which both numerical
values are distinguished by considering variations, and to set
thereof as a reference value.
[0090] In the CNGB3 high expression disease to be evaluated, in a
case where CNGB3 is specifically expressed in the disease-causing
cells, the reference value can be set as a detection limit value of
CNGB3. In a case where CNGB3 is detected from the exosome collected
from the subject animal, it can be evaluated that the subject
animal has a high possibility of developing the CNGB3 high
expression disease, and in a case where the CNGB3 is not detected,
it can be evaluated that the subject animal has a high possibility
of not developing the CNGB3 high expression disease.
[0091] The measurement method of the amount of CNGB3 in the exosome
collected from the subject animal is not particularly limited, and
measurement can be carried out by various methods generally used
when quantitatively or semi-quantitatively measuring expression of
the protein. Examples of the method include methods using immune
reaction such as an ELISA method, immunohistochemistry, and a
Western blotting method. In addition, the amount of CNGB3 in the
exosome collected from the subject animal may be obtained as an
amount of exosome including CNGB3. The CNGB3 is mainly present on a
lipid double membrane surface of the exosome. For this reason, it
is possible to measure the amount of CNGB3 in the exosome also by
applying various methods of measuring exosome having a specific
surface molecule. Examples of devices measuring the exosome having
a specific surface molecule include exosome measurement systems
such as "Exo Counter" (manufactured by JVC Kenwood Corporation),
"dNano" (manufactured by Meiwafosis Co., Ltd.), "Nano Sight"
(manufactured by Quantum Design Japan), and "SP6800" (Sony
Corporation).
[0092] In a measurement method using immune reaction, any
anti-CNGB3 antibody may be used. In addition, instead of the
anti-CNGB3 antibody, it is possible to use a molecule binding to
CNGB3. As the molecule, any of peptide, protein, nucleic acid, low
molecule, and the like may be used. In addition, the molecule is
preferably a labeled product (one in which a labeling substance
directly or indirectly binds to a site binding to CNGB3). The
labeling substance is not particularly limited, and, for example,
may be a low molecule such as biotin, may be a fluorescent
substance, may be an enzyme, or may be a tag peptide such as His
tag, Myc tag, and Flag tag.
[0093] For example, the peptide binding to CNGB3 is preferably the
above-described CNGB3-binding peptide or the labeled product, and
is particularly preferably a Z13 peptide or the labeled product. In
addition, it is also possible to use a peptide bindable to CNGB3
such as a peptide consisting of an amino acid sequence represented
by SEQ ID NO: 41 (MQRTRATPG) (hereinafter, referred to as "Z24
peptide") and a peptide consisting of an amino acid sequence
represented by SEQ ID NO: 42 (VRSSRSTPQ) (hereinafter, referred to
as "Z11 peptide") (for either thereof, refer to NPL 1). As the
peptide binding to CNGB3, a peptide in which all of the peptide
moiety consists of L-amino acids may be used, and it is also
possible to use a peptide consisting of D-amino acids. For example,
it is possible to use a biotinylated Z13 peptide, and a set of
avidin and streptovine labeled with enzyme and fluorescence.
[0094] The exosome used in measurement of the amount of CNGB3 may
be an exosome collected from a subject animal, or may be a
biological sample itself collected from the subject animal, but is
preferably an exosome purified from the biological sample.
Separation of the exosome from the biological sample can be
performed by using a kit for commercially available exosome
separation, for example.
[0095] The biological sample including an exosome is not
particularly limited, but may be blood, plasma, serum, tear,
saliva, peritoneal fluid, urine, and the like, or may be tissue
pieces collected from a mucous membrane such as uterine mucosa and
digestive tract mucous membrane or tissues such as liver, stomach,
small intestine, and large intestine. Among these, blood, plasma,
and serum are widely used as clinical specimens and can be
relatively minimally invasively collected. For this reason, as the
exosome used in measurement of the amount of CNGB3 in the
evaluation method according to the present invention, an exosome
isolated from blood collected from a specimen animal, particularly
plasma or serum, is preferable. Since the evaluation method
according to the present invention can be carried out using the
exosome isolated from serum and the like as a sample, the
evaluation method according to the present invention is also
effective in fast screening of CNGB3 high expression disease such
as health diagnosis.
[0096] The evaluation method according to the present invention can
be used in evaluation of therapeutic effects in treatment of the
CNGB3 high expression disease. For example, the evaluation method
according to the present invention is performed on a patient with
the CNGB3 high expression disease before starting and after
completion of treatment of the CNGB3 high expression disease. In a
case where the CNGB3 high expression disease-causing cells were
decreased, or the physiological activity was lowered in a patient's
body by the treatment, in the patient's body, the amount of the
exosome secreted from the CNGB3 high expression disease-causing
cells is lowered. For this reason, in a case where the amount of
CNGB3 in the exosome is significantly decreased after treatment
compared to before treatment, it can be evaluated that the
therapeutic effect is obtained by the treatment. In addition, it
can be evaluated that the greater the percentage of decrease in the
amount of CNGB3 in the exosome gets, the higher the therapeutic
effect becomes. It is possible to monitor the therapeutic effect by
performing the evaluation method according to the present invention
over time even during the treatment period, in addition to before
starting and after completion of treatment of the CNGB3 high
expression disease.
[0097] In addition, by performing the evaluation method according
to the present invention on an animal affected by the CNGB3 high
expression disease at least once over time, it is possible to
monitor presence or absence of disease onset of the CNGB3 high
expression disease of the affected animal. For example, in a case
where the CNGB3 high expression disease is a disease which has a
high recurrence risk even in a cured case such as cancer or
endometriosis, by performing the evaluation method according to the
present invention on a patient with the CNGB3 high expression
disease over time, it is possible to monitor presence or absence of
recurrence.
EXAMPLES
[0098] Subsequently, the present invention will be further
explained in detail using examples. The present invention is not
limited to the following examples.
[0099] <Endometriosis Model Cell (A431-CNGB3-Myc Cell)>
[0100] A transformed cell (A431-CNGB3-myc cell) obtained by
introducing a gene encoding human CNGB3, in which an myc tag was
fused in a C-terminal, into an A431 cell (human epithelial-like
cell cancer-derived cell strain) and forcibly expressing thereof
was prepared as an endometriosis model cell.
[0101] Culturing of the A431-CNGB3-myc cell was performed at
37.degree. C. in a 5 volume % carbon dioxide environment using a
medium in which 10% of inactivated FBS (bovine fetal serum,
manufactured by Corning Corporation) and 1% of
penicillin-streptomycin (manufactured by Invitrogen Corporation)
were contained in a DMEM High Glucose medium (manufactured by GIBCO
Corporation), as a culture medium. Passage was performed every two
or three days.
[0102] <Endometriosis Model Mouse>
[0103] A431-CNGB3-myc cells were transplanted to a peritoneal
cavity of an immunodeficient mouse (NOD/ShiJic-scid Jcl strain,
supplied by CLEA Japan, Inc.) to prepare an endometriosis model
mouse.
[0104] Specifically, after thawing frozen-stored A431-CNGB3-myc
cells, a cell solution prepared by adding a culture medium to cells
passaged twice using a 10-cm dish (manufactured by Thermo Fisher
Scientific Co., Ltd., Lot No. F3BAXQ103) so as to be
1.times.10.sup.7 cells/0.5 mL/body was used as an administration
solution. The administration solution was intraperitoneally
administered to a 7-week-old female immunodeficient mouse as soon
as possible after preparation to transplant the A431-CNGB3-myc
cells.
[0105] For the mouse, 5 to 10 mice/cage in a polycarbonate cage
(W.times.D.times.H=270.times.440.times.187 (mm)) were bred in an
environment of 19.8.degree. C. to 27.1.degree. C., humidity of 32%
to 75%, and 12 hours of light. Feed (sterilized CRF-1 (solid type),
manufactured by Oriental Yeast Co., Ltd.) and drinking water
(sterile tap water) were freely ingested.
[0106] Model preparation was confirmed by observation and
collection of a peritoneal tumor. One to three weeks after
A431-CNGB3-myc cell transplantation, autopsy was performed on each
two mice a week to visually check whether a tumor (granular shape
of about 1 mm) was observed in the peritoneum and photographed.
Thereafter, the peritoneum was collected, and 4 locations (each on
the left and right sides of the abdomen and back) were cut out,
immersed and fixed in 10% neutral buffer formalin, respectively,
and refrigerated. The peritoneum after formalin fixation was
subjected to immunohistochemical staining using an anti-c-myc
antibody to examine the status of dissemination.
[0107] As a result of visual checking of the mouse after
A431-CNGB3-myc cell transplantation, in the first test, although a
tumor was visually confirmed 1 week after transplantation, no
peritoneal dissemination was observed, but two or three weeks after
transplantation, a tumor and peritoneal dissemination were visually
confirmed. In the second test, peritoneal dissemination was
visually confirmed even one week after transplantation. In
addition, as a result of c-myc staining of the peritoneal tissue
fragment of the mouse after transplantation, it was confirmed that
one week after the transplantation, peritoneal dissemination was
already proceeded.
Reference Example 1
[0108] In a peptide in which a KLAK peptide, an HLAH peptide, and a
CNGB3-binding peptide were linked, the impact of the type of the
amino acid sequence of the CNGB3-binding peptide on the intensity
of the cytotoxicity against endometriosis cells was examined.
[0109] Specifically, two types of peptides consisting of the amino
acid sequences shown in Table 2 were synthesized, and cytotoxicity
against A431-CNGB3-myc cells was compared. In the two types of
peptides, the KLAK sequence and the HLAH sequence moiety were
synthesized from D-amino acids, and the CNGB3-binding peptide
moiety was synthesized with L-amino acids.
