U.S. patent application number 14/354517 was filed with the patent office on 2014-10-09 for cancer stem cell-specific molecule.
This patent application is currently assigned to PHARMALOGICALS RESEARCH PTE. LTD.. The applicant listed for this patent is CHUGAI SEIYAKU KABUSHIKI KAISHA, PHARMALOGICALS RESEARCH PTE. LTD.. Invention is credited to Atsuhiko Kato, Shinta Kobayashi, Koichi Matsubara, Osamu Natori, Hisafumi Okabe, Masami Suzuki, Takeshi Watanabe, Tatsumi Yamazaki.
Application Number | 20140302511 14/354517 |
Document ID | / |
Family ID | 48167907 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302511 |
Kind Code |
A1 |
Yamazaki; Tatsumi ; et
al. |
October 9, 2014 |
CANCER STEM CELL-SPECIFIC MOLECULE
Abstract
An objective of the present invention is to obtain two types of
substantively homogeneous cancer stem cell populations which can be
characterized using the cell surface marker Lgr5, and to provide
cancer therapeutics using an antibody against a cell membrane
molecule specifically expressed in these cancer stem cells by
identifying said cell membrane molecule. A further objective of the
present invention is to provide, using an antibody against a cell
membrane molecule specifically expressed in cancer stem cells, a
reagent for detecting cancer stem cells, and a method for
diagnosing and sorting cancer patients. The present inventors
discovered that highly pure large intestine cancer stem cells (CSC)
can be obtained in a large quantity, and identified the two types
of conditions of large intestine CSCs distinguishable through Lgr5
expression. Moreover, the present inventors discovered that an
antibody against a cell membrane molecule specifically expressed in
said cancer stem cells can damage said cells.
Inventors: |
Yamazaki; Tatsumi; (Tokyo,
JP) ; Okabe; Hisafumi; (Shizuoka, JP) ;
Kobayashi; Shinta; (Helios, SG) ; Watanabe;
Takeshi; (Shizuoka, JP) ; Matsubara; Koichi;
(Helios, SG) ; Natori; Osamu; (Helios, SG)
; Kato; Atsuhiko; (Shizuoka, JP) ; Suzuki;
Masami; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHARMALOGICALS RESEARCH PTE. LTD.
CHUGAI SEIYAKU KABUSHIKI KAISHA |
Helios
Tokyo |
|
SG
JP |
|
|
Assignee: |
PHARMALOGICALS RESEARCH PTE.
LTD.
CHUGAI SEIYAKU KABUSHIKI KAISHA
|
Family ID: |
48167907 |
Appl. No.: |
14/354517 |
Filed: |
October 26, 2012 |
PCT Filed: |
October 26, 2012 |
PCT NO: |
PCT/JP2012/077714 |
371 Date: |
April 25, 2014 |
Current U.S.
Class: |
435/6.12 ;
435/7.23; 436/501; 530/387.3; 530/387.9; 530/391.7 |
Current CPC
Class: |
A61P 1/04 20180101; A61K
39/001102 20180801; A61K 2039/585 20130101; C07K 2317/732 20130101;
G01N 33/574 20130101; A61P 35/04 20180101; C12Q 2600/158 20130101;
C07K 2317/41 20130101; C07K 2317/76 20130101; C07K 14/51 20130101;
A61P 35/00 20180101; G01N 2800/52 20130101; A61K 31/4745 20130101;
C07K 2317/734 20130101; C12Q 1/686 20130101; C12Q 2600/106
20130101; A61K 39/0011 20130101; A61K 39/39558 20130101; G01N
33/57419 20130101; G01N 33/57492 20130101; A61K 2039/55566
20130101; C07K 16/18 20130101; G01N 2333/726 20130101; A61K
2039/505 20130101; C07K 16/28 20130101; C07K 16/3046 20130101; C12Q
1/6886 20130101 |
Class at
Publication: |
435/6.12 ;
435/7.23; 436/501; 530/387.3; 530/387.9; 530/391.7 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/574 20060101 G01N033/574; C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2011 |
JP |
2011-237438 |
Apr 12, 2012 |
JP |
2012-091142 |
Claims
1. A pharmaceutical composition comprising as an active ingredient
at least one antibody that binds to a protein of SEQ ID NOs: 1 to
8.
2. The pharmaceutical composition of claim 1, which is an
anti-cancer agent.
3. The pharmaceutical composition of claim 2, which is an agent for
inhibiting cancer recurrence.
4. The pharmaceutical composition of claim 2, which is an agent for
inhibiting cancer metastasis or an agent for postoperative adjuvant
therapy.
5. The pharmaceutical composition of claim 4, which is an agent for
inhibiting cancer metastasis or an agent for postoperative adjuvant
therapy against Lgr5-positive cancer, which comprises as an active
ingredient at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6.
6. The pharmaceutical composition of claim 2, which is a
therapeutic agent against drug-resistant cancer.
7. The pharmaceutical composition of claim 6, which is a
therapeutic agent against Lgr5-negative cancer, and which comprises
as an active ingredient at least one antibody that binds to a
protein of SEQ ID NOs: 1 to 8.
8. The pharmaceutical composition of claim 7 wherein the
Lgr5-negative cancer is a drug-resistant cancer.
9. The pharmaceutical composition of any one of claims 2 to 8,
which is an agent for inhibiting cancer stem cell proliferation or
an agent for disrupting cancer stem cells.
10. The pharmaceutical composition of any one of claims 2 to 9,
wherein the cancer is a solid cancer.
11. The pharmaceutical composition of any one of claims 2 to 10,
wherein the cancer is a digestive system cancer.
12. The pharmaceutical composition of any one of claims 2 to 11,
wherein the cancer is a colorectal cancer.
13. The pharmaceutical composition of any one of claims 1 to 12,
wherein the antibody is a monoclonal antibody.
14. The pharmaceutical composition of any one of claims 1 to 13,
wherein the antibody is a chimeric antibody, a humanized antibody,
or a human antibody.
15. The pharmaceutical composition of any one of claims 1 to 14,
wherein the antibody is an antibody fragment.
16. The pharmaceutical composition of claim 15, wherein the
antibody is linked to a cytotoxic substance or a proliferation
inhibitor.
17. The pharmaceutical composition of any one of claims 1 to 14,
wherein the antibody has a cytotoxic activity.
18. The pharmaceutical composition of claim 17, wherein the
cytotoxic activity is ADCC.
19. The pharmaceutical composition of claim 17 or 18, which
comprises an antibody with modified sugar chains whose sugar chain
composition has been altered to increase the ratio of defucosylated
antibody or to increase the ratio of antibody attached with
bisecting N-acetylglucosamine.
20. The pharmaceutical composition of claim 17, wherein the
cytotoxic activity is CDC.
21. The pharmaceutical composition of any one of claims 1 to 20,
wherein the antibody has a neutralizing activity.
22. The pharmaceutical composition of any one of claims 2 to 21, or
a pharmaceutical composition comprising the polypeptide of SEQ ID
NO: 9 or a polypeptide resulting from addition, deletion, and/or
substitution of one or several amino acids in the polypeptide of
SEQ ID NO: 9, which is to be used in combination with a
chemotherapeutic agent simultaneously or after chemotherapeutic
treatment.
23. A reagent for detecting a cancer stem cell, which comprises as
an active ingredient at least one antibody that binds to a protein
of SEQ ID NOs: 1 to 8.
24. The reagent of claim 23, wherein it is used in detecting an
Lgr5-positive cancer stem cell, and wherein the antibody is at
least one antibody that binds to a protein of SEQ ID NOs: 1 to
6.
25. The reagent of claim 23, wherein it is used in detecting an
Lgr5-negative cancer stem cell, and wherein the antibody is at
least one antibody that binds to a protein of SEQ ID NOs: 1 to
8.
26. A method for diagnosing cancer or selecting a cancer patient,
wherein the method comprises, by using at least one antibody that
binds to a protein of SEQ ID NOs: 1 to 8, detecting the presence of
at least one of the proteins in a sample isolated from a cancer
patient.
27. The method of claim 26, wherein it is used in diagnosing
Lgr5-positive cancer or selecting a cancer patient, and wherein the
antibody is at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6.
28. The method of claim 26, wherein it is used in diagnosing
Lgr5-negative cancer or selecting a cancer patient, and wherein the
antibody is at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8.
29. A method for assessing the effectiveness of the pharmaceutical
composition of any one of claims 1 to 22, wherein the method
comprises detecting the presence of one or more of the proteins of
SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins in
a sample isolated from a subject administered with the
pharmaceutical composition.
30. The method of claim 29, which uses at least one antibody that
binds to a protein of SEQ ID NOs: 1 to 8.
31. The method of claim 29, which uses polynucleotides encoding the
proteins of SEQ ID NOs: 1 to 8 and/or complementary strands
thereof.
Description
TECHNICAL FIELD
[0001] The present invention relates to cell surface molecules
specific to Lgr5-positive cancer stem cells with a high
proliferative potential or Lgr5-negative cancer stem cells with a
low proliferative potential; and pharmaceutical compositions that
comprise as an active ingredient an antibody against such a cell
surface molecule. The present invention also relates to reagents
for detecting cancer stem cells and methods for selecting cancer
patients, which use an antibody described above.
BACKGROUND ART
[0002] Cancer stem cells (CSCs) are considered to be the origins of
cancer. The reason is that these cells have the ability to
self-renew and differentiate to form a tumor hierarchy (Non-patent
Document 1). Furthermore, CSCs can migrate and be tolerant to
anti-cancer drug therapy (Non-patent Document 1). Since CSCs are
believed to be a rare subset in tumors, there have been numerous
efforts to characterize them based on cell surface markers and
tumor-initiating activity in xenograft transplantations. CSCs have
been reported in several types of cancer, including acute
myelocytic leukemia (AML) (Non-patent Documents 2 and 3), breast
cancer (Non-patent Document 4), glioma (Non-patent Document 5),
head and neck cancer (Non-patent Document 6), pancreatic cancer
(Non-patent Documents 7 and 8), lung cancer (Non-patent Document
9), prostatic cancer (Non-patent Documents 10 and 11), mesenchymal
neoplasm (Non-patent Document 12), and melanoma (Non-patent
Documents 13 and 14). Earlier studies of O'Brien et al. (Non-patent
Document 15) and Ricci-Vitiani et al. (Non-patent Document 16)
reported that CD133 served as a CSC marker for colorectal cancer.
Thereafter, different research groups have reported other markers:
CD44, EpCAM, CD166 (Non-patent Document 17), and ALDH (Non-patent
Documents 18 and 19). Recently, Pang et al. demonstrated that CD26
serves as a marker for a CSC subpopulation with metastatic capacity
(Non-patent Document 20).
[0003] To isolate CSCs, most studies have employed a cell selection
approach using in combination CSC markers such as EpCAM
.sup.high/CD44.sup.+/CD166.sup.+ (Non-patent Document 17),
CD133.sup.+/CD44.sup.+ (Non-patent Document 21),
CD44.sup.high/ALDH.sup.+ (Non-patent Document 18), and
ALDH1.sup.+/CD133.sup.+ (Non-patent Document 19). In vitro spheroid
(cell mass) cultures and direct cancer cell xenograft
transplantation to immunodeficient mice have also been used to
enrich CSCs (Non-patent Document 22). However, there was a
necessity to prepare a large number of cancer stem cells with a
high purity for further understanding the properties of CSCs.
[0004] One challenge in isolating CSCs arises from the phenotypic
heterogeneity and/or instability of these cells (Non-patent
Document 29). Three-dimensional spheroid cultures are often used as
a CSC source. Spheroid cultures are applicable directly to tumor
cells of clinically resected specimens and enable maintenance of
heterogeneous CSC populations, and can have certain potential
advantages compared to xenograft transplantations. Due to the
heterogeneity, however, results of biochemical analyses often show
complicated CSC characteristics. CSC selection using antibodies
against cell surface marker proteins is commonly used to isolate
CSCs, but the number and purity of cells obtained by this method is
limited. On the other hand, phenotypes from xenografts remain
stable even after frequent passages, and using xenografts as a
source of CSCs is also a common approach. However, there is an
argument that xenograft passages in mice only select cells viable
in mice and result in elimination of cells that are hardly affected
by such an environment. It goes without saying that CSCs in
xenograft tumors reflect the characteristics of original CSCs, as
long as they maintain the self-renewability and lineage
differentiation capacity of the original tumor.
[0005] Leucine-rich repeat-containing G-protein-coupled receptor 5
(Lgr5) was originally identified as an orphan G-protein-coupled
receptor of the glycoprotein hormone receptor family (Non-patent
Documents 23 and 24) and was demonstrated to be a Wnt target gene
whose expression is restricted to the crypt (Non-patent Document
25). The discovery that Lgr5-positive columnar cells can regenerate
all epithelial lineages (Non-patent Document 25) and that a single
Lgr5-positive cell can form crypt-villus organoids in vitro without
a mesenchymal niche (Non-patent Document 26), conclusively proves
that Lgr5-positive cells are stem cells in the normal large
intestine. It has also been reported that Lgr5-positive cells form
adenomas in the absence of Apc (Non-patent Document 27) and Lgr5 is
expressed in colorectal cancer cell lines (Non-patent Document 25).