TABLE-US-00001 TABLE 1 KLAK-HLAH Seq. Seq. No. WT 14 + 14
KLAKLAK-KLAKLAK-HLAHLAH- 8 HLAHLAH-VRRADNRPG D5E 14 + 14
KLAKLAK-KLAKLAK-HLAHLAH- 9 HLAHLAH-VRRAENRPG
[0110] <Evaluation of Cytocidal Properties>
[0111] The cytocidal activity of each peptide was evaluated by
measuring an ATP amount in A431-CNGB3-myc cells treated with each
peptide using CellTiter-Glo (registered trademark) assay kit
(manufactured by Promega).
[0112] Specifically, first, A431-CNGB3-myc cells after 2 passages
were disseminated into a 96-well plate (manufactured by
Corning/Costar, Lot No. 00515003) so as to be 1.times.10.sup.4
cells/well by preparing the concentration of the cells. After
culturing for 2 days after the dissemination, each peptide was
added to each well such that a final concentration was 35.5, 75, or
150 .mu.M, respectively, and cultured for 24 hours. Thereafter, the
culture supernatant was removed from each well, and CellTiter-Glo
buffer was added to the remaining cells, and after homogenization,
the supernatant was recovered by centrifugation and used as a
lysate. A double quantity of PBS and a 2.times. CellTiterGlo
Reagent of the equivalent amount of the lysate were added to the
lysate and stirred to obtain a reaction solution, and the reaction
solution was stood at room temperature for 10 minutes. The
luminescence intensity (Luminescence) (RLU: RELATIVE LIGHT UNITS)
of the reaction solution after standing at room temperature was
measured using a Synergy H1 hybrid multi-mode microplate reader
(manufactured by BioTek Corporation). The luminescence intensity of
the reaction solution is an indicator of the amount of ATP. It is
shown that the smaller the luminescence intensity of the reaction
solution is, the amount of ATP is small, and the cytocidal activity
of the administered peptide is strong. All trials were measured in
triplicate and the average was evaluated as the cytocidal activity
at the concentration of each peptide.
[0113] In the evaluation of the cytocidal activity against the
A431-CNGB3-myc cells of each peptide, the measurement result of the
fluorescence intensity (RLU) of each reaction solution is shown in
FIG. 1. In FIG. 1, the "WT" indicates the result of the peptide WT
of Table 1, and the "D5E" indicates the result of the peptide D5E
of Table 1, respectively. Both peptides commonly showed
concentration-dependent cytocidal activity. The peptide WT in which
the CNGB3-binding peptide is a Z13 peptide showed stronger
cytocidal activity than the peptide D5E in which the CNGB3-binding
peptide is a peptide of SEQ ID NO: 4.
Example 1
[0114] In the peptide in which the KLAK peptide, the HLAH peptide,
and the Z13 peptide were linked, a length of the KLAK sequence and
the HLAH sequence was optimized such that the intensity of
cytotoxicity against the endometriosis cells became the
maximum.
[0115] Specifically, 16 types of peptides consisting of an amino
acid sequence shown in Table 2 was synthesized, and cytotoxicity
against the A431-CNGB3-myc cells was compared. In the 16 types of
peptides, the KLAK sequence and the HLAH sequence moiety were
synthesized from D-amino acids, and the Z13 peptide moiety was
synthesized with L-amino acids.
TABLE-US-00002 TABLE 2 Seq. Peptide Seq. No. KLAK-HLAH 1 14 + 14
KLAKLAK-KLAKLAK-HLAHLAH-HLAHLAH-VRRADNRPG 8 2 14 + 13
KLAKLAK-KLAKLAK-HLAHLAH-HLAHLA-VRRADNRPG 10 3 14 + 12
KLAKLAK-KLAKLAK-HLAHLAH-HLAHL-VRRADNRPG 11 4 14 + 11
KLAKLAK-KLAKLAK-HLAHLAH-HLAH-VRRADNRPG 12 5 14 + 10
KLAKLAK-KLAKLAK-HLAHLAH-HLA-VRRADNRPG 13 6 14 + 9
KLAKLAK-KLAKLAK-HLAHLAH-HL-VRRADNRPG 14 7 14 + 8
KLAKLAK-KLAKLAK-HLAHLAH-H-VRRADNRPG 15 8 14 + 7
KLAKLAK-KLAKLAK-HLAHLAH-VRRADNRPG 16 HLAH-KLAK 9 14 + 14
HLAHLAH-HLAHLAH-KLAKLAK-KLAKLAK-VRRADNRPG 17 10 14 + 13
HLAHLAH-HLAHLAH-KLAKLAK-KLAKLA-VRRADNRPG 18 11 14 + 12
HLAHLAH-HLAHLAH-KLAKLAK-KLAKL-VRRADNRPG 19 12 14 + 11
HLAHLAH-HLAHLAH-KLAKLAK-KLAK-VRRADNRPG 20 13 14 + 10
HLAHLAH-HLAHLAH-KLAKLAK-KLA-VRRADNRPG 21 14 14 + 9
HLAHLAH-HLAHLAH-KLAKLAK-KL-VRRADNRPG 22 15 14 + 8
HLAHLAH-HLAHLAH-KLAKLAK-K-VRRADNRPG 23 16 14 + 7
HLAHLAH-HLAHLAH-KLAKLAK-VRRADNRPG 24
[0116] The final concentration of each peptide added to the
A431-CNGB3-myc cells disseminated into the 96-well plate was set to
be 2.34, 4.69, 9.38, 18.8, 37.5, 75, or 150 .mu.M, and the
cytocidal activity of each peptide was evaluated in the same manner
as that of Reference Example 1, except that the culture time after
addition of the peptide was set to 20 hours. In the evaluation of
the cytocidal activity against the A431-CNGB3-myc cells of each
peptide, the measurement result of the fluorescence intensity (RLU)
of each reaction solution is shown in FIG. 2. In the figure, "1" to
"16" show the result of the reaction solution to which peptides 1
to 16 of Table 2 were added, respectively. All of them showed
concentration-dependent cytocidal activity 20 hours after the
addition of the peptide. The peptides 1 to 8 in which the peptide
of the HLAH sequence is linked to a downstream of the peptide of
the KLAK sequence showed stronger cytocidal activity than the
peptides 9 to 16 in which the peptide of the KLAK sequence was
linked to a downstream of the peptide of the HLAH sequence. In
addition, in the peptides 1 to 8, the shortest peptide 8 had
stronger cytocidal activity than the longest peptide 1, and in the
peptides 9 to 16, the shortest peptide 16 had stronger cytocidal
activity than the longest peptide 9.
Example 2
[0117] The peptide 8 showing the strongest cytocidal activity in
Example 1 was further shortened to optimize the intensity of the
cytotoxicity against the endometriosis cells to the maximum.
[0118] Specifically, 14 types of peptides consisting of the amino
acid sequences shown in Table 3 were synthesized, and cytotoxicity
against A431-CNGB3-myc cells was compared. In the 14 types of
peptides, the KLAK sequence and the HLAH sequence moiety were
synthesized from D-amino acids, and the Z13 peptide moiety was
synthesized with L-amino acids.
TABLE-US-00003 TABLE 3 Peptide KLAK-HLAH Seq. Seq. No. 8 14 + 7
KLAKLAK-KLAKLAK-HLAHLAH-VRRADNRPG 16 A1 14 + 6
KLAKLAK-KLAKLAK-HLAHLA-VRRADNRPG 25 A2 14 + 5
KLAKLAK-KLAKLAK-HLAHL-VRRADNRPG 26 A3 14 + 4
KLAKLAK-KLAKLAK-HLAH-VRRADNRPG 27 A4 14 + 3
KLAKLAK-KLAKLAK-HLA-VRRADNRPG 28 A5 14 + 2
KLAKLAK-KLAKLAK-HL-VRRADNRPG 29 A6 14 + 1
KLAKLAK-KLAKLAK-H-VRRADNRPG 30 B1 13 + 7
KLAKLAK-KLAKLA-HLAHLAH-VRRADNRPG 31 B2 12 + 7
KLAKLAK-KLAKL-HLAHLAH-VRRADNRPG 32 B3 11 + 7
KLAKLAK-KLAK-HLAHLAH-VRRADNRPG 33 B4 10 + 7
KLAKLAK-KLA-HLAHLAH-VRRADNRPG 34 B5 9 + 7
KLAKLAK-KL-HLAHLAH-VRRADNRPG 35 B6 8 + 7
KLAKLAK-K-HLAHLAH-VRRADNRPG 36 B7 7 + 7 KLAKLAK-HLAHLAH-VRRADNRPG
37
[0119] The cytocidal activity of each peptide was evaluated in the
same manner as that of Example 1. In evaluation of the cytocidal
activity against the A431-CNGB3-myc cells of each peptide, the
measurement result of the fluorescence intensity (RLU) of the
reaction solution treated with the peptide 8 and the peptides A1 to
A6 was shown in FIG. 3, and the measurement result of the
fluorescence intensity (RLU) of the reaction solution treated with
the peptide 8 and the peptides B1 to B7 was shown in FIG. 4. In the
figures, the "#8" represents the result of the reaction solution to
which the peptide 8 of Table 3 was added, the "A1" to "A6" and "B1"
to "B6" show the result of the reaction solution to which the
peptides A1 to A6 and B1 to B6 of Table 3 were added, respectively.
In the peptides A1 to A6 of which the KLAK sequence consisted of 14
amino acids, the peptides A3 to A6 of which the HLAH sequence
consisted of 4 amino acids or less showed cytocidal activity even
when the peptide final concentration was 75 .mu.M (FIG. 3). In
addition, in the peptides B1 to B7 of which the HLAH sequence
consisted of 7 amino acids, the peptides B3 to B7 of which the KLAK
sequence consisted of 11 amino acids or less showed cytocidal
activity even when the peptide final concentration was 75 .mu.M
(FIG. 4). In addition, only the peptide A2 showed stronger
cytocidal activity than the peptide 8. From the results, it was
recognized that as the effector peptide, a peptide in which a
peptide of which the KLAK sequence consists of 14 amino acids and a
peptide of which the HLAH sequence consists of 5 amino acids were
linked showed the highest cytocidal activity.