When considered together, the findings described above suggest that
Lgr5-positive cells are an origin of colorectal cancer (Non-patent
Document 25). It has been proven that, as in stem cells of the
normal large intestine, Wnt activity is essential for in vitro and
in vivo proliferation of CSCs and that exogenous HGF enhances Wnt
activity (Non-patent Document 28).
[0006] Lgr5 was identified as a marker for normal large intestine
stem cells, and has been demonstrated to serve as a marker for
origins of colorectal cancer (Patent Document 1 and Non-patent
Document 30). Furthermore, Lgr5 was reported to be a protein that
is over-expressed in colorectal cancer stem cells (Patent Document
2). The biological role of Lgr5 in the development of large
intestine cancer remains poorly understood.
[0007] To date, various anti-cancer drugs and cancer therapeutic
methods have been developed, but there are still issues to be
solved, such as poor effectiveness, adverse effects, or being
effective in only a limited number of patients. In recent years,
therapeutic methods for targeting cancer stem cells have drawn
attention, but their effectiveness and adverse effects remain
poorly understood (Non-patent Document 31).
PRIOR ART DOCUMENTS
Patent Documents
[0008] Patent Document 1: US20100275280 [0009] Patent Document 2:
WO09/005,809
Non-patent Documents
[0009] [0010] Non-patent Document 1: Reya T, Morrison S J, Clarke M
F, Weissman I L (2001) Stem cells, cancer, and cancer stem cells.
Nature 414:105-111. [0011] Non-patent Document 2: Bonnet D, Dick J
E (1997) Human acute myeloid leukemia is organized as a hierarchy
that originates from a primitive hematopoietic cell. Nat Med
3:730-737. [0012] Non-patent Document 3: Lapidot T, et al. (1994) A
cell initiating human acute myeloid leukaemia after transplantation
into SCID mice. Nature 367:645-648. [0013] Non-patent Document 4:
Al-Hajj M, Wicha M S, Benito-Hernandez A, Morrison S J, Clarke M F
(2003) Prospective identification of tumorigenic breast cancer
cells. Proc Natl Acad Sci USA 100:3983-3988. [0014] Non-patent
Document 5: Singh S K, et al. (2004) Identification of human brain
tumour initiating cells. Nature 432:396-401. [0015] Non-patent
Document 6: Prince M E, et al. (2007) Identification of a
subpopulation of cells with cancer stem cell properties in head and
neck squamous cell carcinoma. Proc Natl Acad Sci USA 104:973-978.
[0016] Non-patent Document 7: Hermann P C, et al. (2007) Distinct
populations of cancer stem cells determine tumor growth and
metastatic activity in human pancreatic cancer. Cell Stem Cell 1:
313-323. [0017] Non-patent Document 8: Li C, et al. (2007)
Identification of pancreatic cancer stem cells. Cancer Res
67:1030-1037. [0018] Non-patent Document 9: Eramo A, et al. (2008)
Identification and expansion of the tumorigenic lung cancer stem
cell population. Cell Death Differ 15:504-514. [0019] Non-patent
Document 10: Collins A T, Berry P A, Hyde C, Stower M J, Maitland N
J (2005) Prospective identification of tumorigenic prostate cancer
stem cells. Cancer Res 65:10946-10951. [0020] Non-patent Document
11: Patrawala L, et al. (2006) Highly purified CD44+ prostate
cancer cells from xenograft human tumors are enriched in
tumorigenic and metastatic progenitor cells. Oncogene 25:1696-1708.
[0021] Non-patent Document 12: Wu C, et al. (2007) Side population
cells isolated from mesenchymal neoplasms have tumor initiating
potential. Cancer Res 1:8216-8222. [0022] Non-patent Document 13:
Schatton T, et al. (2008) Identification of cells initiating human
melanomas. Nature 451:345-349. [0023] Non-patent Document 14: Boiko
A D, et al. (2010) Human melanoma-initiating cells express neural
crest nerve growth factor receptor CD271. Nature 446:133-137.
[0024] Non-patent Document 15: O'Brien C A, Pollett A, Gallinger S,
Dick J E (2007) A human colon cancer cell capable of initiating
tumour growth in immunodeficient mice. Nature 445:106-110. [0025]
Non-patent Document 16: Ricci-Vitiani L, et al. (2007)
Identification and expansion of human colon-cancer-initiating
cells. Nature 445:111-115 [0026] Non-patent Document 17: Dalerba P,
et al. (2007) Phenotypic characterization of human colorectal
cancer stem cells. Proc Natl Acad Sci USA 104:10158-10163. [0027]
Non-patent Document 18: Chu P, et al. (2009) Characterization of a
subpopulation of colon cancer cells with stem cell-like properties.
Int J Cancer 124:1312-1321. [0028] Non-patent Document 19: Huang E
H, et al. (2009) Aldehyde dehydrogenase 1 is a marker for normal
and malignant human colonic stem cells (SC) and tracks SC
overpopulation during colon tumorigenesis. Cancer Res 69:3382-3389.
[0029] Non-patent Document 20: Pang R, et al. (2010) A
subpopulation of CD26+ cancer stem cells with metastatic capacity
in human colorectal cancer. Cell Stem Cell 6:603-615. [0030]
Non-patent Document 21: Haraguchi N, et al. (2008) CD133+ CD44+
population efficiently enriches colon cancer initiating cells. Ann
Surg Oncol 15:2927-2933. [0031] Non-patent Document 22: Ishizawa K,
et al. (2010) Tumor-initiating cells are rare in many human tumors.
Cell Stem Cell 7:279-282. [0032] Non-patent Document 23: McDonald
T, et al. (1998) Identification and cloning of an orphan G
protein-coupled receptor of the glycoprotein hormone receptor
subfamily. Biochem Biophys Res Commun 247:266-270. [0033]
Non-patent Document 24: Hsu S Y, Liang S G, Hsuch A J (1998)
Characterization of two LGR genes homologous to gonadotropin and
thyrotropin receptors with extracellular leucine-rich repeats and a
G protein-coupled, seven-transmembrane region. Mol Endcrinol
12:1830-1845. [0034] Non-patent Document 25: Barker N, et al.
(2007) Identification of stem cells in small intestine and colon by
marker gene Lgr5. Nature 449:1003-1007. [0035] Non-patent Document
26: Sato T, et al. (2009) Single Lgr5 stem cells build crypt-villus
structures in vitro without a mesenchymal niche. Nature
459:262-265. [0036] Non-patent Document 27: Barker N, et al. (2009)
Crypt stem cells as the cells-of-origin of intestinal cancer.
Nature 457:608-611. [0037] Non-patent Document 28: Vermeulen L, et
al. (2010) Wnt activity defines colon cancer stem cells and is
regulated by the microenvironment. Nat Cell Biol 12:468-476. [0038]
Non-patent Document 29: Clevers H (2011) The cancer stem cell:
premises, promises and challenges. Nat Med 17:313-319. [0039]
Non-patent Document 30: Barker N, et al. (2007) Identification of
stem cells in small intestine and colon by marker gene Lgr5. Nature
449:1003-1007. [0040] Non-patent Document 31: Park C Y, et al.
(2009) Cancer stem cell-directed therapies:recent data from the
laboratory and clinic. Mol Ther 17(2):219-230.
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0041] The present invention was achieved in view of the
circumstances described above. The present invention isolates two
types of cancer stem cell populations, which are substantially
homogeneous and characterized with Lgr5, a cell surface marker, and
identifies cell membrane molecules expressed specifically in the
cancer stem cells. An objective of the present invention is to
provide agents for treating cancer which use antibodies to those
cell membrane molecules. Another objective of the present invention
is to provide reagents for detecting cancer stem cells, methods for
diagnosing and selecting cancer patients, which use antibodies to
cell membrane molecules expressed specifically in cancer stem
cells.
Means for Solving the Problems
[0042] In order to prove the stem cell theory that can explain the
mechanism of oncogenesis, maximum efforts have been made to
identify, isolate, and characterize cancer stem cells (CSCs).
However, obtaining highly pure CSCs in an amount sufficient to
characterize them remained difficult. It was therefore also
difficult to identify cell membrane molecules specifically
expressed on cancer stem cells. The present inventors conducted
dedicated studies to achieve the above-described objectives.
[0043] To isolate large intestine CSCs from human colorectal cancer
xenografts maintained in NOG mice, the present inventors
established, for the first time, a method for preparing a large
quantity of highly pure large intestine CSCs using an in vitro
monolayer culture (also simply referred to as adherent culture)
with serum-free stem cell media. Specifically, the present
inventors demonstrated that a large number of large intestine CSCs
can be obtained with high purity by performing an adherent culture
of isolated cells derived from moderately-differentiated human
colorectal cancer xenografts maintained in NOD/Shi-scid,
IL-2R.gamma. null (NOG) mice. Under these conditions, only large
intestine CSCs can grow, survive, and expand, and thus, the present
inventors were able to obtain substantially homogeneous large
intestine CSCs with high purity. When passaged by adherent culture
using serum-free stem cell media, large intestine CSCs prepared by
the above method were maintained stably without phenotypic
alterations over a month or more. The cells expressed various
previously-reported colorectal cancer stem cell markers (CD133,
CD44, EpCAM, CD166, CD24, CD26, and CD29), and exhibited
tumor-initiating activity at a rate of almost 100%. Furthermore,
the cells formed tumors having the same histopathological features
(hierarchical organization) as the original primary tumor. The
cells are also characterized by being highly proliferative under
adherent culture conditions and positive for Lgr5, a cell surface
marker. Furthermore, the high proliferative Lgr5-positive cancer
stem cells, when administered via the caudal vein to mice, formed
tumor masses in organs such as lung and liver, which indicates that
the cells play an essential role in cancer metastasis.
[0044] On the other hand, low proliferative Lgr5-negative cancer
stem cells were isolated by treating with anti-cancer agents such
as irinotecan and 5-FU cancer stem cells that are positive for cell
surface marker Lgr5 and highly proliferative under adherent culture
conditions. Furthermore, the isolated low proliferative
Lgr5-negative cancer stem cells were revealed to be converted to
high proliferative Lgr5-positive cancer stem cells by re-culturing
under adherent culture conditions. This demonstrates that high
proliferative Lgr5-positive cancer stem cells and low proliferative
Lgr5-negative cancer stem cells are interconvertible to each other
and thus have an intrinsic interchangeability. Due to this ability,
actively proliferating Lgr5-positive large intestine CSCs, when
cultured under an altered culture condition or in the presence of
an anti-cancer drug, were converted into the Lgr5-negative
quiescent state. Alternatively, Lgr5-negative CSCs, when isolated
and then cultured again under an adherent culture condition, were
converted to Lgr5-positive CSCs that proliferate actively. These
cells also exhibited tumor-initiating activity at a rate of almost
100%. Furthermore, the high proliferative Lgr5-positive cancer stem
cells, when inoculated via the caudal vein to mice, formed tumor
masses in organs such as lung and liver, which indicates that the
cells play an essential role in cancer metastasis.
[0045] The interconversion of CSCs between the two types of
conditions due to environmental changes may be helpful in
explaining drug resistance and recurrence of cancer. The
involvement of Lgr5-negative CSCs in oncogenesis can be correlated
with the basic nature of stem cells. A plausible hypothesis is that
CSCs can use intrinsic means to convert into a different subset of
a cell population under environmental changes such as by aggressive
treatment with anti-cancer agents. CSCs, when exposed to stress
such as anti-cancer drugs or changes in the culturing environment,
convert themselves to low proliferative Lgr5-negative CSCs in order
to survive avoiding the stress. Once the stress is removed, the
cells can change again into high proliferative Lgr5-positive CSCs
and start to proliferate. This implies that CSCs have a
self-defense ability based on an intrinsic mechanism to adapt to
new environments (FIG. 36).
[0046] The above-described research findings by the present
inventors demonstrate that high proliferative Lgr5-positive and low
proliferative Lgr5-negative cancer stem cells both play important
roles in cancer development, formation, metastasis, recurrence,
drug resistance, etc., and can be major target cells in the
development of anti-cancer agents. In particular, high
proliferative Lgr5-positive cancer stem cells are considered to be
involved in oncogenesis and metastasis while low proliferative
Lgr5-negative cancer stem cells are thought to be involved in
cancer recurrence. Thus, if cell surface molecules that are
expressed specifically on high proliferative Lgr5-positive cancer
stem cells and low proliferative Lgr5-negative cancer stem cells
can be identified, such molecules would enable therapies using
antibodies and discovery of new anti-cancer agents and reagents for
detecting cancer stem cells.
[0047] Specifically, the present invention provides:
[1] a pharmaceutical composition comprising as an active ingredient
at least one antibody that binds to a protein of SEQ ID NOs: 1 to
8; [2] the pharmaceutical composition of [1], which is an
anti-cancer agent; [3] the pharmaceutical composition of [2], which
is an agent for inhibiting cancer recurrence; [4] the
pharmaceutical composition of [2], which is an agent for inhibiting
cancer metastasis or an agent for postoperative adjuvant therapy;
[5] the pharmaceutical composition of [4], which is an agent for
inhibiting cancer metastasis or an agent for postoperative adjuvant
therapy against Lgr5-positive cancer, which comprises as an active
ingredient at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6; [6] the pharmaceutical composition of [2], which is a
therapeutic agent against drug-resistant cancer; [7] the
pharmaceutical composition of [6], which is a therapeutic agent
against Lgr5-negative cancer, and which comprises as an active
ingredient at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8; [8] the pharmaceutical composition of [7] wherein the
Lgr5-negative cancer is a drug-resistant cancer; [9] the
pharmaceutical composition of any one of [2] to [8], which is an
agent for inhibiting cancer stem cell proliferation or an agent for
disrupting cancer stem cells; [10] the pharmaceutical composition
of any one of [2] to [9], wherein the cancer is a solid cancer;
[11] the pharmaceutical composition of any one of [2] to [10],
wherein the cancer is a digestive system cancer; [12] the
pharmaceutical composition of any one of [2] to [11], wherein the
cancer is a colorectal cancer; [13] the pharmaceutical composition
of any one of [1] to [12], wherein the antibody is a monoclonal
antibody; [14] the pharmaceutical composition of any one of [1] to
[13], wherein the antibody is a chimeric antibody, a humanized
antibody, or a human antibody; [15] the pharmaceutical composition
of any one of [1] to [14], wherein the antibody is an antibody
fragment; [16] the pharmaceutical composition of [15], wherein the
antibody is linked to a cytotoxic substance or a proliferation
inhibitor; [17] the pharmaceutical composition of any one of [1] to
[14], wherein the antibody has a cytotoxic activity; [18] the
pharmaceutical composition of [17], wherein the cytotoxic activity
is ADCC; [19] the pharmaceutical composition of [17] or [18], which
comprises an antibody with modified sugar chains whose sugar chain
composition has been altered to increase the ratio of defucosylated
antibody or to increase the ratio of antibody attached with
bisecting N-acetylglucosamine; [20] the pharmaceutical composition
of [17], wherein the cytotoxic activity is CDC; [21] the
pharmaceutical composition of any one of [1] to [20], wherein the
antibody has a neutralizing activity; [22] the pharmaceutical
composition of any one of [2] to [21], or a pharmaceutical
composition comprising the polypeptide of SEQ ID NO: 9 or a
polypeptide resulting from addition, deletion, and/or substitution
of one or several amino acids in the polypeptide of SEQ ID NO: 9,
which is to be used in combination with a chemotherapeutic agent
simultaneously or after chemotherapeutic treatment; [23] a reagent
for detecting a cancer stem cell, which comprises as an active
ingredient at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8; [24] the reagent of [23], wherein it is used in
detecting an Lgr5-positive cancer stem cell, and wherein the
antibody is at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6; [25] the reagent of [23], wherein it is used in
detecting an Lgr5-negative cancer stem cell, and wherein the
antibody is at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8; [26] a method for diagnosing cancer or selecting a
cancer patient (a method for testing and/or selecting cancer),
wherein the method comprises, by using at least one antibody that
binds to a protein of SEQ ID NOs: 1 to 8, detecting the presence of
at least one of the proteins in a sample isolated from a cancer
patient; [27] the method of [26], wherein it is used in diagnosing
Lgr5-positive cancer or selecting a cancer patient, and wherein the
antibody is at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6; [28] the method of [26], wherein it is used in
diagnosing Lgr5-negative cancer or selecting a cancer patient, and
wherein the antibody is at least one antibody that binds to a
protein of SEQ ID NOs: 1 to 8; [29] a method for assessing the
effectiveness of the pharmaceutical composition of any one of [1]
to [22], wherein the method comprises detecting the presence of one
or more of the proteins of SEQ ID NOs: 1 to 8 and/or
polynucleotides encoding the proteins in a sample isolated from a
subject administered with the pharmaceutical composition; [30] the
method of [29], which uses at least one antibody that binds to a
protein of SEQ ID NOs: 1 to 8; and [31] the method of [29], which
uses polynucleotides encoding the proteins of SEQ ID NOs: 1 to 8
and/or complementary strands thereof.
[0048] The present invention also provides:
[A1] a method for treating cancer, comprising administering to a
subject at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8; [A2] at least one antibody that binds to a protein of
SEQ ID NOs: 1 to 8 for use in the treatment of cancer; [A3] use of
at least one antibody that binds to a protein of SEQ ID NOs: 1 to 8
for producing an anti-cancer agent; and [A4] a process for
manufacturing an anti-cancer agent, which comprises the step of
using at least one antibody that binds to a protein of SEQ ID NOs:
1 to 8.
[0049] In a non-limiting embodiment of the present invention,
treatment of cancer includes inhibition of cancer recurrence,
inhibition of cancer metastasis, postoperative adjuvant therapy,
treatment of drug-resistant cancer, inhibition of cancer stem cell
proliferation, and disruption of cancer stem cells; and anti-cancer
agents include agents for inhibiting cancer recurrence, agents for
inhibiting cancer metastasis, agents for postoperative adjuvant
therapy, agents for treating drug-resistant cancer, agents for
inhibiting cancer stem cell proliferation, and agents for
disrupting cancer stem cells.
[0050] Furthermore, the present invention provides:
[B1] a reagent for detecting the presence of one or more of the
proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the
proteins, preferably, a reagent for detecting a cancer stem cell, a
reagent for cancer diagnosis, a reagent for selecting a cancer
patient, or a reagent for testing the effectiveness of the
pharmaceutical compositions of [1] to [22], which contains at least
one antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a
portion of a polynucleotide encoding the protein of SEQ ID NOs: 1
to 8 and/or the complementary strand thereof; [B2] a method for
detecting a cancer stem cell, diagnosing cancer, selecting a cancer
patient, or testing the effectiveness of the pharmaceutical
compositions of [1] to [22], which comprises detecting the presence
of one or more of the proteins of SEQ ID NOs: 1 to 8 and/or
polynucleotides encoding the proteins in a sample isolated from a
cancer patient using, preferably, at least one antibody that binds
to a protein of SEQ ID NOs: 1 to 8, or a portion of a
polynucleotide encoding the protein of SEQ ID NOs: 1 to 8 and/or
the complementary strand thereof; [B3] a reagent for detecting the
presence of one or more of the proteins of SEQ ID NOs: 1 to 8
and/or polynucleotides encoding the proteins, preferably at least
one antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a
portion of a polynucleotide encoding the protein of SEQ ID NOs: 1
to 8 and/or the complementary strand thereof, which is for use in
detecting a cancer stem cell, diagnosing cancer, selecting a cancer
patient, or testing the effectiveness of pharmaceutical
compositions of [1] to [22]; [B4] use of a reagent for detecting
the presence of one or more of the proteins of SEQ ID NOs: 1 to 8
and/or polynucleotides encoding the proteins, preferably at least
one antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a
portion of a polynucleotide encoding the protein of SEQ ID NOs: 1
to 8 and/or the complementary strand thereof, which is for
producing a reagent for detecting a cancer stem cell, a reagent for
diagnosing cancer, a reagent for selecting a cancer patient, or a
reagent for testing the effectiveness of the pharmaceutical
compositions of [1] to [22]; and [B5] a process for producing a
reagent for detecting a cancer stem cell, a reagent for diagnosing
cancer, a reagent for selecting a cancer patient, or a reagent for
testing the effectiveness of the pharmaceutical compositions of [1]
to [22], which comprises using a reagent for detecting the presence
of one or more of the proteins of SEQ ID NOs: 1 to 8 and/or
polynucleotides encoding the proteins, preferably at least one
antibody that binds to a protein of SEQ ID NOs: 1 to 8, or a
portion of a polynucleotide encoding the protein of SEQ ID NOs: 1
to 8 and/or the complementary strand thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows photographs depicting histological images (HE
stain) of colorectal cancer xenografts PLR59 and PLR123 derived
from moderately-differentiated colorectal cancer. Even after 15
passages, cells derived from xenografts PLR59 and PLR123 formed
tumors with a morphology (hierarchical organization) very similar
to the original tumor, and had budding clusters (arrow) and ductal
structures with goblet cells (inset). "Original" indicates tumors
obtained by surgical resection; "Early passage" indicates
xenografts PLR59 and PLR123 after 4 passages in NOG mice; and "Late
passage" indicates xenograft PLR59 after 15 passages and PLR123
after 19 passages in MOG mice. Scale bar represents 100 .mu.m.
[0052] FIG. 2 is a diagram showing a result of flow cytometry
analysis of cells from xenografts PLR59 and PLR123 passaged in NOG
mice for known CSC markers. The cells were stained with antibodies
against the markers indicated and then analyzed with flow
cytometry. Gray area indicates the ALDH activity or fluorescence
intensity of cells after staining with the indicated antibodies.
White area indicates the ALDH activity in the presence of an ALDH
inhibitor or the fluorescence intensity of cells after staining
with isotype antibodies as a control.
[0053] FIG. 3 shows photographs depicting histological images (HE
stain) of tumors formed by injection of 100 cells each of PLR59 and
PLR123 cells. The morphologies of the tumors derived from 100 cells
each of PLR59 and PLR123 cells were highly similar to the original
tumors. Arrows indicate budding clusters. Scale bar represents 100
.mu.m.
[0054] FIG. 4 shows photographs depicting a result of phase
contrast microscopic observation of non-adherent and adherent cells
(PLR123 cells). The cells were cultured in serum-free media
supplemented with EGF and FGF. The non-adherent cells closely
interacted together to form a spheroid-like structure, whereas the
adherent cells proliferated without forming cell clusters. Scale
bar represents 25 .mu.m.
[0055] FIG. 5 is a diagram showing the proliferation of
non-adherent and adherent CSCs (PLR123 cells). The viable cell
count after three days of culture (black column) is shown in
percentage to the count on day 0 (white column). The results were
averaged from three experiments. The bar at the top of each column
represents standard deviation.
[0056] FIG. 6 is a diagram showing a result of flow cytometry
analysis of non-adherent cells and adherent cells (PLR123 cells)
for known CSC markers. Both types of cells were positive for known
CSC markers such as CD133, CD44, EpCAM, CD166, CD24, CD26, and
CD29, while the adherent cells alone were positive for Lgr5 and
ALDH activity. Gray area indicates the ALDH activity or
fluorescence intensity of cells after staining with the indicated
antibodies. White area indicates the ALDH activity in the presence
of an ALDH inhibitor or the fluorescence intensity of cells after
staining with isotype antibodies as a control.
[0057] FIG. 7 is a photograph showing a result of Western blot
analysis of primary cells of PLR123 cells, non-adherent CSCs, and
adherent CSCs for .beta.-catenin, TCF1, TCF3, TCF4, and
phosphorylated c-JUN protein. Expression of all of the proteins was
up-regulated in Lgr5-positive adherent CSCs as compared to the
primary cells. GAPDH was also visualized as a reference protein for
protein loading.
[0058] FIG. 8 is a diagram showing the inhibition of growth of
Lgr5-positive adherent CSCs (PLR123 cells) by FH535 (50 .mu.M) and
Cardamonin (50 .mu.M). The viable cell count after three days of
culture in the presence of FH535 (gray column) or Cardamonin (black
column) is shown in percentage to the count in the presence of DMSO
alone (white column). The results were averaged from three
experiments. The bar at the top of each column represents standard
deviation.
[0059] FIG. 9 is a diagram showing the proliferation of PLR123
cells in the presence or absence of EGF and FGF. Adherent CSCs were
cultured for three days in the presence or absence of EGF and FGF
(black column). The viable cell count is shown in percentage to the
count on day 0 (white column). The results were averaged from three
experiments. The bar at the top of each column represents standard
deviation.
[0060] FIG. 10 is a diagram showing the effect of chemotherapeutic
agents on the proliferation of Lgr5-positive adherent CSCs and
Lgr5-negative non-adherent CSCs (PLR123 cells). The viable cell
count after treatment with 5-FU (10 .mu.g/ml; gray column) or
irinotecan (10 .mu.g/ml; black column) is shown in percentage to
the viable cell count after culturing without the chemotherapeutic
agents (white column). The results were averaged from three
experiments. The bar at the top of each column represents standard
deviation.
[0061] FIG. 11 is a diagram showing a change in Lgr5 expression
after treatment of adherent CSCs (PLR123 cells) with a
chemotherapeutic agent. This figure shows a result of flow
cytometry. The upper panels show the result in the absence of
chemotherapeutic agent (control); the middle panels show cells
treated with 5-FU; and the bottom panels show cells treated with
irinotecan. Gray area indicates the ALDH activity or fluorescence
intensity of cells after staining with the indicated antibodies.