Example 3
[0120] In the peptide in which the KLAK peptide, the HLAH peptide,
and the Z13 peptide were linked, the intensity of the cytocidal
activity in a case where the constituent amino acid was D-amino
acid and in a case where the constituent amino acid was L-amino
acid was examined.
[0121] Specifically, intensity of the cytocidal activity of the
peptide A2 (both of the KLAK sequence consisting of 14 amino acids
and the HLAH sequence consisting of 5 amino acids include D-amino
acids. It is referred to as a peptide (14D+5D)) described in Table
3, a peptide (14D+5L) in which in the amino acids of the peptide 8,
the KLAK sequence consisting of 14 amino acids includes D-amino
acids, and the HLAH sequence consisting of 5 amino acids include
L-amino acids, a peptide (14L+5D) in which in the amino acids of
the peptide 8, the KLAK sequence consisting of 14 amino acids
include L-amino acids, and the HLAH sequence consisting of 5 amino
acids include D-amino acids, and a peptide (14L+5L) in which all of
the amino acids of the peptide 8 include L-amino acids was examined
in the same manner as that of Example 1. All of the Z13 peptide
moiety of these peptides include only L-amino acids.
[0122] In the evaluation of the cytocidal activity against the
A431-CNGB3-myc cells of each peptide, the measurement result of the
fluorescence intensity (RLU) of the reaction solution treated with
each peptide was shown in FIG. 5. The cytocidal activity of the
peptide (14L+5L) in which all of the escape peptides include
L-amino acids was the lowest, and the cytocidal activity of the
peptide A2 (peptide (14D+5D)) in which all of the escape peptides
include D-amino acids was the highest. It was estimated that the
peptide consisting of D-amino acids was hardly digested in the
endosome or the cytoplasm than the peptide consisting of L-amino
acids, and the endosome escape activity and the apoptosis-inducing
activity were sufficiently exhibited.
Example 4
[0123] The cytocidal activity of the peptide A2 in which the
cytocidal activity was the highest was examined in Examples 2 and
3.
[0124] <Quantitative Analysis of Alive Cells>
[0125] The A431-CNGB3-myc cells disseminated on a cover glass
placed in a multi-dish for cell culture were added such that the
final concentration of the peptide A2 was 0 (control), 37.5, 75, or
150 .mu.M, and cultured. After 24, 48, or 72 hours from the
addition of the peptide, the number of cells (alive cells) adhered
and spread onto the cover glass were counted. The measurement
result of the number of the alive cells (Cells/view) per single
visual field is shown in FIG. 6. As a result, it was determined
that the number of the alive cells of the A431-CNGB3-myc cells was
decreased dependent on the concentration of the added peptide A2
over time.
[0126] <Analysis of Apoptosis-Inducing Activity>
[0127] The A431-CNGB3-myc cells disseminated on a cover glass
placed in a multi-dish for cell culture were added such that the
final concentration of the peptide A2 was 0 (control), 37.5, 75, or
150 .mu.M, and cultured. After 24, 48, or 72 hours from the
addition of the peptide, apoptosis was detected by using ApopTag
(registered trademark) Fluorescein In Situ Apoptosis Detection Kit.
FIG. 7 shows the result in which Apop-Tag assay was performed on
the cells after 48 hours from the addition of the peptide. In the
figure, the "Hoechst" represents the result of nuclear staining
with a Hoechst 33342 solution. As a result, dependent on the
concentration of the added peptide A2, it was confirmed that the
number of the Apop-Tag stained cells was large, and apoptosis was
induced by the peptide A2.
Example 5
[0128] The peptide A2 which has the highest cytocidal activity in
Examples 2 and 3 was administered to the endometriosis model mouse,
and a therapeutic effect on endometriosis was examined.
[0129] <Single Administration of Peptide into Peritoneal
Cavity>
[0130] 0.5 mL of a physiological saline solution heated to
37.degree. C. in the peritoneum was put into the endometriosis
model mouse after 7 days from transplanting the A431-CNGB3-myc
cells into the peritoneal cavity under isoflurane anesthesia, and
immediately after massaging, the peptide A2 was dissolved in the
physiological saline solution and the solution was administered
into the peritoneum. The peptide A2 was administered such that an
administration amount per mouse weight was 0 mg/10 mL/kg (control),
5 mg/10 mL/kg, or 10 mg/10 mL/kg (n=3).
[0131] <Multiple Administration of Peptide to Peritoneal
Cavity>
[0132] 0.5 mL of a physiological saline solution heated to
37.degree. C. in the peritoneum was put into the endometriosis
model mouse after 7 days from transplanting the A431-CNGB3-myc
cells into the peritoneal cavity under isoflurane anesthesia, and
immediately after massaging, the peptide A2 was dissolved in the
physiological saline solution and the solution was administered
into the peritoneum (single administration). Thereafter, after 8
and 9 days from transplanting the A431-CNGB3-myc cells into the
peritoneal cavity, the peptide A2 solution was administered in the
same manner. That is, the peptide A2 solution was administered once
a day, a total of three times. The peptide A2 was administered such
that an administration amount per mouse weight was 0 mg/10 mL/kg
(control), 2.5 mg/10 mL/kg, 5.0 mg/10 mL/kg, or 7.5 mg/10 mL/kg
(n=3).
[0133] <Collection of Peritonea (Material Collection)>
[0134] The endometriosis model mouse into which the peptide A2 was
administered died of bleeding under isoflurane anesthesia after 24
hours or 48 hours from the final administration to collect the
peritoneum. The peritoneum for ATP measurement was frozen with
liquid nitrogen, and stored in a deep freezer until the
measurement. A peritoneum for pathological specimen preparation was
immersed and fixed in 10% neutral buffer formalin, respectively,
and refrigerated.
[0135] A weight of each mouse was measured before being died of
bleeding. There was no particular change in the weight of each
mouse regardless of the administration amount or the number of
administrations of the peptide A2.
[0136] <Measurement of ATP Amount>
[0137] The measurement of the ATP amount was performed by using a
CellTiter-Glo (registered trademark) assay kit (manufactured by
Promega). The weight of the frozen peritoneum was measured, and 10
times the quantity of CellTiter-Glo buffer of the frozen tissue was
added, and after homogenization, the supernatant was recovered by
centrifugation and used as a lysate. A double quantity of PBS and a
2.times.CellTiterGlo Reagent of the equivalent amount of the lysate
were added to the lysate and stirred to obtain a reaction solution,
and the reaction solution was stood at room temperature for 10
minutes. The luminescence intensity (RLU) of the reaction solution
after standing at room temperature was measured using a Synergy H1
hybrid multi-mode microplate reader (manufactured by BioTek
Corporation). It is shown that the luminescence intensity of the
reaction solution is an indicator of the amount of ATP, and the
smaller the luminescence intensity of the reaction solution is, the
amount of ATP is small, and the cytocidal activity of the
administered peptide is strong. All trials were measured in
triplicate (n=3) and the average was evaluated as the cytocidal
activity of each peptide.
[0138] The measurement result of the luminescence intensity (RLU)
of the peritoneum of the endometriosis model mouse into which the
peptide A2 was administered once is shown in FIG. 8. In addition,
the measurement result of the luminescence intensity (RLU) of the
peritoneum of the endometriosis model mouse into which the peptide
A2 was repeatedly administered three times is shown in FIG. 9. As
shown in FIGS. 8 and 9, in any of a mouse to which administration
was performed once and a mouse to which administration was
performed multiple times, it was confirmed that the luminescence
intensity (RLU) of the peritoneum was decreased dependent on the
administered peptide A2 amount, and the amount of ATP in the
peritoneum was decreased. The decrease in the amount of ATP in the
peritoneum means reduction of the number of the alive cells of the
A431-CNGB3-myc cells transplanted into the peritoneum. From the
result, it can be said that the A431-CNGB3-myc cells in the
peritoneal cavity was killed by the peptide A2, that is, the
peptide A2 can exhibit cytocidal effect in an in-vivo environment,
and is particularly useful as a therapeutic agent for
endometriosis.
[0139] <TUNEL Staining>
[0140] HE staining and TUNEL staining were performed on the
peritoneum for pathological specimen preparation fixed with 10%
neutral buffer formalin. As a result, it was confirmed that a
peptide A2 administration group is TUNEL method (TdT-mediated Dutp
nick end labeling) positive (not illustrated), and the peptide A2
induces apoptosis and shows a cytocidal effect.
Example 6
[0141] A cytocidal activity of the peptide A2 (peptide (14D+5D)) to
the A431-CNGB3-myc cells and a cell strain derived from various
uterine cancer cells was examined.
[0142] As the cancer-derived cell strain, an Ishikawa cell derived
from human endometrium gland cancer, an SNG-II cell derived from
human endometrium cancer, an Hec-1A cell derived from human uterine
body cancer, and a RL95-2 cell derived from human endometrium
cancer were used. The cells were cultured in the same manner as the
A431-CNGB3-myc cells.
[0143] As the peptide treating each cell, three types of peptides
consisting of amino acid sequences shown in Table 4 were used. In
the three types of peptides, the KLAK sequence and the HLAH
sequence moiety were synthesized from D-amino acids, and the Z13
peptide moiety was synthesized with L-amino acids.
TABLE-US-00004 TABLE 4 Seq. KLAK-HLAH-Z13 Seq. No. A2 14D + 5D +
Z13 KLAKLAK-KLAKLAK-HLAHL- 26 VRRADNRPG A2-1 14D + Z13
KLAKLAK-KLAKLAK-VRRADNRPG 38 A2-2 5D + Z13 HLAHL-VRRADNRPG 39
[0144] The cytocidal activity of each peptide was evaluated in the
same manner as that of Example 1, except that the final
concentration of each peptide added to each cell disseminated into
the 96-well plate was set to be 0 (peptide non-added), 2.34, 4.69,
9.38, 18.8, 37.5, 75.0, 150.0, or 300.0 .mu.M. A relative
fluorescence intensity (%) of each reaction solution in a case
where the fluorescence intensity (RLU) of the reaction solution to
which no peptide was added was set to 100% was calculated as a
relative ATP amount (%). The calculation result is shown in FIGS.