White area indicates the ALDH activity in the presence of an ALDH
inhibitor or the fluorescence intensity of cells after staining
with isotype antibodies as a control.
[0062] FIG. 12 is a diagram showing Lgr5 mRNA levels in PLR123
cells before and after switching to adherent culture or suspension
culture (normalized to 1). F.fwdarw.A represents the switching from
suspension culture to adherent culture, while A.fwdarw.F represents
the switching from adherent culture to suspension culture. The
results were averaged from three experiments. The bar at the top of
each column represents standard deviation.
[0063] FIG. 13 shows photographs depicting a result of Western blot
analysis of Lgr5-negative non-adherent CSCs and Lgr5-positive
adherent CSCs (PLR123 cells) for E-cadherin and Snail. Non-adherent
CSCs expressed E-cadherin at a high level, while adherent CSCs
expressed Snail at a high level. GADPH was used as a loading
control.
[0064] FIG. 14 shows photographs depicting a result of
immunocytochemistry of Lgr5-negative non-adherent CSCs and
Lgr5-positive adherent CSCs (PLR123 cells) using E-cadherin
antibody, Snail antibody, and .beta.-catenin antibody. Non-adherent
CSCs were epithelium-like cells expressing cell-surface E-cadherin
and .beta.-catenin at high levels, while adherent CSCs were
mesenchyme-like cells with nuclear localization of Snail and
.beta.-catenin. Scale bar represents 25 .mu.m.
[0065] FIG. 15 shows photographs depicting a result of
immunohistochemistry of PLR123-derived xenograft tissues using
anti-Lgr5 antibody and anti-Snail antibody. The concomitant
expression of nuclear Snail and cytoplasmic Lgr5 was detected in
EMT-like cells of budding areas (left panel), while such expression
was not observed in the ducts (right panel). Arrows indicate
Lgr5-positive budding cells. Scale bar represents 10 .mu.m.
[0066] FIG. 16 shows photographs depicting histopathological
features of xenograft tissues. Histopathological PDCC xenografts
derived from a poorly-differentiated colorectal cancer (PDCC)
xenograft reconstructed almost the same histopathological
morphology as the original tumor. The PDCC xenografts did not have
apparent epithelial duct structures (4 and 13 passages). Scale bar
represents 100 .mu.m.
[0067] FIG. 17 shows photographs depicting a histopathological
result on xenograft tumors that originated from a single or ten
Lgr5-positive cells derived from PLR123, or ten Lgr5-negative cells
derived from PLR123. The hierarchical organization was observed in
all tumors, and their histopathological features were highly
similar to the original tumor. Scale bar represents 100 .mu.m.
[0068] FIG. 18 shows photographs depicting a result of phase
contrast microscopic observation of non-adherent and adherent large
intestine CSCs (PLR59 cells). The cells were cultured in serum-free
media supplemented with EGF and FGF. The non-adherent cells closely
interacted together to form a spheroid-like structure, whereas the
adherent cells proliferated without forming cell clusters. Scale
bar represents 25 .mu.m.
[0069] FIG. 19 is a diagram showing the proliferation of
non-adherent and adherent CSCs (PLR59 cells). The viable cell count
after three days of culture (black column) is shown in percentage
to the count on day 0 (white column). The results were averaged
from three experiments. The bar at the top of each column
represents standard deviation.
[0070] FIG. 20 is a diagram showing a result of flow cytometry
analysis of non-adherent and adherent cells (PLR59 cells) for known
CSC markers. Adherent cells were positive for all markers reported,
whereas non-adherent cells were negative for Lgr5 and ALDH. Gray
area indicates the ALDH activity or fluorescence intensity of cells
after staining with the indicated antibodies. White area indicates
the ALDH activity in the presence of an ALDH inhibitor or the
fluorescence intensity of cells after staining with isotype
antibodies as a control.
[0071] FIG. 21 shows photographs depicting a result of Western blot
analysis of primary cells of PLR59 cells, non-adherent CSCs, and
adherent CSCs for .beta.-catenin, TCF1, TCF3, TCF4, and
phosphorylated c-JUN protein. Expression of all of the proteins was
up-regulated in Lgr5-positive adherent CSCs as compared to the
primary cells. GAPDH was also visualized as a reference for protein
loading.
[0072] FIG. 22 is a diagram showing growth inhibition of
Lgr5-positive adherent CSCs (PLR59 cells) by FH535 (50 .mu.m) and
Cardamonin (50 .mu.m). The viable cell count after three days of
culture in the presence of FH535 (gray column) or Cardamonin (black
column) is shown in percentage to the count on day 0 (white
column).
[0073] FIG. 23 is a diagram showing the proliferation of PLR59
cells in the presence or absence of EGF and FGF. Adherent CSCs were
cultured for three days in the presence or absence of EGF and FGF
(black column). The viable cell count is shown in percentage to the
count on day 0 (white column).
[0074] FIG. 24 is a diagram showing the effect of 5-FU (10
.mu.g/ml) and irinotecan (10 .mu.g/ml) on the growth of
Lgr5-positive adherent CSCs and Lgr5-negative non-adherent CSCs
(PLR59 cells). The viable cell count after treatment with 5-FU
(gray column) or irinotecan (black column) is shown in percentage
to that after culturing without the agents (white column).
[0075] FIG. 25 is a diagram showing a result of flow cytometry
analysis of adherent CSCs (PLR59 cells) for CSC markers after
treatment with 5-FU or irinotecan. The upper panels show cells
treated with 5-FU, and the bottom panels show cells treated with
irinotecan. Gray area indicates the ALDH activity or fluorescence
intensity of cells after staining with the indicated antibodies.
White area indicates the ALDH activity in the presence of an ALDH
inhibitor or the fluorescence intensity of cells after staining
with isotype antibodies as a control.
[0076] FIG. 26 shows photographs depicting a result of Western blot
analysis of Lgr5-negative non-adherent CSCs and Lgr5-positive
adherent CSCs (PLR59 cells) for E-cadherin and Snail. Non-adherent
CSCs expressed E-cadherin at a high level and adherent CSCs
expressed Snail at a high level. GADPH was used as a loading
control.
[0077] FIG. 27 is a diagram showing the expression levels of Lgr5
mRNA in Lgr5-negative non-adherent CSCs and Lgr5-positive CSCs
(PLR59 cells). Levels of Lgr5 mRNA in adherent and non-adherent
CSCs are shown as a ratio to the levels in primary cells from
xenograft tumors PLR59 and PLR123. The level of Lgr5 mRNA in
adherent CSCs (right, white column) was remarkably increased as
compared to the level in the primary cells from xenografts (left,
black column), while the level was not increased in non-adherent
CSCs (middle, gray column). Lgr5 mRNA levels were determined by
quantitative PCR and normalization with the expression of GAPDH and
ACTB. All experiments were performed in triplicate. Error bars
represent standard deviation.
[0078] FIG. 28 shows photographs depicting a result of specificity
assessment of anti-human Lgr5 monoclonal antibodies (mAbs) 2U2E-2
and 2T15E-2 by immunofluorescence microscopy observation of DG44
cells transfected with Lgr4, Lgr5, or Lgr6 cDNA. The transfectants
and non-transfected parental cells were fixed and treated with 5
.mu.g/ml antibodies. Intense fluorescence (green signals at right)
was observed in cells containing Lgr5 cDNA but not in parental
cells and cells containing Lgr4 or Lgr6 cDNA. Scale bar represents
5 .mu.m.
[0079] FIG. 29 is a diagram showing a result of specificity
assessment of anti-human Lgr5 monoclonal antibody (mAb) 2T15E-2 by
flow cytometry of DG44 cells transfected with Lgr4, Lgr5, or Lgr6
cDNA. The transfectants and non-transfected parental cells were
incubated with monoclonal antibody 2T15E-2 and analyzed by FACS.
Antibody 2T15E-2 reacted with cells containing Lgr5 cDNA but not
with parental cell and cells containing Lgr4 or Lgr6 cDNA. The
expression of Lgr4, Lgr5, and Lgr6 in the transfectants was
assessed by Western blot analysis.
[0080] FIG. 30 is a diagram showing a result of flow cytometry
analysis of adherent CSCs derived from xenografts PLR59 and PLR123.
The cells were cultured for one month and analyzed for known cancer
stem cell markers. Even after one month of in vitro culture,
adherent CSCs derived from PLR59 and PLR123 were positive for all
of known cancer stem cell markers. Gray area indicates the ALDH
activity or fluorescence intensity of cells after staining with the
indicated antibodies. White area indicates the treatment of cells
by the ALDH activity in the presence of an ALDH inhibitor or the
fluorescence intensity of cells after staining with isotype
antibodies as a control.
[0081] FIG. 31 After culturing for one month, adherent CSCs derived
from xenografts PLR59 and PLR123 were analyzed by flow cytometry
(FIG. 30), and injected to NOG mice. The indicated numbers of
adherent CSCs were injected subcutaneously in the lateral abdomen
of NOG mice to assess the tumor-initiating activity in NOG mice.
This figure is a diagram showing the result of assessment of
tumorigenesis 47 days after inoculation. Even subcutaneous
injection of 10 adherent CSCs allowed tumor formation at all of the
injection sites. The tumors were highly similar in
histopathological morphology to the original tumors.
[0082] FIG. 32 shows photographs depicting the phenotypic
interconversion of large intestine CSCs depending on the culture
condition or chemotherapeutic treatment. Lgr5-positive CSCs were
tested for the sensitivity to 5-FU and irinotecan. Both 5-FU and
irinotecan significantly inhibited the proliferation of
Lgr5-positive large intestine CSCs. After three days of exposure to
the 5-FU or irinotecan, cells resistant to the chemotherapeutic
drugs arose. The drug-resistant cells exhibited a dense,
agglutinative morphology. Scale bar represents 25 .mu.m.
[0083] FIG. 33 shows photographs depicting the morphological
interconversion of CSCs. When Lgr5-negative large intestine CSCs
were dispersed and then cultured in a flat-bottomed plate, some of
the cells adhered to the plate bottom, became positive for Lgr5,
and showed a mesenchymal cell-like morphology (at left). On the
other hand, when Lgr5-positive adherent large intestine CSCs were
cultured in an ultra-low adherent plate, some of the cells halted
their growth and formed a spheroid-like structure. Scale bar
represents 10 .mu.m.
[0084] FIG. 34 is a diagram showing the tumor-initiating activity
of adherent CSCs in various organs. 5.times.10.sup.5 adherent CSCs
from PLR123 were injected to the tail vein (n=5). The tumor
formation frequency on day 40 after administration is shown for
various organs.
[0085] FIG. 35 shows photographs depicting a result of
histopathological experiments on tumors in the lungs, liver, lymph
nodes, and subcutaneous tissues. In the lungs, tumor cells formed
undifferentiated tumor foci. Meanwhile, in the liver and other
organs, tumor cells formed a ductal structure involving multiple
differentiation stages. Scale bar represents 100 .mu.m.
[0086] FIG. 36 is a schematic diagram for the proposed CSC model.
CSCs undergo an intrinsic interconversion between two types of
independent states in response to environmental changes such as the
presence of anti-cancer drugs. According to previous findings,
normal stem cells expressing Lgr5 are assumed to transform into
CSCs via mutation in multiple genes. High proliferative CSCs
express Lgr5, and undergo EMT. Under a specific stressful
environment, the cells can change into the Lgr5-negative quiescent
state. Niche environment is involved in stimulating the transition
of CSCs to the differentiation stage.
[0087] FIG. 37 is a diagram showing a result of flow cytometric
analysis of primary cells from PLR59 and PLR123, Lgr5.sup.+ cancer
stem cells, and Lgr5.sup.- cancer stem cells with EREG.
[0088] FIG. 38 shows graphs depicting the ADCC activity of
anti-EREG antibody against Lgr5-positive and Lgr5-negative cells
derived from PLR59 and Lgr5-positive and Lgr5-negative cells
derived from PLR123 cells.
[0089] FIG. 39 shows photographs depicting immunostaining of tumor
(PLR123) obtained by surgical resection, and a PLR123-derived
xenograft model (5 passages (FIG. 39B), 10 passages (FIG. 39C), and
15 passages (FIG. 39D)) in terms of Lgr5. Tissue sections were
stained with anti-Lgr5 antibody. "Original" indicates the tumor
obtained from a patient by surgical resection (FIG. 39A). Scale bar
represents 25 .mu.m.
[0090] FIG. 40 shows photographs depicting a histopathological
result (HE stain) on xenograft tumors derived from Lgr5-positive
cells of PLR59 (FIGS. 40A and 40B) and PLR123 (FIGS. 40C and 40D).
NOG mice were subcutaneously injected with ten (FIGS. 40A and 40C)
or a single (FIGS. 40B and 40D) Lgr5-positive cell(s) obtained from
PLR59 or PLR123 by adherent culture. All tumors showed
histopathological features highly similar to the original tumors.
Scale bar represents 50 .mu.m.