10 to 14. In the figure, the "14D-Z13+5D-Z13" represents a relative
ATP amount of the reaction solution to which both of a peptide A2-1
and a peptide A2-2 were added, the "14D-Z13" represents a relative
ATP amount of the reaction solution to which only the peptide A2-1
was added, the "5D-Z13" represents a relative ATP amount of the
reaction solution to which only the peptide A2-2 was added, and the
"14D-5D-Z13" represents a relative ATP amount (%) of the reaction
solution to which only the peptide A2 was added, respectively. As
shown in FIGS. 10 to 14, it was confirmed that the peptide A2 has
cytocidal activity to not only the A431-CNGB3-myc cells but also
various cell strains derived from a uterine cancer cell. In
addition, in the cell treated with both of a peptide in which the
KLAK peptide and the Z13 peptide were linked and a peptide in which
the HLAH peptide and the Z13 peptide were linked, similar to the
cell treated only with the peptide in which the KLAK peptide and
the Z13 peptide were linked and the cell treated only with the
peptide in which the HLAH peptide and the Z13 peptide were linked,
the cytocidal activity was hardly observed or significantly
weak.
Example 7
[0145] Since the A431 cells are epithelial-like cell cancer-derived
cell strains, the endometriosis model mouse in which the
A431-CNGB3-myc cells were transplanted into the peritoneal cavity
is a cancer-bearing mouse. Here, a peptide
(IFLLWQR-RR-KLAKLAK-KLAKLAK-HLAHL, SEQ ID NO: 40) in which an
effector peptide consisting of an amino acid sequence represented
by SEQ ID NO: 1 and an IF7 peptide are linked (hereinafter,
referred to as "IF7 (RR)-(K+H) peptide") was administered to the
peritoneal cavity of the endometriosis model mouse, and the
cytocidal activity to cancer cells was examined. In the IF7
(RR)-(K+H) peptide, the KLAK sequence and the HLAH sequence moiety
were synthesized with D-amino acids, and other peptide moieties
were synthesized with L-amino acids.
[0146] <Tail Vein Administration of Peptide>
[0147] 50 .mu.L of a solution obtained by dissolving the IF7
(RR)-(K+H) peptide in a physiological saline solution was
administered into an endometriosis model mouse after 14 days from
transplanting the A431-CNGB3-myc cells into the peritoneal cavity
once a day for six consecutive days via a tail vein. The IF7
(RR)-(K+H) peptide was administered such that an administration
amount per mouse weight was 0 .mu.g/body (control), 10 .mu.g/body,
or 50 .mu.g/body.
[0148] As a result, in the control group into which a peptide was
not administered (n=8), the number of dead individuals after 24
days from transplanting the A431-CNGB3-myc cells into the
peritoneal cavity was 1, and the alive rate was 88.9%. With respect
to this, in a group into which the IF7 (RR)-(K+H) peptide was
administered such that the IF7 (RR)-(K+H) peptide was 10
jag/body/day or 50 .mu.g/body (n=6), the alive rate was 100%. In
addition, at the time of the end of the test, the spread of cancer
cells (A431-CNGB3-myc cells) in the peritoneal cavity of all mice
was examined. In contrast, in the control group, the cancer cells
relatively spread throughout the whole peritoneum, and in the group
into which the IF7 (RR)-(K+H) peptide was administered, the spread
of cancer cells in many mice was relatively limited. There was no
particular change in the weight of each mouse regardless of the
administration amount or the number of administrations of the IF7
(RR)-(K+H) peptide. From the result, it was recognized that the
intravenously administered IF7 (RR)-(K+H) peptide has cytocidal
activity to cancer cells and is useful as an anti-cancer drug.
Example 8
[0149] The IF7 (RR)-(K+H) peptide used in Example 7 was
intravenously administered to a cancer-bearing mouse in which a
tumor was formed in the back, and cytocidal activity to cancer
cells was examined.
[0150] <Luciferase Gene-Transferred Ovarian Cancer
Cancer-Bearing Subcutaneous Tumor Mouse (OVCAR3-Luc Mouse)>
[0151] As the tumor tissue to be transplanted into a mouse, a tumor
tissue obtained by culturing a luciferase gene-introduced ovarian
cancer cell strain (OVCAR3-Luc cells, transferred from another
facility) was used. Culturing of OVCAR3-Luc cells was performed at
37.degree. C. in a 5 volume % carbon dioxide environment using RPMI
medium 1640 (11875-093, gibe by life technologies) as a culture
medium.
[0152] Approximately 1.times.10.sup.6 OVCAR3-Luc cells were
transplanted into the back of an eight-week female SCID mouse
(C.B-17/Icr-scid/scid Jcl family, supplied by CLEA Japan, Inc.) to
prepare an OVCAR3-Luc mouse.
[0153] <Tail Vein Administration of Peptide>
[0154] In the same manner as that of Example 7, 50 .mu.L of a
solution obtained by dissolving the IF7 (RR)-(K+H) peptide in a
physiological saline solution was administered into the OVCAR3-Luc
mouse once a day for six consecutive days via a tail vein. The IF7
(RR)-(K+H) peptide was administered such that an administration
amount per mouse weight was 0 .mu.g/body (control) or 10
.mu.g/body.
[0155] <Measurement of Size of Tumor Tissue>
[0156] Each mouse was subjected to a luminescence imaging test, and
the number of photons of the tumors on the back and the size of the
tumor tissue were measured over time.
[0157] (1) Measurement of the Number of Photons
[0158] Measurement was performed using an in vivo luminescence
imaging apparatus (Xenogen IVIS-200, manufactured by Caliper
Corporation) using a luciferin-luciferase luminescence mechanism.
First, 100 .mu.L of a 30 mg/mL potassium D-luciferin (126-05116,
manufactured by Wako Pure Chemical Industries, Ltd.) solution was
administered to the peritoneal cavity of the OVCAR 3-Luc mouse.
After 15 minutes from the administration, the mouse was measured
with the in vivo luminescence imaging apparatus, and the number of
photons was counted.
[0159] (2) Measurement of Tumor Volume
[0160] The estimated tumor volume (mm.sup.3) on the back of each
OVCAR3-Luc mouse was obtained from the long diameter of the tumor
by the following equation. The long diameter (mm) and the short
diameter (mm) of the tumor were measured using a caliper.
[Estimated tumor volume (mm3)]=[long diameter (mm)].times.[short
diameter (mm)].times.[short diameter (mm)].times.1/2
[0161] FIG. 15 shows a temporal change in an increase rate (%) in
the number of the photons of the tumor on the back of each mouse,
and FIG. 16 shows a temporal change in an increase rate (%) of the
tumor volume (mm.sup.3) on the back of each mouse. Both of the
increase rate of the number of the photons and the increase rate of
the tumor volume were based on the value of the peptide solution on
the day before the administration starting date (100%). In the
figure, the "treatment" represents a treatment period in which the
peptide solution was intravenously administered. As shown in FIG.
15, in the mouse treated with the IF7 (RR)-(K+H) peptide, the
number of the photons was hardly increased during the peptide
treatment period, and the increase rate after the peptide treatment
period was also smaller than the control group (Saline). In
addition, although there was no difference in the tumor volume
between the control group and the IF7 (RR)-(K+H) peptide
administration group during the peptide treatment period, the
increase rate after the treatment period of the IF7 (RR)-(K+H)
peptide administration group was apparently smaller. From the
result, it can be said that administration of the IF7 (RR)-(K+H)
peptide can kill the tumor tissue in vivo, and the peptide is
useful as an anti-cancer drug.
Reference Example 2
[0162] Paraffin-embedded sections of cancer tissues of various
organs were immunostained with an anti-CNGB3 antibody to examine
presence or absence of the expression of CNGB3. As the tissue
sections, two types (OV20811 and OV2088) among four types of
commercially available human ovarian cancer tissue array (Ovary
cancer tissue array (manufactured by US Biomax) and two types
(FDA800a and MC964a) among cancer tissue arrays (Multiple organ
tumor tissue array) of various organs were used. In addition, as
the anti-CNGB3 antibody, among commercially available antibodies,
two types of Biorbyt (Catalog No. orb 156415 BRT 100UG)
(hereinafter, referred to as "antibody orb" sometimes) and Osenses
(Code; OSC00253W) (hereinafter, referred to as "antibody 253W" were
used).
[0163] Immunostaining of tissue sections was performed as follows.
First, a paraffin-embedded tissue section was reacted with a
blocking agent for liquid immunity test (Immunoblock, manufactured
by DS Pharma Biomedical Co., Ltd.) for 30 minutes to block
non-specific reaction, and then washing treatment was performed
twice with TBST (Tris buffer physiological saline containing Tween
20) for 5 minutes. Then, the tissue section was immersed in 0.3%
hydrogen peroxide water, reacted for 5 minutes, blocked for
endogenous peroxidase activity, and washed twice for 5 minutes with
TBST. Subsequently, the tissue section was immersed in a 0.3%
hydrogen peroxide solution, reacted for 5 minutes, and then
subjected to blocking treatment of internal peroxidase activity.
Thereafter, washing treatment was performed with TBST twice for 5
minutes. Subsequently, the tissue section was immersed in a primary
antibody solution obtained by diluting the anti-CNGB3 antibody 100
times with REAL Antibody Diluent (Code 52022, manufactured by Dako
Corporation) and reacted at room temperature for 30 minutes, and
then washing treatment was performed twice with TBST for 5 minutes.
In addition, the tissue section was reacted with a labeled
secondary antibody (EnVision+System-HRP-labeled Polymer anti-rabbit
antibody, manufactured by Dako Corporation) for 30 minutes at room
temperature, and then washing treatment was performed twice with
TBST for 5 minutes. Thereafter, the tissue section was treated with
a DAB coloring reagent [DAB+Liquid (large size), RUO (K3468),
manufactured by Dako Corporation] for 5 minutes to develop color.
The tissue section after DAB coloring was washed with water,
treated with Mayer's hematoxylin for 2 minutes to stain nuclei, and
then sealed.
[0164] The staining result of OV20811 among human ovarian cancer
tissue arrays is shown in Tables 5 to 10, and the staining result
of OV2088 is shown in Tables 11 to 16, respectively. The staining
result of FDA800a among cancer tissue arrays of various organs is
shown in Tables 17 and 18, and the staining result of MC964a is
shown in Tables 19 to 21, respectively. In the tables, in the
column of each anti-CNGB3 antibody, "+" means the result of
immunostaining with the antibody, and "-" means the result of no
immunostaining with the antibody, respectively. It was recognized
that most tissue sections were immunostained with the anti-CNGB3
antibody and CNGB3 was expressed.
TABLE-US-00005 TABLE 5 OV20811 Grade Stage orb 253W 1 Serous
adenocarcinoma 1 Ia + + 2 Serous adenocarcinoma I - - 3
Adenocarcinoma (sparse) -- Ia + + 4 Serous papillary adenocarcinoma
2 II + + 5 Serous papillary adenocarcinoma Ia - + 6 Mucinous
papillary adenocarcinoma 1 Ic - + 7 Mucinous papillary
adenocarcinoma -- Ia + - (fibrous tissue and blood vessel) 8
Mucinous papillary adenocarcinoma 1 + - 9 - - 10 + + 11 IIa + + 12
Serous papillary adenocarcinoma 2 Ic + + 13 Ib + - 14 IIIc + + 15 +
- 16 IV + - 17 3 Ic + + 18 Serous papillary adenocarcinoma -- IIIa
+ + (carcinoma sparse necrosis) 19 Serous papillary adenocarcinoma
3 Ic + + 20 Ib + + 21 2 - + 22 IIb + + 23 Ia + + 24 3 IIb + + 25 Ib
+ + 26 Mucinous papillary adenocarcinoma + - 27 Ia + + 28 IIa + +
29 Ib + + 30 Serous papillary adenocarcinoma 2 Ia + + 31 IIa + + 32
-- Ib + + 33 3 Ia + + 34 2 IIIc + + 35 2-3 II + + 36 Mucinous
papillary adenocarcinoma 2 I + + 37 Mucinous papillary
adenocarcinoma -- Ib - - (tumoral necrosis)
TABLE-US-00006 TABLE 6 OV20811 Grade Stage orb 253W 38 Serous
papillary adenocarcinoma 3 IIa + + 39 IV + + 40 + + 41 2 Ib + + 42
2-3 Ia - + 43 Serous papillary adenocarcinoma -- IIIc + + (fibrous
tissue and blood vessel) 44 Serous papillary adenocarcinoma 3 IV +
+ 45 Mucinous papillary adenocarcinoma IIIc + + 46 Serous papillary
adenocarcinoma Ia + + 47 + + 48 -- + + 49 1 IIIb + + 50 3 Ia + + 51
IIb + + 52 Serous adenocarcinoma 2 II + + 53 Serous papillary
adenocarcinoma 3 Ia + + 54 2 + + 55 3 IIa + + 56 Mucinous papillary
adenocarcinoma Ia + + 57 Serous papillary adenocarcinoma II + + 58
Serous adenocarcinoma 2 IIIc + + 59 Serous papillary adenocarcinoma
3 Ib + + 60 Serous papillary adenocarcinoma IIIc + + 61 Mucinous
papillary adenocarcinoma Ia + + with necrosis 62 Serous papillary
adenocarcinoma IIa + + 63 Ia + + 64 -- Ila + + 65 Serous
adenocarcinoma 3 Ia + + 66 Serous papillary adenocarcinoma + + 67
II + + 68 Ib + + 69 Ic + + 70 2 Ia + + 71 Mucinous papillary
adenocarcinoma 3 + + 72 Mucinous papillary adenocarcinoma -- Ib + +
(corpus albicans tissue) 73 Serous papillary adenocarcinoma -- Ia +
+ (fibrous tissue) 74 Serous papillary adenocarcinoma 3 III + +
TABLE-US-00007 TABLE 7 OV20811 Grade Stage orb 253W 75 Serous
papillary adenocarcinoma -- Ic - + (ovary tisuue) 76 Serous
papillary adenocarcinoma 3 IIa + + 77 + + 78 IIb + + 79 Mucinous
papillary adenocarcinoma II + + with necrosis 80 Serous papillary
adenocarcinoma Ib + + 81 + + 82 Serous papillary adenocarcinoma --
II + + (ovary tisuue) 83 Serous papillary adenocarcinoma 3 Ia + +
84 Serous papillary adenocarcinoma -- + + (chronic inflammation of
fibrous tissue and blood vessel) 85 Serous papillary adenocarcinoma
3 Ib + + 86 I + + 87 Ia + + 88 Serous papillary adenocarcinoma I +
+ with necrosis 89 Serous papillary adenocarcinoma 2 + + 90 3 II +
+ 91 Ic - + 92 Mucinous papillary adenocarcinoma Ib + + with
necrosis 93 Adenocarcinoma (sparse) -- III - + 94 Serous papillary
adenocarcinoma 3 I + + 95 Ia + + 96 Mucinous papillary
adenocarcinoma Ib + + 97 Mucinous adenocarcinoma IIa - + 89 Ib + +
99 Adenocarcinoma Ia + + 100 II + + 101 2 Ia + + 102 Endometrioid
adenocarcinoma 1 Ib + + 103 Endometrioid adenocarcinoma -- II - -
(tumoral necrosis) 104 Endometrioid adenocarcinoma 1 + + 105
Endometrioid adenocarcinoma -- Ic - - (tumoral necrosis)
TABLE-US-00008 TABLE 8 OV20811 Grade Stage orb 253W 106
Endometrioid adenocarcinoma 1 Ia + + 107 Endometrioid
adenocarcinoma (sparse) IIa - + 108 Endometrioid adenocarcinoma Ia
+ + 109 I - + 110 + + 111 Ib + + 112 Ia + + 113 IIa + + 114 I + +
115 Ia + + 116 3 I + + 117 2 Ia + + 118 I + + 119 3 IIa + + 120 IV
+ + 121 2 Ic + + 122 IIc + + 123 3 Ia + + 124 Ic + + 125 Ib + + 126
IIa + + 127 Clear cell carcinoma -- I - - 128 + + 129 Clear cell
carcinoma (sparse) IIIc - + with massive necrosis 130 Clear cell
carcinoma (sparse) I + + with necrosis 131 Clear cell carcinoma + +
132 + + 133 Clear cell carcinoma II - - (tumoral necrosis) 134
Clear cell carcinoma Ic + + 135 I + + 136 + + 137 Undifferentiated
carcinoma + + 138 Ia + + 139 Squamous cell carcinoma from 1 I + +
140 malignant transformation of teratoma 2 Ia + + 141 IIa + + 142
IIIc + + 143 Ia + + 144 3 + +
TABLE-US-00009 TABLE 9 OV20811 Grade Stage orb 253W 145 Squamous
cell carcinoma -- Ib + + 146 Transitional cell carcinoma + + 147
IIIc + + 148 Ia - + 149 + + 150 Endodermal sinus carcinoma - + 151
IV - + 152 Ic - - 153 - + 154 Ib - + 155 I - + 156 - + 157 II + +
158 Ib - + 159 II - + 160 Ib - + 161 IV - - 162 Ia + - 163 + - 164
Granular cell tumor I - + 165 Ic - + 166 Ib - - 167 I - + 168 Ia -
+ 169 Ib - - 170 Ia - + 171 IIb + - 172 Ic - - 173 I - + 174 Ia - -
175 - - 176 - + 177 + + 178 - -
TABLE-US-00010 TABLE 10 OV20811 Grade Stage orb 253W 179 Granular
cell tumor -- Ib + - 180 - - 181 Ia - - 182 II - + 183 Ib N.T. +
184 Dysgerminoma Ia + + 185 Ib + + 186 Ia + + 187 + + 188 + - 189
IIb + - 190 Ia - - 191 I - + 192 Ia - - 193 + + 194 Interstitial
cell tumor - - 195 Sertoli cell tumor + + 196 Ic - - 197 I - - 198
Theca cell tumor Ia - - 199 - - 200 Malignant theca cell tumor Ib -
+ 201 III - - 202 Malignant teratoma Ia - + 203 Immature teratoma
Ib - - 204 - - 205 I - - 206 Gynandroblastoma Ia - - 207 Malignant
fibroepithelioma - - 208 Malignant non-specificity - - steroid cell
tumor
TABLE-US-00011 TABLE 11 OV2088 Grade Stage orb 253W 1 Serous
papillary adenocarcinoma 1 Ia + + 2 + + 3 2 Ib + + 4 - - 5 1 Ia - +
6 - + 7 2 Ib + + 8 + + 9 1 Ia - - 10 - - 11 - + 12 - + 13 - - 14
Serous papillary adenocarcinoma -- - - (fibrous tissue and blood
vessel) 15 Serous papillary adenocarcinoma 1 Ic - - 16 - - 17
Serous adenocarcinoma 2 I + + 18 + + 19 Serous papillary
adenocarcinoma 1 + + 20 - + 21 Ia - + 22 + - 23 2 Ic - + 24 + + 25
III - - 26 - - 27 Ia - + 28 - + 29 - + 30 - - 31 - - 32 - - 33 Ib +
+ 34 + + 35 Serous adenocarcinoma with necrosis IIIa - - 36 Serous
adenocarcinoma (sparse) + - 37 Serous papillary adenocarcinoma I +
+ 38 + + 39 Ib + + 40 + -
TABLE-US-00012 TABLE 12 OV2088 Grade Stage orb 253W 41 Serous
adenocarcinoma (sparse) 2 Ib - - 42 Serous adenocarcinoma (fibrous
-- - - tissue and blood vessel) 43 Serous adenocarcinoma 3 Ia - -
44 - - 45 2 - + 46 - + 47 Serous adenocarcinoma (fibrous -- - - 48
tissue and blood vessel) - - 49 Serous adenocarcinoma 3 Ib + + 50 +
+ 51 Serous adenocarcinoma (ovarian tissue) -- + + 52 - + 53 Serous
papillary adenocarcinoma 3 Ia - + 54 - + 55 2 II + + 56 + + 57 3 Ib
- + 58 - + 59 2 + + 60 + - 61 III - - 62 - - 63 I + + 64 - + 65