[0091] FIG. 41 shows photographs depicting symmetrical cell
division of Lgr5-positive cells. Lgr5-positive cells stained with
PKH67 dye were cultured for 72 hours, and then observed under a
fluorescent microscope. FIG. 41A, B, and C show stained images of
the cells for 0, 48, and 72 hours, respectively. Scale bar
represents 20 .mu.m.
[0092] FIG. 42 shows photographs depicting symmetrical cell
division of Lgr5-positive cells in the absence of matrigel and
serum (FIGS. 42A and 42B), and asymmetrical cell division of
Lgr5-positive cells in the presence of matrigel and serum (FIGS.
42C and 42D). FIGS. 42A and 42C show images after a single
division, while FIGS. 42B and 42D show images after second or third
division.
[0093] FIG. 43-1 shows photographs depicting immunostained images
of colon CSCs that varied to negative for Lgr5 after three days of
exposure to irinotecan. The cells were stained with antibodies
specific to HLA-DMA (A) and TMEM173 (B).
[0094] FIG. 43-2 shows photographs depicting immunostained images
of colon CSCs that varied to negative for Lgr5 after three days of
exposure to irinotecan. The cells were stained with antibodies
specific to ZMAT3 (C) and GPR110 (D).
[0095] FIG. 44 shows photographs depicting immunostained images of
irinotecan-treated Lgr5-positive CSCs (PLR123). The cells were
immunostained for Lgr5. The immunostained images include those
before irinotecan treatment (FIG. 44A) and after irinotecan
treatment (FIG. 44B). From Lgr5-negative cells inoculated again and
cultured in the absence of irinotecan, Lgr5-positive cells appeared
at the latest by four days after reinoculation (FIG. 44C), and
expanded by eight days after reinoculation (FIG. 44D). Scale bar
represents 50 .mu.m.
[0096] FIG. 45 shows graphs depicting transcript levels for the
Lgr5 gene determined by quantitative real-time PCR. The level of
Lgr5 mRNA was high in Lgr5-positive cells prepared by adherent
culture. The level was decreased under the spheroid culture
condition and was almost undetectable in Lgr-negative cells after
irinotecan treatment. Meanwhile, in Lgr5-positive and -negative
cells prepared by adherent culture, the mRNA level for the CK20
gene was below the detection limit. The level was increased in
Lgr5-positive cells of the spheroid culture condition.
[0097] FIG. 46 shows photographs depicting the expression of Lgr5
and CK20 proteins assessed by immunohistochemical staining.
Spheroid cultures of Lgr5-positive CSCs (PLR59 (FIG. 46A) and
PLR123 (FIG. 46B)) were fixed and sliced into thin sections and
then reacted with Lgr5 antibody (2L36) and CK20 antibody (DAKO).
The spheroids contained a small number of Lgr5-positive cells as
well as a large number of CK20-positive cells that were negative
for Lgr5.
[0098] FIG. 47 shows graphs depicting the percentage of viable
cells, relative to non-treated control cells, of Lgr5-positive
(black line) and -negative (gray line) CSCs (PLR59 and PLR123)
cultured for three days in the absence or presence of irinotecan or
5-FU at each concentration indicated on the horizontal axis.
Lgr5-negative cells were fully resistant to both growth
inhibitors.
[0099] FIG. 48 is a diagram showing the expression of CSC markers.
"Before treatment" indicates the expression of CSC markers in
Lgr5-positive cells prepared by an adherent culture, which are
derived from a PLR123 xenograft model. "After irinotecan treatment"
indicates the expression of CSC markers in Lgr5-negative cells
prepared via irinotecan treatment. "Re-inoculation after irinotecan
removal" indicates the expression of CSC markers in Lgr5-negative
cells re-inoculated to an irinotecan-free medium. Gray area
indicates the ALDH activity or fluorescence intensity of cells
after staining with the indicated antibodies. White area indicates
the ALDH activity in the presence of an ALDH inhibitor.
[0100] FIG. 49 is a diagram showing interconversion between
Lgr5-positive and -negative cells. Lgr5-positive cells were
collected by FACS. After limiting dilution, the cells were
inoculated and cultured for three days in the presence of
irinotecan under adherent culture conditions. On the other hand,
irinotecan-treated Lgr5-negative cells were diluted by limiting
dilution, and then inoculated and cultured for four days in the
absence of irinotecan under adherent culture conditions. Lgr5
expression was visualized with PE-labeled anti-mouse IgG antibody
(indicated in red) or AlexaFluo 488-labeled anti-mouse IgG antibody
(indicated in green).
[0101] FIG. 50 shows diagrams depicting heat maps for (A) seven
genes the expression of which was significantly up-regulated in
Lgr5-negative cells as compared to Lgr5-positive cells and for (B)
20 genes the expression levels of which were elevated in
Lgr5-positive and Lgr5-negative cells as compared to primary cells
derived from xenograft animals. RNAs were prepared from
Lgr5-positive and -negative CSCs derived from PLR59 and PLR123, and
primary cells isolated from xenograft animals. RNAs were analyzed
using Affymetrix U133.
[0102] FIG. 51 shows photographs depicting the binding of
anti-HLA-DMA antibody and anti-EREG antibody to Lgr5-positive and
Lgr5-negative CSCs with immunohistochemical staining. CSCs (PLR123)
were fixed and treated with anti-HLA-DMA antibody (Dako) and
anti-EREG antibody (EP27). Intense fluorescence signals (red for
both HLA-DMA and EREG) were observed on Lgr5-negative cells treated
with anti-HLA-DMA antibody, whereas weak fluorescence (green) or no
fluorescence was detected on Lgr5-positive cells. Fluorescence
signals were detected on both Lgr5-negative and -positive cells
treated with anti-EREG antibody.
[0103] FIG. 52 shows photographs depicting the transition from
Lgr5-negative CSCs to Lgr5-positive CSCs at an early stage of tumor
formation. In NOG mice injected with PLR123 xenograft
animal-derived Lgr5-negative CSCs, tumors derived from
Lgr5-negative CSCs were stained with antibodies against Lgr5
(green), HLA-DMA (red), and EREG (green). Lgr5-weakly-expressing,
HLA-DMA-positive, EREG-positive cells, and Lgr5-positive,
HLA-DMA-negative, EREG-positive cells were observed to be present
on day 5. Scale bar represents 10 .mu.m.
[0104] FIG. 53 shows photographs depicting the reconstitution of
tumor hierarchy from Lgr5-negative CSCs. The tissue structure (FIG.
53A) is shown along with an image obtained by immunofluorescence
microscopic observation using anti-Lgr5 antibody and
anti-E-cadherin antibody (FIG. 53B). Green and red indicate the
presence of Lgr5 and E-cadherin, respectively. Scale bar represents
50 .mu.m.
[0105] FIG. 54 shows photographs depicting histopathology (HE)
(first row), immunostaining using Lgr5 antibody (green) and
E-cadherin antibody (red) (second row), immunostaining using Lgr5
antibody (green) and HLA-DMA antibody (red) (third row), and
immunostaining using EREG antibody (red) (fourth row), of tumors
after irinotecan treatment. Mice bearing tumors derived from
Lgr5-positive CSCs (PLR123) were treated with irinotecan, and their
tumors were observed. Irinotecan or vehicle was administered to
mice at days 12, 15, and 18 after tumor grafting. Scale bar
represents 25 .mu.m.
[0106] FIG. 55 Irinotecan was administered at a dose of 120
mg/kg/day to NOG mice at days 12, 15, and 18 after grafting tumors
derived from Lgr5-positive CSCs (PLR123). This figure is a graph
showing tumor volumes in control mice administered with vehicle
(closed diamond) and mice administered with irinotecan (closed
square or triangle). Each value represents mean+standard
deviation.
[0107] FIG. 56 is a graph showing the numbers of Lgr5-positive and
HLA-DMA-positive cells in xenograft tumor tissues. Thin sections of
tissues were treated with Lgr5 antibody and HLA-DMA antibody, and
then the numbers of Lgr5-positive and HLA-DMA-positive cells were
measured. Values represent the total numbers of cells counted for
respective groups (n=3).
[0108] FIG. 57 is a graph showing the anti-tumor effect of EREG
antibody after irinotecan treatment. PLR123-derived Lgr5-positive
cells were injected to the peritoneal cavities of SCID mice, and
irinotecan was administered at a dose of 100 mg/kg/day at days 6,
10, and 13 after injection of Lgr5-positive cells. Then, EREG
antibody was administered at a dose of 10 mg/kg/day 17 and 24 days
after injection of Lgr5-positive cells. Each value represents
mean.+-.standard deviation (n=4).
[0109] FIG. 58 shows photographs and diagrams depicting the
anti-tumor effect of EREG antibody. In FIG. 58A, the first row
shows histological staining (HE) of tumors from xenograft models
prepared by intravenously injecting PLR123-derived Lgr5-positive
cells to NOG mice, and the second row shows immunohistochemical
staining using EREG antibody. Arrows in the panels indicate
EREG-expressing cells at nodules (second row) and corresponding HE
stain images (first row). FIGS. 58B and 58C show the number of
tumors formed in the lungs of SCID-beige mice intravenously
injected with Lgr5-positive cells derived from PLR123 xenograft
models. In the EREG antibody administration group, EREG antibody
was administered once a week for five times from three days after
injection of Lgr5-positive cells. The number of tumor nodules per
animal, found on lung thin sections, are shown in FIG. 58B. Each
symbol (circle) indicates the number of tumor nodules in an animal
in a group tested (FIG. 58B). The number of tumor nodules
categorized by size in the antibody administration group and
control group are shown in FIG. 58C. White columns indicate tumors
that are smaller than 100 .mu.m; gray columns indicate tumors of
100 to 200 .mu.m; and black columns indicate tumors larger than 200
.mu.m (FIG. 58C). Tissue staining (HE stain) of tumors in the EREG
antibody administration and non-administration groups is shown in
FIG. 58D. Scale bar represents 200 .mu.m.
[0110] FIG. 59 shows photographs depicting the same tissue sections
from primary and liver metastatic colorectal cancers isolated from
patients, which were stained with HE (second and fourth rows), and
antibodies against Lgr5 (green), HLA-DMA (red), and EREG (green)
(first and third rows). Positivity for Lgr5 indicates proliferating
CSCs, while positivity for EREG and HLA-DMA implies Lgr5-negative
quiescent CSCs. Both Lgr5-negative and -positive CSCs were detected
in ductal structures and budding areas of primary and liver
metastatic tumors. Lgr5-positive CSCs were also found as single
cells in stromal regions. The same staining patterns were also
observed in multiple tumor tissues isolated from different
patients. Arrows indicate CSCs. Scale bar represents 10 .mu.m.
[0111] FIG. 60 is a diagram showing the anti-tumor effect of
anti-CD70 antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-CD70 antibody, respectively.
[0112] FIG. 61 is a diagram showing the anti-tumor effect of
anti-EDAR antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-EDAR antibody, respectively.
[0113] FIG. 62 is a diagram showing the anti-tumor effect of
anti-FAS antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-FAS antibody, respectively.
[0114] FIG. 63 is a diagram showing the anti-tumor effect of
anti-PVRL4 antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-PVRL4 antibody, respectively.
[0115] FIG. 64 is a diagram showing the anti-tumor effect of
anti-TNFSF9 antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-TNFSF9 antibody,
respectively.
[0116] FIG. 65 is a diagram showing the anti-tumor effect of
anti-PROCR antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-PROCR antibody, respectively.
[0117] FIG. 66 is a diagram showing the anti-tumor effect of
anti-EPCAM antibody on irinotecan non-treated CSCs. The horizontal
and vertical axes indicate the antibody concentration and %
inhibition of cell growth by anti-EPCAM antibody, respectively.
[0118] FIG. 67 is a diagram showing the anti-tumor effect of
anti-FAS antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-FAS antibody, respectively.
[0119] FIG. 68 is a diagram showing the anti-tumor effect of
anti-PROM2 antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-PROM2 antibody, respectively.
[0120] FIG. 69 is a diagram showing the anti-tumor effect of
anti-PVRL4 antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-PVRL4 antibody, respectively.
[0121] FIG. 70 is a diagram showing the anti-tumor effect of
anti-TNFSF9 antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-TNFSF9 antibody, respectively.
[0122] FIG. 71 is a diagram showing the anti-tumor effect of
anti-PROCR antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-PROCR antibody, respectively.
[0123] FIG. 72 is a diagram showing the anti-tumor effect of
anti-EPCAM antibody on irinotecan treated CSCs. The horizontal and
vertical axes indicate the antibody concentration and % inhibition
of cell growth by anti-EPCAM antibody, respectively.
[0124] FIG. 73 is a diagram showing the expression levels of
differentiation marker CK20 in CSCs cultured in the presence of
BMP4. FIGS. 73A and 73B show the expression levels of CK20 in PLR59
and PLR123, respectively.