Serous adenocarcinoma -- - - 66 (necrotic tissue) - - 67 Serous
adenocarcinoma 3 Ib - + 68 + + 69 + + 70 Serous papillary
adenocarcinoma -- + + (fibrous tissue and blood vessel) 71 Serous
adenocarcinoma (necrotic tissue) Ic - + 72 Serous adenocarcinoma 3
- + 73 III - + 74 - - 75 II + + 76 + + 77 Ic - + 78 - + 79 Ia - +
80 - +
TABLE-US-00013 TABLE 13 OV2088 Grade Stage orb 253W 81 Serous
adenocarcinoma 3 IIc + + 82 + + 83 Ia + + 84 - + 85 2 Ib + + 86
Serous adenocarcinoma -- - + (ovarian tissue) 87 Serous
adenocarcinoma 3 II + + 88 + + 89 I - + 90 - + 91 II - + 92 - + 93
IV + - 94 + - 95 II - + 96 - + 97 IIa - + 89 - + 99 Ia - - 100 + +
101 Serous adenocarcinoma -- + + (chronic inflammation of fibrous
tissue and blood vessel) 102 Serous adenocarcinoma 3 + + 103 Serous
adenocarcinoma (fibrous -- Ic + + 104 tissue and blood vessel) - +
105 Serous adenocarcinoma 3 Ic + + 106 - - 107 Serous papillary
adenocarcinoma Ib - - 108 - - 109 Serous adenocarcinoma Ia - - 110
- - 111 Ib - + 112 Serous adenocarcinoma -- - + (ovarian tissue)
113 Serous adenocarcinoma 3 Ia + - 114 + - 115 I - - 116 - + 117 -
- 118 - + 119 - + 120 - -
TABLE-US-00014 TABLE 14 OV2088 Grade Stage orb 253W 121 Serous
adenocarcinoma with 3 Ia - + 122 clear cell carcinoma - - 123
Mucinous papillary adenocarcinoma 1 I - - 124 - - 125 2 Ic - - 126
- + 127 Serous papillary adenocarcinoma 1-2 II - - 128 - - 129
Mucinous papillary adenocarcinoma 1 I + + 130 + + 131 Ia + + 132 +
+ 133 1-2 + + 134 + + 135 Mucinous adenocarcinoma 2 - - 136 - - 137
I - - 138 - - 139 Adenocarcinoma 3 Ia - - 140 - - 141
Adenocarcinoma from malignant - - 142 transformation of teratoma -
- 143 Adenocarcinoma - - 144 - - 145 Endometrioid carcinoma
(sparse) -- Ib + + 146 + + 147 Squamous cell carcinoma from IIIc
N.T. N.T. 148 malignant transformation + + of teratoma (mature
teratoma sparse) 149 Squamous celll carcinoma from 3 Ib + - 150
malignant transformation of teratoma - - 151 Squamous celll
carcinoma - + 152 - + 153 Squamous cell carcinoma from Ia + - 154
malignant transformation of teratoma + - 155 Endodermal sinus
carcinoma -- - + 156 - + 157 Ib - + 158 - + 159 - - 160 - -
TABLE-US-00015 TABLE 15 OV2088 Grade Stage orb 253W 161
Endometrioid carcinoma 2 Ia + + 162 + + 163 Granular cell tumor --
- - 164 - + 165 + + 166 + + 167 Ib - - 168 + + 169 - - 170 - - 171
Granulosa-theca cell tumor - - 172 - - 173 Granular cell tumor - -
174 - - 175 Ia - + 176 - + 177 + - 178 + - 179 Clear cell carcinoma
2 I + + 180 + + 181 3 + + 182 + + 183 Clear cell carcinoma 2 IIIc +
+ 184 with necrosis - - 185 Dysgerminoma -- I - + 186 - + 187 Ia +
+ 188 + + 189 Ib - - 190 - - 191 Immature teratoma - + 192 - - 193
Ia - + 194 - - 195 Strumal carcinoid (sparse) Ib + + 196 + + 197
Undifferentiated carcinoma + - 198 with neruoendocrine feature + -
199 Transitional cell carcinoma 2 + + 200 + N.T.
TABLE-US-00016 TABLE 16 OV2088 Grade Stage orb 253W 201
Carcinosarcoma -- Ia - - 202 - - 203 Cellular fibroma -- - - 204 -
- 205 Embryonal carcinoma Ia - - 206 N.T. N.T. 207 Diffuse B-cell
lymphoma -- - - 208 - -
TABLE-US-00017 TABLE 17 FDA800a orb 253W 1 Skin Basal cell
carcinoma of occipitalia skin + - 2 Squamous cell carcinoma of left
chest wall + - 3 Lung Small cell carcinoma - - 4 Adenocarcinoma + -
5 Squamous cell carcinoma - + 6 Large cell carcinoma + + 7
Bronchoalveolar carcinoma - - 8 Testis Spermatocytoma + - 9
Spermatocytoma - - 10 Embryonal carcinoma + + 11 Prostate
Adenocarcinoma (Gleason grade: 4; + + Gleason score: 4 + 5) 12
Rhabdomyosarcoma - - 13 Gall Adenocarcinoma + + 14 bladder
Adenosquamous carciinoma + - 15 Small Adenocarcinoma + - 16
intestine Low garde malignant interstitialoma - + 17 Pancreas Duct
Adenocarcinoma + + 18 Islet cell carcinoma - + 19 Colon
Adenocarcinoma + + 20 Low garde malignant interstitialoma + + 21
Rectum Adenocarcinoma + + 22 Low garde malignant interstitialoma -
- 23 Malignant melanoma + + 24 Esophagus Squamous cell carcinoma +
+ 25 Adenocarcinoma + - 26 Appendix Adenocarcinoma + + 27 Tongue
Squamous cell carcinoma + + 28 Parotid Adenoid cystic carcinoma + +
gland 29 Stomach Mucinous adenocarcinoma + + 30 Liver
Hepatoblastoma + - 31 Hepatocellular carcinoma + + 32 Breast Ductal
carcinoma in situ + + 33 Invasive ductal carcinoma + + 34 Duvtal
carcinoma in situ + - 35 Medullary carcinoma N.T. N.T. 36 Uterus
Adenocarcinoma endometrium + + 37 Clear cell carcinoma with
necrosis (sparse) + + 38 Leiomyoma - - 39 Squamous cell carcinoma +
+ 40 Cervix Endocervical type adenocarcinoma + +
TABLE-US-00018 TABLE 18 FDA800a orb 253W 41 Ovary Mucinous
adenocarcinoma - + 42 Serous adenocarcinoma + + 43 Mediastinum
Thymoma (type B3) + + 44 Thyroid Medullary carcinoma - - 45 Papilly
carcinoma + + 46 Brain Atypical meningioma - + 47 Malignant
ependymoma of - - right occipital lobe 48 Anaplastic
oligodendoroglioma - - with calcification (sparse) 49 Pantmorphic
glioblastoma of + - right occipital lobe 50 Medulloblastoma of
cerebullum + - 51 Bladder Transitional cell carcinoma - - 52 Low
garde malignant + - leiomysarcoma 53 Kidney Transitional cell
carcinoma - + 54 Papillary renal cell carcinoma - - 55 Clear cell
carcinoma + + 56 Lymph node Diffuse B-cell lymphoma + - 57 Diffuse
B-cell lymphoma of - - right thigh 58 Diffuse B-cell lymphoma - -
of mandible 59 Diffuse B-cell lymphoma - - of spleen 60 Hodgkin's
lymphoma of - - left clavicle 61 Soft tissue Leiomyosarcoma of
chest wall - - 62 Embryonal rhabdomyosarcoma - - of left leg 63
Retroperitoneum Rhabdomyosarcoma - - 64 Primitive neuroectodermal
tumor - - 65 Abdominal Clear cell sarcoma + + wall 66
Retroperitoneum Neuroblastoma - - 67 Peritoneal Mucinous
liposarcoma - - cavity 68 Pelvic cavity Chordoma + + 69 Soft tissue
Neurofibroma of right low back N.T. N.T. 70 Pelvic cavity Synovial
sarcoma of right - - chest wall 71 Bone Osteosarcoma of right femur
- - 72 Peritoneum Malignant mesothelioma - -
TABLE-US-00019 TABLE 19 MC964a orb 253W 1 Cerebrum Astrocytoma + -
2 Anaplastic oligogendroglioma - - 3 Malignant ependymoma + - 4
Astrocytoma + - 5 Astrocytoma + - 6 Glioblastoma + + 7 Esophagus
Squamous cell carcinoma (sparse) + + 8 Squamous cell carcinoma
(sparse) + + 9 Squamous cell carcinoma + + 10 Stomach
Adenocarcinoma - - 11 Adenocarcinoma + + 12 Interstitialoma + + 13
Liver Hepatocellular carcinoma + + 14 Cholangioma + + 15
Hepatoblastoma + + 16 Small Adenocarcinoma + + 17 intestine Diffuse
B-cell lymphoma - - 18 Interstitialoma + + 19 Colon Adenocarcinoma
+ + 20 Interstitialoma - + 21 Diffuse B-cell lymphoma - - 22 Rectum
Adenocarcinoma + + 23 Interstitialoma + + 24 Interstitialoma + - 25
Pancreas Islet cell carcinoma + + 26 Adenocarcinoma + + 27
Adenocarcinoma + + 28 Omentum Metastatic adenocarcinoma + + 29
majus Metastatic dysgerminoma - + 30 Interstitialoma - - 31 Lung
Small cell undifferentiated carcinoma - - 32 Small cell
undifferentiated carcinoma - - 33 Atypical carcinoid + - 34
Squamous cell carcinoma + + 35 Adenocarcinoma + + 36 Squamous cell
carcinoma + +
TABLE-US-00020 TABLE 20 MC964a orb 253W 37 Uterine Squamous cell
carcinomaa + + 38 cervix Squamous cell carcinoma + + 39 Squamous
cell carcinoma + + 40 Uterus Endometrioid adenocarcinoma + + 41
Endometrioid adenocarcinoma + + 42 Clear cell carcinoma - + 43
Breast Invasive lobular carcinoma + - 44 Invasive ductal carcinoma
- + 45 Cystosarcoma phyllodes - - 46 Ovary Serous adenocarcinoma -
- 47 Serous adenocarcinoma - + 48 Serous adenocarcinoma + + 49
Spleen Diffuse B-cell lymphoma + - 50 Diffuse non-Hodgkin's
lymphoma - - 51 Diffuse B-cell lymphoma + + 52 Prostate
Adenocarcinoma (Gleason grade: 3; + + Gleason score: 3 + 4) 53
Adenocarcinoma (Gleason grade: 3; + + Gleason score: 3 + 3) 54
Adenocarcinoma (Gleason grade: 3; - + Gleason score: 2 + 3) 55
Testis Seminoma with necrosis - + 56 Diffuse B-cell lymphoma - - 57
Embryonal carcinoma + + 58 Kidney Clear cell carcinoma - + 59 Clear
cell carcinoma - + 60 Sarcomatoid carcinoma - - 61 Bladder
Transitional cell carcinoma + + 62 Mucinous adenocarcinoma + + 63
Leiomyosarcoma + +
TABLE-US-00021 TABLE 21 MC964a orb 253W 64 Lymph Hodgkin's lymphoma
of neck + + 65 node Hodgkin's lymphoma of neck - + 66 Diffuse
B-cell lymphoma of left groin - - 67 Diffuse B-cell lymphoma of
light elbow joint - - 68 Mediastinum Diffuse T-cell lymphoma of
mediastinum - - 69 Mesentery Diffuse B-cell lymphoma of mesentery +
- 70 Bone Osteosarcoma of left leg + + 71 Osteosarcoma of right
femur - + 72 Osteosarcoma of right femur - + 73 Dedifferentiation
chondrosarcoma - - of pars sacralis 74 Well-differentiated
chondrosarcoma + + of right femur 75 Mesenchymal chondrosarcoma + -
of pars sacralis 76 Skin Squamous cell carcinoma of cheek + + 77
Tongue Squamous cell carcinoma of lip + + 78 Nose Squamous cell
carcinoma of nose + + 79 Tongue Squamous cell carcinoma of pharynx
+ + 80 Larynx Squamous cell carcinoma of larynx + + 81 Squamous
cell carcinoma of larynx + + 82 Pharynx Diffuse B-cell lymphoma of
pharynx - - 83 Pharynx Squamous cell carcinoma of larynx + + 84
Nose Squamous cell carcinoma of nasopharynx - - 85 Tongue Squamous
cell carcinoma + + 86 Embryonal rhabdomyosarcoma - + 87
Mucoepidermoid carcinoma + + 88 Fatty tissue Mucoid liposarcoma of
left leg + + 89 Fibrous Isolated fibroma of chest wall - - 90
tissue Fibrosarcoma of left forearm + + 91 Thyroid Medullary
carcinoma + + 92 Papillary carcinoma + + 93 Diffuse B-cell lymphoma
- - 94 Skin Malignant melanoma of anus margin + + 95 Basal cell
carcinoma of scalp - - 96 Squamous cell carcinoma of left chest
wall + +
Example 9
[0165] The amount of CNGB3 in exosomes isolated from blood was
measured to examine whether it is possible to distinguish a patient
with a CNGB3 high expression disease from a person without a CNGB3
high expression disease. The following tests were performed with an
approval of the Hamamatsu Medical University Institutional Review
Board.
[0166] <Serum Sample>
[0167] Blood collected from two healthy persons (persons with no
tumor, who have not been confirmed to be affected by any disease
and are expected to be healthy) and patients from Hamamatsu Medical
University School of Medicine (three patients with ovarian cancer,
three patients with uterine cancer, three patients with breast
cancer, one patient with cervical cancer, two patients with colon
cancer, three patients with endometriosis, and one patient with
uterine adenomyosis patient) was centrifuged and the obtained serum
aliquots were stored at -80.degree. C. The patients with uterine
adenomyosis are patients who have developed both of endometriosis
and fibroids.
[0168] <Exosome Separation>
[0169] The exosome was isolated from the serum using Exosome
Isolation Kit (manufactured by Fujifilm Wako Pure Chemical
Industries, Ltd.) according to the manufacturer's instruction.
Specifically, first, the serum was centrifuged at 1,000.times.g at
4.degree. C..degree. for 20 minutes, and the supernatant was
recovered. The collected supernatant was centrifuged at
10,000.times.g at 4.degree. C. for 30 minutes, and the supernatant
was transferred to a new tube. Subsequently, 1.0 mL of the
supernatant was mixed with exosome capture beads with a rotator at
4.degree. C. for 180 minutes. Subsequently, after washing the
capture beads, the exosome was eluted with 100 .mu.L of an elution
buffer and stored at -20.degree. C.
[0170] <Peptide Synthesis>
[0171] As a biotinylated Z13 peptide (peptide consisting of L-amino
acids) in which biotin bound to a Z13 peptide (VRRADNRPG: SEQ ID
NO: 3), one synthesized by GenScript Inc. was used.
[0172] <Avidin Blotting>
[0173] An exosome isolated from 800 .mu.L of serum was mixed with a
concentrated Laemmli buffer to prepare a measurement sample. The
measurement sample was isolated by SDS-PAGE and then transferred to
a PVDF membrane. The PVDF membrane was blocked in 5% BSA-containing
TBST (0.1 v/v % Tween-20) at room temperature for 60 minutes. The
PVDF membrane after blotting was washed and incubated overnight at
4.degree. C..degree. in 1.0 .mu.g/mL of biotinylated Z13
peptide-containing TBST. Thereafter, the membrane was washed with
TBST, and subsequently incubated at room temperature for 60 minutes
in 0.1 .mu.g/mL TBST containing HRP (horseradish
peroxidase)-binding avidin (manufactured by Thermo Fisher
Scientific). The CNGB3 on the membrane was detected using a CCD
camera system (manufactured by ATTO) and ECL prime (manufactured by
GE Healthcare). Densitometry analysis was performed using an image
analysis software "CS Analyzer 4" (manufactured by ATTO).
[0174] <Statistical Analysis>
[0175] All analysis was performed using a medial statistics
software "GraphPad Prism 6" (manufactured by GraphPad Software
Corporation).
[0176] The signal intensity of the band of CNGB 3 and the relative
value thereof (relative value with the signal intensity of healthy
person 1 being 1) obtained in the result of avidin blotting of each
measurement sample are shown in Table 22. However, since breast
cancer patients 1 and 2 could secure only a small amount of serum,
exosomes isolated from 100 .mu.L of serum were used, and a breast
cancer patient 3 used exosomes isolated from 37.5 .mu.L of serum.
For this reason, the signal intensity of breast cancer patient
samples was corrected by multiplying by the reciprocal of a serum
use amount.
TABLE-US-00022 TABLE 22 Subject sample Signal intensity Relative
signal intensity Healthy person 1 1082305 1.00 Healthy person 2
1188517 1.10 Ovarian cancer patient 1 4377691 4.04 Ovarian cancer
patient 2 3907260 3.61 Ovarian cancer patient 3 3620987 3.35
Uterine body cancer patient 1 2681313 2.48 Uterine body cancer
patient 2 2939504 2.72 Uterine body cancer patient 3 14049406 12.98
Breast cancer patient 1 18864576 17.43 Breast cancer patient 2
41851784 38.67 Breast cancer patient 3 20114133 18.58 Uterine
cervix cancer patient 1 3480924 3.22 Large intestine cancer patient
1 6057912 5.60 Large intestine cancer patient 2 2285673 2.11
Endometriosis patient 1 13117100 12.75 Endometriosis patient 2
4358049 4.24 Endometriosis patient 3 3454806 3.36 Uterine
adenomyosis patient 1 5835780 5.67
[0177] As a result, in all the samples, a band of CNGB3 was
detected, and it was confirmed that CNGB3 was contained in the
exosomes. The CNGB3 amount of exosomes isolated from cancer
patients, endometriosis patients and uterine adenomyosis patients
was more than two times the exosomes isolated from healthy persons.
From the result, it was confirmed that in exosomes isolated from
the serum of patients with high expression of CNGB3 in
disease-causing cells of the patients, such as tumor patients,
endometriosis patients, and uterine adenomyosis patients, a
significantly larger amount of CNGB3 is contained than in the
exosomes isolated from serum, and therefore, by setting an
appropriate reference value (cutoff value), it is possible to
evaluate a possibility of onset of a CNGB3 high expression disease
based on the amount of CNGB3 in the exosomes.
[0178] In addition, it was confirmed that CNGB3 was even present in
the exosomes of the healthy persons, although in a very small
amount. This is presumably because a molecule having a high binding
property to CNGB3 such as CNGB3-binding peptide was used to detect
CNGB3 of exosomes in serum with a very high sensitivity. Since it
was possible to detect exosomes including CNGB3 released from an
extremely small amount of cancer cells in the bodies of the healthy
persons, it could be expected that it is also possible to detect
not only an advanced cancer but also an early cancer even in a case
where the exosomes in serum are set as specimens by using a
CNGB3-binding peptide.