MODE FOR CARRYING OUT THE INVENTION
[0125] The present invention relates to cell surface molecules that
are specifically expressed on cancer stem cells, and pharmaceutical
compositions (anti-cancer agents, etc.) and reagents for detecting
cancer stem cells, which use antibodies against the cell surface
molecules.
[0126] Herein, "cancer" refers to the physiological condition in
mammals, which is typically characterized by unregulated cell
growth, or such a physiological condition. Herein, cancer types are
not particularly limited, and include those listed below.
Carcinomas (epithelial cancers) include pancreatic cancer,
prostatic cancer, breast cancer, skin cancer, cancers of the
digestive tract, lung cancer, hepatocellular carcinoma, cervical
cancer, uterine cancer, ovary cancer, fallopian tube cancer,
vaginal cancer, liver cancer, bile duct cancer, bladder cancer,
ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, and
cancers of other glandular tissues. Sarcomas (non-epithelial
tumors) include liposarcoma, leiomyosarcoma, rhabdomyosarcoma,
synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral
nerve sheath tumor, gastrointestinal stromal tumor, desmoid tumor,
Ewing's sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma,
myeloma, tumors of other parenchymal organs, for example, melanoma
and brain tumor (Kumar V, Abbas A K, Fausio N. Robbins and Cotran
Pathologic Basis of Disease. 7th Ed. Unit I: General Pathology, 7:
Neoplasia, Biology of tumor growth: Benign and malignant neoplasms.
269-342, 2005).
[0127] Herein, "tumor" refers to arbitrary benign (non-cancerous)
and malignant (cancerous) tissue masses, including pre-cancerous
lesions, which result from overgrowth or overexpansion of
cells.
[0128] Herein, cancer stem cell (CSC) refers to cells having the
abilities described in (i) and/or (ii) below.
(i) The ability to self-renew. The self-renewal ability refers to
the ability of either or both of the divided daughter cells to
produce cells which maintain the same capacity and the degree of
differentiation as the parental cell in terms of cell lineage. (ii)
The ability to differentiate into various types of cancer cells
that constitute a cancer cell mass. Like normal stem cells, various
types of cancer cells differentiated from cancer stem cells
generate a hierarchical organization with cancer stem cells at the
top in terms of cell lineage. Various types of cancer cells are
generated in a sequential manner from cancer stem cells. This
results in the formation of a cancer cell mass that exhibits a
variety of features.
[0129] Cancer stem cell refers to a cancer cell that has the
ability to form cancers as well as, like normal stem cell,
pluripotency and self-renewal ability. Cancer stem cells generate a
hierarchical organization with cancer stem cells at the top.
Various types of cancer cells are generated in a sequential manner
from cancer stem cells. This results in the formation of a cancer
cell mass that exhibits a variety of features. Cancer cell mass
refers to, not a group of individual cells, but a mass formed by
the adhesion of cells etc. as in human tumor tissue, which is built
with cancer cells, and other cells such as stromal cells and blood
cells, extracellular matrix such as collagen and laminin, and so
on.
[0130] The origin of cancer stem cells, which are the target in
therapy using pharmaceutical compositions of the present invention,
is not particularly limited; it is possible to use cancer stem
cells derived from mammals such as humans, monkeys, chimpanzees,
dogs, bovines, pigs, rabbits, rats, and mice. However, cancer stem
cells derived from humans are preferred, and those derived from
human tumor tissues are more preferred.
[0131] Cancer stem cells to be detected using the present invention
are preferably those which reconstitute the hierarchical structure
of cancer tissues. For example, cancer cell lines are prepared by
grafting cancer tissues from which the detected cancer stem cells
have been collected, into, preferably, nonhuman animals, and
passaging them in such animals, and one can test whether the
established cancer cell lines reconstitute the hierarchical
structure of the cancer tissues. One can test whether the
hierarchical structure of cancer tissues is reproduced by
NOG-established cancer cell lines prepared by grafting and
passaging cancer tissues in nonhuman animals, more preferably
immunodeficient animals, and still more preferably NOG mice which
lack functional T cells, B cells, and natural killer cells.
[0132] Alternatively, cancer stem cells to be detected using the
present invention can be a spheroid (cell mass) formed by spheroid
culture. "Spheroid culture" means that cancer stem cells are
inoculated in a culture vessel such as non-adherent or low-adherent
cell culture flasks, plates, or dishes using a medium capable of
culturing cancer stem cells, and then the cells are cultured under
a three-dimensionally floating condition. A cell mass formed by
this method is called "spheroid".
[0133] NOG-established cancer cell lines can be generated by a
method known to those skilled in the art, for example, the method
described in Fujii E. et al., Pathol int. 2008; 58: 559-567. Human
colorectal cancer, stomach cancer, lung cancer, breast cancer,
pancreatic cancer, or the like is resected surgically. After
mechanically mincing it with scissors, the cancer is grafted
subcutaneously in NOG mice and passaged to establish cell lines.
Even after passages, NOG-established cancer cell lines maintain the
properties of the original human cancer tissues.
[0134] In the present invention, cancer stem cells can be selected
by using cell markers. Cell markers used in the present invention
include, for example, leucine-rich repeat-containing
G-protein-coupled receptor 5 (Lgr5), CD133, CD44, EpCAM, CD166,
CD24, CD26, and CD29.
[0135] The present invention relates to molecules expressed in
cancer stem cells that are positive for the expression of the cell
marker Lgr5, and are adherent and highly proliferative under
serum-free culture conditions. Hereinafter, such cancer stem cells
are also referred to as "high proliferative Lgr5-positive cancer
stem cells".
[0136] The present invention also relates to molecules expressed in
cancer stem cells that are negative for the expression of the cell
marker Lgr5, and non-adherent and poorly proliferative under
serum-free culture conditions. Hereinafter, such cancer stem cells
are also referred to as "low proliferative Lgr5-negative cancer
stem cells".
[0137] Any culture media or liquids can be used to culture cancer
stem cells of the present invention as long as they are serum-free
media and capable of culturing cancer stem cells. There is no
particular limitation on the culture media or liquids. For example,
it is possible to use conventional basal media or mixtures thereof
that are supplemented with EGF, bFGF, hLIF, HGF, NGF, NSF-1, TGF
.beta., TNF.alpha., heparin, BSA, insulin, transferrin, putrescine,
selenite, progesterone, hydrocortisone, D-(+)-glucose, sodium
bicarbonate, HEPES, L-glutamine, or N-acetylcysteine. The
concentration of EGF is not particularly limited; however, it
ranges from 0.1 to 100 ng/ml, preferably from 0.5 to 50 ng/ml, and
more preferably from 1 to 20 ng/ml. The concentration of bFGF is
not particularly limited; however, it ranges from 0.1 to 100 ng/ml,
preferably from 0.5 to 50 ng/ml, and more preferably from 1 to 20
ng/ml. The concentration of hLIF is not particularly limited;
however, it ranges from 0.1 to 100 ng/ml, preferably from 0.5 to 50
ng/ml, and more preferably from 1 to 20 ng/ml. The concentration of
HGF is not particularly limited; however, it ranges from 0.1 to 100
ng/ml, preferably from 1 to 50 ng/ml. The concentration of NGF is
not particularly limited; however, it ranges from 0.1 to 100 ng/ml,
preferably from 1 to 50 ng/ml. The concentration of NSF-1 is not
particularly limited; however, it ranges from 0.1 to 100 ng/ml,
preferably from 1 to 50 ng/ml. The concentration of TGF.beta. is
not particularly limited; however, it ranges from 0.1 to 100 ng/ml,
preferably from 1 to 50 ng/ml. The concentration of TNF.alpha. is
not particularly limited; however, it ranges from 0.1 to 100 ng/ml,
preferably from 1 to 50 ng/ml. The concentration of heparin is not
particularly limited; however, it ranges from 10 ng/ml to 10
.mu.g/ml, preferably from 2 to 5 .mu.g/ml. The concentration of BSA
is not particularly limited; however, it ranges from 0.1 to 10
mg/ml, preferably from 1 to 8 mg/ml. The concentration of insulin
is not particularly limited; however, it ranges from 1 to 100
.mu.g/ml, preferably from 10 to 50 .mu.g/ml. The concentration of
transferrin is not particularly limited; however, it ranges from 10
to 500 .mu.g/ml, preferably from 50 to 200 .mu.g/ml. The
concentration of putrescine is not particularly limited; however,
it ranges from 1 to 50 .mu.g/ml, preferably from 10 to 20 .mu.g/ml.
The concentration of selenite is not particularly limited; however,
it ranges from 1 to 50 nM, preferably from 20 to 40 nM. The
concentration of progesterone is not particularly limited; however,
it ranges from 1 to 50 nM, preferably from 10 to 30 nM. The
concentration of hydrocortisone is not particularly limited;
however, it ranges from 10 ng/ml to 10 .mu.g/ml, preferably from
100 ng/ml to 1 .mu.g/ml. The concentration of D-(+)-glucose is not
particularly limited; however, it ranges from 1 to 20 mg/ml,
preferably from 5 to 10 mg/ml. The concentration of sodium
bicarbonate is not particularly limited; however, it ranges from
0.1 to 5 mg/ml, preferably from 0.5 to 2 mg/ml. The concentration
of HEPES is not particularly limited; however, it ranges from 0.1
to 50 mM, preferably from 1 to 20 mM. The concentration of
L-glutamine is not particularly limited; however, it ranges from
0.1 to 10 mM, preferably from 1 to 5 mM. The concentration of
N-acetylcysteine is not particularly limited; however, it ranges
from 1 to 200 .mu.g/ml, preferably from 10 to 100 .mu.g/ml. Known
basal culture liquids, which are not particularly limited as long
as they are suitable for culturing cancer cells from which cancer
stem cells are derived, include, for example, DMEM/F 12, DMEM, F10,
F12, IMDM, EMEM, RPMI-1640, MEM, BME, Mocoy's 5A, and MCDB131. Of
them, DMEM/F12 is preferred.
[0138] The most preferred stem cell media include DMEM/F12 medium
supplemented with 20 ng/ml human EGF, 10 ng/ml human bFGF, 4
.mu.g/ml heparin, 4 mg/ml BSA, 25 .mu.g/ml human insulin, and 2.9
mg/ml glucose where each concentration a final concentration.
[0139] As described herein, high proliferative Lgr5-positive cancer
stem cells have the properties of mesenchymal cells. Meanwhile, as
described herein, low proliferative Lgr5-negative cancer stem cells
have the properties of epithelial cells. Herein, "epithelial cells"
refers to cells that constitute epithelial tissues in the living
body.
[0140] In the present invention, the origin of cancer stem cells is
not particularly limited; however, they are preferably derived from
a solid cancer, more preferably a gastrointestinal cancer.
Gastrointestinal cancers include, for example, esophageal cancer,
stomach cancer, duodenal cancer, pancreatic cancer, bile duct
cancer, gallbladder cancer, biliary tract cancer, colorectal
cancer, colon cancer, and rectal cancer. A preferred
gastrointestinal cancer is colorectal cancer.
[0141] Furthermore, in the present invention, cancer stem cells are
preferably positive for one or more of the cell markers CD133,
CD44, EpCAM, CD166, CD24, CD26, and CD29, more preferably positive
for CD133, CD44, EpCAM, CD166, CD24, CD26, and CD29.
[0142] In addition, in the present invention, acetaldehyde
dehydrogenase (ALDH) activity can be used as a cell marker. In the
present invention, Lgr5-positive adherent cancer stem cells are
positive for the ALDH activity cell marker, whereas Lgr5-negative
cancer stem cells are negative for ALDH activity.
[0143] In the present invention, one or more of HLA-DMA, TMEM173,
ZMAT3, and GPR110 can also be used as cell markers. Lgr5-positive
adherent cancer stem cells are negative for any of the cell markers
HLA-DMA, TMEM173, ZMAT3, and GPR110, while Lgr5-negative cancer
stem cells are positive for any of the cell markers HLA-DMA,
TMEM173, ZMAT3, and GPR110.
[0144] In the present invention, cancer stem cells preferably have
the feature of reconstituting the hierarchical structure of cancer
tissues.
[0145] Herein, "hierarchical structure" means that some of the
unique and characteristic structures observed in a normal tissue
are detected histopathologically in the structure of a tumor
originated from the tissue. In general, highly-differentiated
cancers reconstitute the hierarchical structure to a high degree.
For example, lumen formation and mucous cells are observed in the
case of tumors of glandular lumen-forming organs (stomach cancer,
colorectal cancer, pancreatic cancer, liver cancer, bile duct
cancer, breast cancer, lung adenocarcinoma, prostatic cancer,
etc.). In the case of tumors that form squamous epithelial
structures (squamous cell carcinoma of lung, skin, vaginal mucosa,
etc.), layer structure formation, the tendency to keratosis, and
such are observed in the epithelium. On the other hand,
poorly-differentiated cancers insufficiently reconstitute the
hierarchical structure, and they are said to be highly atypical
(Kumar V, Abbas A K, Fausio N. Robbins and Cotran Pathologic Basis
of Disease. 7th Ed. Unit I: General Pathology, 7: Neoplasia,
Biology of tumor growth: Benign and malignant neoplasms. 272-281,
2005). Since the hierarchical structure is considered to be
reconstituted as a result of various biological reactions, cancer
stem cells that reconstitute it are thought to be highly
useful.