Sequence CWU 1
1
42119PRTArtificial SequenceEffector peptide 1Lys Leu Ala Lys Leu
Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala His
Leu29PRTArtificial SequenceCNGB3 binding
peptidemisc_feature(5)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino
acid 2Val Arg Arg Ala Xaa Asn Xaa Pro Gly1 539PRTArtificial
SequenceZ13 peptide 3Val Arg Arg Ala Asp Asn Arg Pro Gly1
549PRTArtificial SequenceCNGB3 binding peptide 4Val Arg Arg Ala Glu
Asn Arg Pro Gly1 559PRTArtificial SequenceCNGB3 binding peptide
5Val Arg Arg Ala Asn Asn Leu Pro Gly1 569PRTArtificial
SequenceCNGB3 binding peptide 6Val Arg Arg Ala Asn Asn Arg Pro Gly1
577PRTArtificial SequenceIF7 peptide 7Ile Phe Leu Leu Trp Gln Arg1
5837PRTArtificial SequenceKLAK(14)-HLAH(14)-Z13 peptide 8Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala
His Leu Ala His His Leu Ala His Leu Ala His Val Arg Arg Ala 20 25
30Asp Asn Arg Pro Gly 35937PRTArtificial
SequenceKLAK(14)-HLAH(14)-D5E peptide 9Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala His Leu Ala His His
Leu Ala His Leu Ala His Val Arg Arg Ala 20 25 30Glu Asn Arg Pro Gly
351036PRTArtificial SequenceKLAK(14)-HLAH(13)-Z13 peptide 10Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala
His Leu Ala His His Leu Ala His Leu Ala Val Arg Arg Ala Asp 20 25
30Asn Arg Pro Gly 351135PRTArtificial SequenceKLAK(14)-HLAH(12)-Z13
peptide 11Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys
His Leu1 5 10 15Ala His Leu Ala His His Leu Ala His Leu Val Arg Arg
Ala Asp Asn 20 25 30Arg Pro Gly 351234PRTArtificial
SequenceKLAK(14)-HLAH(11)-Z13 peptide 12Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala His Leu Ala His His
Leu Ala His Val Arg Arg Ala Asp Asn Arg 20 25 30Pro
Gly1333PRTArtificial SequenceKLAK(14)-HLAH(10)-Z13 peptide 13Lys
Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10
15Ala His Leu Ala His His Leu Ala Val Arg Arg Ala Asp Asn Arg Pro
20 25 30Gly1432PRTArtificial SequenceKLAK(14)-HLAH(9)-Z13 peptide
14Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1
5 10 15Ala His Leu Ala His His Leu Val Arg Arg Ala Asp Asn Arg Pro
Gly 20 25 301531PRTArtificial SequenceKLAK(14)-HLAH(8)-Z13 peptide
15Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1
5 10 15Ala His Leu Ala His His Val Arg Arg Ala Asp Asn Arg Pro Gly
20 25 301630PRTArtificial SequenceKLAK(14)-HLAH(7)-Z13 peptide
16Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1
5 10 15Ala His Leu Ala His Val Arg Arg Ala Asp Asn Arg Pro Gly 20
25 301737PRTArtificial SequenceHLAH(14)-KLAK(14)-Z13 peptide 17His
Leu Ala His Leu Ala His His Leu Ala His Leu Ala His Lys Leu1 5 10
15Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Val Arg Arg Ala
20 25 30Asp Asn Arg Pro Gly 351836PRTArtificial
SequenceHLAH(14)-KLAK(13)-Z13 peptide 18His Leu Ala His Leu Ala His
His Leu Ala His Leu Ala His Lys Leu1 5 10 15Ala Lys Leu Ala Lys Lys
Leu Ala Lys Leu Ala Val Arg Arg Ala Asp 20 25 30Asn Arg Pro Gly
351935PRTArtificial SequenceHLAH(14)-KLAK(13)-Z13 peptide 19His Leu
Ala His Leu Ala His His Leu Ala His Leu Ala His Lys Leu1 5 10 15Ala
Lys Leu Ala Lys Lys Leu Ala Lys Leu Val Arg Arg Ala Asp Asn 20 25
30Arg Pro Gly 352034PRTArtificial SequenceHLAH(14)-KLAK(13)-Z13
peptide 20His Leu Ala His Leu Ala His His Leu Ala His Leu Ala His
Lys Leu1 5 10 15Ala Lys Leu Ala Lys Lys Leu Ala Lys Val Arg Arg Ala
Asp Asn Arg 20 25 30Pro Gly2133PRTArtificial
SequenceHLAH(14)-KLAK(13)-Z13 peptide 21His Leu Ala His Leu Ala His
His Leu Ala His Leu Ala His Lys Leu1 5 10 15Ala Lys Leu Ala Lys Lys
Leu Ala Val Arg Arg Ala Asp Asn Arg Pro 20 25
30Gly2232PRTArtificial SequenceHLAH(14)-KLAK(13)-Z13 peptide 22His
Leu Ala His Leu Ala His His Leu Ala His Leu Ala His Lys Leu1 5 10
15Ala Lys Leu Ala Lys Lys Leu Val Arg Arg Ala Asp Asn Arg Pro Gly
20 25 302331PRTArtificial SequenceHLAH(14)-KLAK(13)-Z13 peptide
23His Leu Ala His Leu Ala His His Leu Ala His Leu Ala His Lys Leu1
5 10 15Ala Lys Leu Ala Lys Lys Val Arg Arg Ala Asp Asn Arg Pro Gly
20 25 302430PRTArtificial SequenceHLAH(14)-KLAK(13)-Z13 peptide
24His Leu Ala His Leu Ala His His Leu Ala His Leu Ala His Lys Leu1
5 10 15Ala Lys Leu Ala Lys Val Arg Arg Ala Asp Asn Arg Pro Gly 20
25 302529PRTArtificial SequenceKLAK(14)-HLAH(6)-Z13 peptide 25Lys
Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10
15Ala His Leu Ala Val Arg Arg Ala Asp Asn Arg Pro Gly 20
252628PRTArtificial SequenceKLAK(14)-HLAH(5)-Z13 peptide 26Lys Leu
Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala
His Leu Val Arg Arg Ala Asp Asn Arg Pro Gly 20 252727PRTArtificial
SequenceKLAK(14)-HLAH(4)-Z13 peptide 27Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala His Val Arg Arg Ala
Asp Asn Arg Pro Gly 20 252827PRTArtificial
SequenceKLAK(14)-HLAH(3)-Z13 peptide 28Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Ala His Val Arg Arg Ala
Asp Asn Arg Pro Gly 20 252925PRTArtificial
SequenceKLAK(14)-HLAH(2)-Z13 peptide 29Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala Lys His Leu1 5 10 15Val Arg Arg Ala Asp Asn
Arg Pro Gly 20 253024PRTArtificial SequenceKLAK(14)-HLAH(1)-Z13
peptide 30Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys
His Val1 5 10 15Arg Arg Ala Asp Asn Arg Pro Gly 203129PRTArtificial
SequenceKLAK(13)-HLAH(7)-Z13 peptide 31Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu Ala His Leu Ala1 5 10 15His Leu Ala His Val Arg
Arg Ala Asp Asn Arg Pro Gly 20 253228PRTArtificial
SequenceKLAK(12)-HLAH(7)-Z13 peptide 32Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys Leu His Leu Ala His1 5 10 15Leu Ala His Val Arg Arg
Ala Asp Asn Arg Pro Gly 20 253327PRTArtificial
SequenceKLAK(11)-HLAH(7)-Z13 peptide 33Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala Lys His Leu Ala His Leu1 5 10 15Ala His Val Arg Arg Ala
Asp Asn Arg Pro Gly 20 253426PRTArtificial
SequenceKLAK(10)-HLAH(7)-Z13 peptide 34Lys Leu Ala Lys Leu Ala Lys
Lys Leu Ala His Leu Ala His Leu Ala1 5 10 15His Val Arg Arg Ala Asp
Asn Arg Pro Gly 20 253525PRTArtificial SequenceKLAK(9)-HLAH(7)-Z13
peptide 35Lys Leu Ala Lys Leu Ala Lys Lys Leu His Leu Ala His Leu
Ala His1 5 10 15Val Arg Arg Ala Asp Asn Arg Pro Gly 20
253624PRTArtificial SequenceKLAK(8)-HLAH(7)-Z13 peptide 36Lys Leu
Ala Lys Leu Ala Lys Lys His Leu Ala His Leu Ala His Val1 5 10 15Arg
Arg Ala Asp Asn Arg Pro Gly 203723PRTArtificial
SequenceKLAK(7)-HLAH(7)-Z13 peptide 37Lys Leu Ala Lys Leu Ala Lys
His Leu Ala His Leu Ala His Val Arg1 5 10 15Arg Ala Asp Asn Arg Pro
Gly 203823PRTArtificial SequenceKLAK(14D)-Z13 peptide 38Lys Leu Ala
Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys Val Arg1 5 10 15Arg Ala
Asp Asn Arg Pro Gly 203914PRTArtificial
SequenceKLAK(14)-HLAH(5)-Z13 peptide 39His Leu Ala His Leu Val Arg
Arg Ala Asp Asn Arg Pro Gly1 5 104028PRTArtificial
SequenceIF7(RR)-(K+H) peptide 40Ile Phe Leu Leu Trp Gln Arg Arg Arg
Lys Leu Ala Lys Leu Ala Lys1 5 10 15Lys Leu Ala Lys Leu Ala Lys His
Leu Ala His Leu 20 25419PRTArtificial SequenceZ24 peptide 41Met Gln
Arg Thr Arg Ala Thr Pro Gly1 5429PRTArtificial SequenceZ11 peptide
42Val Arg Ser Ser Arg Ser Thr Pro Gln1 5
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