[0146] "Reconstitution of the hierarchical structure" means that
the unique and characteristic structure possessed by the original
cancer stem cells is also observed even after isolation or
induction of cancer stem cells.
[0147] Furthermore, in the present invention, cancer stem cells
preferably have the ability of epithelial-mesenchymal transition
(EMT). Herein, the ability of epithelial-mesenchymal transition
means both that epithelial cells transition into mesenchymal cells
by obtaining their characteristics, and that mesenchymal cells
transition into epithelial cells by obtaining their
characteristics. EMT does not occur in normal cells except during
the process of embryogenesis. Epithelial cells, which are bound
together tightly and exhibit polarity, change into mesenchymal
cells that are bound together more loosely, exhibit a loss of
polarity, and have the ability to move. These mesenchymal cells can
spread into tissues around the primary tumor, and also separate
from the tumor, invade blood and lymph vessels, and move to new
locations where they divide and form additional tumors. Drug
resistance, metastasis, or recurrence of cancer can be explained by
such additional tumor formation.
[0148] Furthermore, the present invention provides pharmaceutical
compositions comprising as an active ingredient an antibody that
binds to a molecule expressed in a substantially homogeneous cancer
stem cell population comprising the above cancer stem cells of the
present invention. "Substantially homogeneous" means that, when
immunodeficient animals are grafted with 1000 cells, 100 cells, or
10 cells and analyzed for the frequency of formation of cancer cell
populations using Extreme Limiting Dilution Analysis (Hu Y &
Smyth G K., J Immunol Methods. 2009 Aug. 15; 347(1-2): 70-8)
utilizing, for example, the method described in Hu Y & Smyth G
K., J Immunol Methods. 2009 Aug. 15; 347 (1-2):70-8 or Ishizawa K
& Rasheed Z A. et al., Cell Stem Cell. 2010 Sep. 3;
7(3):279-82, the frequency of cancer stem cells is 1/20 or more,
preferably 1/10 or more, more preferably 1/5 or more, even more
preferably 1/3 or more, still more preferably 1/2 or more, and yet
more preferably 1/1.
[0149] In the present invention, cancer stem cell populations can
be prepared, for example, by culturing cells or a group of cells
containing the cancer stem cells described herein.
[0150] Herein, "adherent culture" means that, after seeding cells
into culture vessels for adherent culture, the adhered cells are
cultured and passaged while non-adherent cells are removed. The
cells grown to confluency are detached with Accutase and passaged
into fresh adherent culture flasks, adherent culture plates, or
adherent culture dishes for further culture. Culture vessels for
adherent culture are not particularly limited as long as they are
used for adherent culture. It is possible to appropriately select
and use flasks for adherent culture or highly adherent flasks,
plates for adherent culture or highly adherent plates,
flat-bottomed plates for adherent culture or highly adherent
flat-bottomed plates, dishes for adherent culture or highly
adherent dishes, etc.
[0151] Media used for adherent culture are not particularly
limited; however, it is preferable to use serum-free stem cell
culture media.
[0152] Herein, "adherent" refers to the property of cells to adhere
to culture vessels for adherent culture when they are cultured in
the vessels.
[0153] Herein, "suspension culture" means that, after seeding cells
into culture vessels for suspension culture, the floating cells are
cultured and passaged while adherent cells are removed. The cells
grown to confluency are passaged into fresh low adherent cell
culture flasks, ultra low adherent cell culture flasks, low
adherent plates, ultra low adherent plates, low adherent dishes, or
ultra low adherent dishes for further culture. Culture vessels for
suspension culture are not particularly limited as long as they are
used for suspension culture. It is possible to appropriately select
and use low adherent cell culture flasks, ultra low adherent cell
culture flasks, low adherent plates, ultra low adherent plates, low
adherent dishes, ultra low adherent dishes, etc.
[0154] Media used for suspension culture are not particularly
limited; however, it is preferable to use serum-free stem cell
culture media. A cell group containing cancer stem cells are
preferably expanded before performing adherent or suspension
culture. Herein, "non-adherent" refers to the property of cells to
be cultured in a floating state without adherence to culture
vessels for suspension culture when the cells are cultured in the
vessels.
[0155] "Expansion of a cell group" means, for example,
proliferation by spheroid culture or grafting and passaging in
nonhuman animals, but is not particularly limited thereto.
[0156] As described herein, for nonhuman animals, immunodeficient
animals can be used for grafting since they are unlikely to have
rejection reactions. Immunodeficient animals preferably used
include nonhuman animals that lack functional T cells, for example,
nude mice and nude rats, and nonhuman animals that lack both
functional T and B cells, for example, SCID mice and NOD-SCID mice.
It is more preferably to use mice that lack T, B, and NK cells and
have excellent transplantability, including, for example, NOG
mice.
[0157] Regarding the weekly age of nonhuman animals, for example, 4
to 100-week-old athymic nude mice, SCID mice, NOD-SCID mice, or NOG
mice are preferably used. NOG mice can be prepared, for example, by
the method described in WO 2002/043477, and are available from the
Central Institute for Experimental Animals or the Jackson
Laboratory (NSG mice).
[0158] Cells to be grafted may be any cells, including cell masses,
tissue fragments, individually dispersed cells, cells cultured
after isolation, and cells isolated from a different animal into
which the cells have been grafted; however, dispersed cells are
preferred. The number of grafted cells may be 10.sup.6 or less;
however, it is acceptable to graft more cells.
[0159] With respect to the grafting site, subcutaneous grafting is
preferred because the graft technique is simple. The grafting site
is not particularly limited, and it is preferable to select an
appropriate grafting site depending on the animal used. There is no
particular limitation on the grafting operation of NOG-established
cancer cell lines, and the cells can be grafted by conventional
grafting operations.
[0160] Cancer stem cells or a cancer stem cell population can be
prepared, for example, by collecting cancer tissues from patients
and culturing the tissues in a serum-free stem cell culture medium
under adherent or floating culture conditions. Alternatively,
cancer tissues collected from patients can be spheroid-cultured,
and then cultured in a serum-free stem cell culture medium under
adherent or floating culture conditions to prepare cancer stem
cells or a cancer stem cell population Alternatively, cancer
tissues collected from patients can be grafted and passaged in
nonhuman animals, and then cultured in a serum-free stem cell
culture medium under adherent or floating culture conditions to
prepare cancer stem cells or a cancer stem cell population.
Alternatively, it is possible to use a method in which cancer
tissues collected from patients are grafted and passaged in NOG
mice to prepare NOG-established cancer cell lines, and they are
cultured in a serum-free stem cell culture medium under adherent or
suspension culture conditions.
[0161] Cancer stem cells and cancer stem cell populations of the
present invention can be used in methods of screening for
pharmaceutical agents, anti-cancer agents, or the like.
[0162] In an embodiment of methods of screening for pharmaceutical
agents, the present invention provides methods comprising the steps
of:
(a) preparing a substantially homogeneous cancer stem cell
population comprising an Lgr5-positive adherent cancer stem cell;
(b) contacting a test substance with the cancer stem cell
population or a cancer stem cell comprised in the cancer stem cell
population; and (c) detecting a change in a biological property of
the cancer stem cell population or cancer stem cell contacted with
the test substance.
[0163] In these methods, first, a substantially homogeneous cancer
stem cell population containing Lgr5-positive adherent cancer stem
cells or Lgr5-negative cancer stem cells is prepared. Then, a test
substance is contacted with the prepared cancer stem cell
population or cancer stem cells contained in the cancer stem cell
population. In these methods, there is no particular limitation on
the method for contacting a test substance with a cancer stem cell
population or cancer stem cells contained in the cancer stem cell
population. For example, a test substance may be contacted with
cultured cells of a cancer stem cell population or cancer stem
cells contained in the cancer stem cell population. This treatment
can be carried out by adding a test substance to a cell culture
medium or cell extract. When a test substance is a protein, this
treatment can be performed, for example, as follows: a vector
comprising a DNA encoding the protein is introduced into a cancer
stem cell population or cancer stem cells contained in the cancer
stem cell population; or the vector is added to a cell extract of a
cancer stem cell population or cancer stem cells contained in the
cancer stem cell population. Alternatively, it is possible, for
example, to use the two-hybrid method utilizing yeast, animal
cells, or the like.
[0164] In these methods, then, a change in a biological property of
the cancer stem cell population or cancer stem cells treated with
the test substance is detected. Such a change in a biological
property includes, for example, a change in the proliferation
ability, a change in the viable cell count, a change in a tissue
structure characteristic of the process of cancer progression of
the cancer stem cell population or cancer stem cells, and a change
in the expression of a DNA, RNA, protein, or metabolite in the
cancer stem cell population or cancer stem cells. A change in a
biological property can be detected, for example, by the methods
described below.
[0165] There is no particular limitation on the assessment of the
expression of DNAs, RNAs, proteins, peptides, and metabolites; the
expression can be assessed by conventional expression assessment
methods. RNAs include microRNAs, siRNAs, tRNAs, snRNAs, mRNAs, and
non-coding RNAs. For example, mRNAs of a gene are extracted
according to a conventional method. Using the mRNAs as a template,
the transcriptional level of the gene can be determined using the
Northern hybridization or RT-PCR method. DNA array techniques can
also be used to determine the expression level of the gene.
Alternatively, fractions containing a protein encoded by a gene are
collected according to a conventional method. The translational
level of the gene can be determined by detecting the protein
expression by an electrophoresis method such as SDS-PAGE. The
translational level of a gene can also be determined by performing
the Western blotting method using an antibody against a protein and
detecting the protein expression. These methods can be used to
screen for pharmaceutical agents (pharmaceutical compositions).
[0166] The DNAs, RNAs, and proteins that are contained in a cancer
stem cell population or cancer stem cells and which are
characteristic of the process of cancer progression of the cancer
stem cell population or cancer stem cells, preferably include the
proteins or polypeptides of any one of SEQ ID NOs: 1 to 6, and
polynucleotides encoding the proteins or polypeptides.
[0167] For example, when there is no change in a biological
property of a cancer stem cell population or cancer stem cells, or
the degree of the change is reduced after treatment with a test
substance compared to before the treatment, the test substance is
expected to be useful as a pharmaceutical agent (pharmaceutical
composition) that has the activity of suppressing cancer recurrence
or metastasis (for example, an agent for suppressing cancer
recurrence, an agent for post-chemotherapy adjuvant therapy, an
agent for postoperative adjuvant therapy, an anti-cancer agent, or
an agent for suppressing cancer metastasis). Such test substances
can be selected as effective substances that have the therapeutic
or preventive effect against cancerous diseases. Such
pharmaceutical agents (pharmaceutical compositions) having the
activity of suppressing cancer progression are used as an agent for
suppressing cancer recurrence, an agent for post-chemotherapy
adjuvant therapy, an agent for postoperative adjuvant therapy, an
anti-cancer agent, or an agent for suppressing cancer metastasis.
Anti-cancer agents of the present invention may be used against,
for example, cancers resistant to pharmaceutical agents or
chemotherapeutic agents. Specifically, pharmaceutical agents
(pharmaceutical compositions) of the present invention also include
therapeutic agents against drug-resistant or chemotherapeutic
agent-resistant cancers.
[0168] In the present invention, the above pharmaceutical agents
(pharmaceutical compositions) are not particularly limited to
anti-cancer agents or agents for suppressing metastasis or
recurrence, and they can also be used as an agent for inhibiting
angiogenesis or cell growth. Furthermore, the pharmaceutical agents
(pharmaceutical compositions) of the present invention may be used
simultaneously with chemotherapeutic agents or after treatment with
chemotherapeutic agents. The pharmaceutical agents are not
particularly limited, and they include proteinaceous agents,
nucleic acid agents, low-molecular-weight agents, and cellular
agents.
[0169] In another embodiment of the screening methods, the present
invention provides methods of screening for pharmaceutical agents
(pharmaceutical compositions), which comprise the steps of:
(a) preparing a substantially homogeneous cancer stem cell
population comprising an Lgr5-negative non-adherent cancer stem
cell; (b) contacting a test substance with the cancer stem cell
population or a cancer stem cell comprised in the cancer stem cell
population; and (c) detecting a change in a biological property of
the cancer stem cell population or cancer stem cell contacted with
the test substance.
[0170] In these methods, first, a substantially homogeneous cancer
stem cell population containing Lgr5-negative non-adherent cancer
stem cells is prepared. Then, the prepared cancer stem cell
population or cancer stem cells contained in the cancer stem cell
population are treated with a test substance. Next, a change in a
biological property of the cancer stem cell population or cancer
stem cells treated with the test substance is detected.
[0171] The DNAs, RNAs, and proteins that are contained in such a
cancer stem cell population or cancer stem cells, and which are
characteristic of the process of cancer progression of the cancer
stem cell population or cancer stem cells, preferably include the
proteins or polypeptides of any one of SEQ ID NOs: 1 to 8, and
polynucleotides encoding the proteins or polypeptides. In a
non-limiting embodiment of the present invention, the proteins or
polypeptides of SEQ ID NOs: 1 to 6, and polynucleotides encoding
the proteins or polypeptides may be used. In another non-limiting
embodiment of the present invention, the protein or polypeptide of
SEQ ID NO: 7 or 8, and polynucleotides encoding the protein or
polypeptide may be used.
[0172] Pharmaceutical agents (pharmaceutical compositions) that are
obtained by the screening methods are not particularly limited, and
they can be used as anti-cancer agents. Specifically, when there is
no change in a biological property of a cancer stem cell population
or cancer stem cells, or the degree of the change is reduced after
treatment with a test substance compared to before the treatment,
the test substance is expected to be useful as a pharmaceutical
agent that has the activity of suppressing cancer recurrence or
metastasis (for example, an agent for suppressing cancer
recurrence, an agent for post-chemotherapy adjuvant therapy, an
agent for postoperative adjuvant therapy, an anti-cancer agent, or
an agent for suppressing cancer metastasis). Such test substances
may be selected as effective substances that have the therapeutic
or preventive effect against cancerous diseases. Such
pharmaceutical agents (pharmaceutical compositions) having the
activity of suppressing cancer progression are used as an agent for
suppressing cancer recurrence, an agent for post-chemotherapy
adjuvant therapy, an agent for postoperative adjuvant therapy, an
anti-cancer agent, or an agent for suppressing cancer
metastasis.
[0173] Furthermore, pharmaceutical agents (pharmaceutical
compositions) of the present invention include cancer therapeutic
agents against Lgr5-negative cancers, which contain as an active
ingredient at least an antibody that binds to a protein of SEQ ID
NOs: 1 to 8. The Lgr5-negative cancers include those resistant to
drugs or chemotherapeutic agents.
[0174] Still another embodiment of the screening methods of the
present invention includes methods that use nonhuman animals
administered with a test substance, and a cancer stem cell
population of the present invention or cancer stem cells contained
in the cancer stem cell population. Specifically, the present
invention provides methods of screening for pharmaceutical agents
(pharmaceutical compositions), which comprise the steps of:
(a) preparing a substantially homogeneous cancer stem cell
population comprising an Lgr5-positive adherent cancer stem cell;
(b) administering a nonhuman animal with a test substance and the
cell population or a cancer stem cell comprised in the cancer stem
cell population; and (c) detecting tumor formation in the nonhuman
animal.
[0175] In these methods, first, a substantially homogeneous cancer
stem cell population containing Lgr5-positive adherent cancer stem
cells is prepared. Then, nonhuman animals are administered with a
test substance, and the cancer stem cell population prepared or
cancer stem cells contained in the cancer stem cell population.
[0176] In these methods, the method for administering a test
substance to nonhuman animals is not particularly limited. Oral
administration, or parenteral administration such as subcutaneous,
intravenous, local, transdermal, or transintestinal (transrectal)
administration can be appropriately selected depending on the type
of a test substance to be administered.
[0177] Furthermore, in these methods, there is no particular
limitation on the method for administering a cancer stem cell
population or cancer stem cells to nonhuman animals, and an
appropriate method can be selected depending on the cell population
to be administered. The preferred method is subcutaneous or
intravenous administration.
[0178] In these methods, then, tumor formation is detected in the
nonhuman animals.
[0179] The assessment of a test substance can be performed as
follows: tissues administered with a test substance and a cancer
stem cell population or cancer stem cells are excised from nonhuman
animals, and then histological features of the tissues are observed
to determine the presence or absence of tumor formation. When tumor
formation is not detected, the test substance is expected to be
useful as a pharmaceutical agent having the activity of suppressing
cancer progression or metastasis (for example, an anti-cancer agent
or an agent for suppressing cancer metastasis or recurrence), and
the test substance can be selected as an effective substance that
has the therapeutic or preventive effect against cancerous
diseases. That is, pharmaceutical agents (pharmaceutical
compositions) obtained by the screening methods are not
particularly limited, and can be used as an anti-cancer agent, or
an agent for suppressing cancer metastasis or recurrence.
[0180] "Test substances" used in the methods of the present
invention are not particularly limited, and include, for example,
single compounds such as natural compounds, organic compounds,
inorganic compounds, proteins, antibodies, peptides, and amino
acids, as well as compound libraries, expression products of gene
libraries, cell extracts, cell culture supernatants, products of
fermenting microorganisms, extracts of marine organisms, plant
extracts, prokaryotic cell extracts, unicellular eukaryote
extracts, and animal cell extracts. These may be purified products
or crude purified products such as extracts of plants, animals, and
microorganisms. Also, methods for producing test substances are not
particularly limited; test substances may be isolated from natural
materials, synthesized chemically or biochemically, or prepared by
genetic engineering. It is also possible to appropriately use
antisense and RNAi molecules that are designed by known methods
based on partial sequences of polynucleotides encoding the protein
of any one of SEQ ID NOs: 1 to 8. If needed, the above test
substances can be appropriately labeled and used. Labels include,
for example, radiolabels and fluorescent labels. Mixtures of an
above-mentioned test substance and multiple kinds of such labels
are included in the test substances of the present invention.
[0181] Furthermore, the present invention provides pharmaceutical
agents such as vaccines comprising a partial peptide of the protein
of any one of SEQ ID NOs: 1 to 8, and methods of screening for
vaccines. Such screening methods preferably include methods for
determining the cytotoxic activity targeted to cancer stem cells
disclosed in the present application using cytotoxic T cells (CTL)
or the like induced with a cancer vaccine of the present invention
in vitro. Specifically, adherent and non-adherent cells are
separated from peripheral blood mononuclear cells (PBMCs) collected
by centrifugation of human peripheral blood in Ficoll-Conray
density gradient. The adherent cells are incubated with 100 ng/ml
GM-CSF (Novartis) and 10 IU/ml IL-4 (GIBCO-BRL) in AIM-V (GIBCO),
and then the cells are used as antigen-presenting cells (APC).
Meanwhile, the non-adherent cells are incubated with 30 to 100
IU/ml recombinant IL-4 (Ajinomoto) in AIM-V. On day 7 to 10, a
partial peptide of the protein of any one of SEQ ID NOs: 1 to 8
provided by the present invention is added (at a final
concentration of 30 .mu.g/ml) to APC. On the following day,
recombinant TNF-.alpha. and IFN-.alpha. (Sumitomo Pharma Co.) are
added for APC maturation. Then, CD8-positive cells isolated from
autologous non-adherent cells are mixed with irradiated APC in
IL-2-free AIM-V. After two days of incubation, IL-2 (Takeda
Pharmaceutical Company) is added at a final concentration 100 IU/ml
to the culture. The CD8-positive cells are stimulated every seven
days using, as APC, autologous PHA blasts (PHA-stimulated T cells)
that have been stimulated with the T cell mitogen PHA. A fresh
medium containing 100 IU/ml IL-2 is added to the culture at every
time point of stimulation. CTL on day 28 is used for the activity
assay. High proliferative Lgr5-positive cancer stem cells and low
proliferative Lgr5-negative cancer stem cells that are provided by
the present invention can be used as target cells of CTL. The
cytotoxic activity can be assessed by determining .sup.51Cr-sodium
chromate uptake activity by a measurement method similar to that of
ADCC activity.
[0182] Furthermore, pharmaceutical agents selected by the screening
methods of the present invention may be further screened as
necessary for more effective and practical preventive or
therapeutic active substances by conducting additional drug
effectiveness tests and safety tests, and further conducting
clinical tests in human cancer patients. Based on results of
structural analysis of pharmaceutical agents thus selected, they
can be industrially manufactured by chemical synthesis, biochemical
synthesis (fermentation), or genetic engineering.
[0183] "High proliferative ability" means that the doubling time is
6 days or less, preferably 4 days or less, and more preferably 3
days or less when cells are cultured in a serum-free medium
supplemented with EGF and FGF using the method described
herein.
[0184] "Low proliferative ability" means that the doubling time is
7 days or more, preferably 14 days or more, and more preferably
there is no significant proliferation when cells are cultured in a
serum-free medium supplemented with EGF and FGF using the method
described herein.
[0185] When preparing such high proliferative Lgr5-positive cancer
stem cells and low proliferative Lgr5-negative cancer stem cells,
the cells can be separated using the cell marker Lgr5. The
separation methods include the following: methods in which a cell
population containing cancer stem cells is isolated by using an
anti-Lgr5 antibody;
methods in which a substantially homogeneous cancer stem cell
population is first prepared by culturing a population containing
cancer stem cells under adherent or suspension culture conditions,
and then the population is isolated by using an anti-Lgr5 antibody;
and methods in which a substantially homogeneous cancer stem cell
population is first prepared by culturing a population containing
cancer stem cells in a medium with or without a growth inhibitor
under adherent culture conditions, and then the population is
isolated by using an anti-Lgr5 antibody. Any of the above methods
may be used in the present invention. Preferably, cells are
isolated from cancer tissues after three or more passages in NOG
mice, and cultured in a serum-free stem cell culture media under
adherent culture conditions to prepare high proliferative
Lgr5-positive cancer stem cells. Then, low proliferative
Lgr5-negative cancer stem cells can be prepared as follows. The
resulting Lgr5-positive cancer stem cells are maintained under
various stresses such as a contact with a growth inhibitor, for
example, treatment with irinotecan (culture for three days in a
serum-free stem cell medium supplemented with 10 .mu.g/ml
irinotecan).
[0186] Furthermore, the present invention provides methods of
screening for pharmaceutical agents, which comprise contacting a
test substance with cancer stem cells that differ in the
proliferation ability, which are induced by the methods provided by
the present invention. Specifically, the present invention provides
methods of screening for pharmaceutical agents, which comprises
detecting a change in a biological property of cancer stem cells by
contacting a test substance with high or low proliferative cancer
stem cells induced by a method for converting low proliferative
cancer stem cells to high proliferative cancer stem cells, or
converting high proliferative cancer stem cells to low
proliferative cancer stem cells.
[0187] Specifically, as described herein, low proliferative cancer
stem cells can be prepared by maintaining high proliferative cancer
stem cells under various stresses such as a suspension culture or
in contact with a growth inhibitor. For example, high proliferative
cancer stem cells can be converted to low proliferative cancer stem
cells by culturing high proliferative cancer stem cells under
suspension culture conditions. Alternatively, high proliferative
cancer stem cells can be converted to low proliferative cancer stem
cells by culturing high proliferative cancer stem cells in low
adherent or ultra low adherent cell culture vessels such as low
adherent plates, ultra low adherent plates, low adherent dishes,
ultra low adherent dishes, low adherent flasks, or ultra low
adherent cell culture flasks. In other words, low proliferative
cancer stem cells can be prepared by culturing high proliferative
cancer stem cells in low adherent or ultra low adherent cell
culture vessels such as low adherent plates, ultra low adherent
plates, low adherent dishes, ultra low adherent dishes, low
adherent flasks, or ultra low adherent cell culture flasks.
[0188] In a non-limiting embodiment, high proliferative cancer stem
cells can be converted to low proliferative cancer stem cells by
using a growth inhibitor such as 5-FU or irinotecan. Specifically,
low proliferative cancer stem cells can be produced by exposing
high proliferative cancer stem cells to a growth inhibitor such as
5-FU or irinotecan. Exposure to a growth inhibitor can be achieved
under any condition such as in vitro culture or inside the body of
grafted nonhuman animals. In this case, those skilled in the art
can select an appropriate exposure dose of a cell growth inhibitor
for cancer stem cells. Alternatively, high proliferative cancer
stem cells can be prepared by re-seeding low proliferative cancer
stem cells into a medium without a growth inhibitor such as 5-FU or
irinotecan. In another non-limiting embodiment, high proliferative
cancer stem cells can be produced by discontinuing administration
of a growth inhibitor to nonhuman animals having low proliferative
cancer stem cells.
[0189] Furthermore, low proliferative cancer stem cells can be
cultured under adherent culture conditions to convert them to high
proliferative cancer stem cells. Alternatively, low proliferative
cancer stem cells can be converted to high proliferative cancer
stem cells by culturing low proliferative cancer stem cells in a
non-low-adherent but highly adherent cell culture vessel such as a
flat-bottomed plate, plate, adherent culture plate, adherent
culture flask, dish, or adherent culture dish. That is, high
proliferative cancer stem cells can be produced by culturing low
proliferative cancer stem cells in a non-low-adherent but highly
adherent cell culture vessel such as a flat-bottomed plate, plate,
adherent culture plate, adherent culture flask, dish, or adherent
culture dish.
[0190] The present invention also relates to cancer cell detection
reagents. Cancer cell detection reagents of the present invention
preferably contain as an active ingredient at least one antibody
that binds to a protein of SEQ ID NOs: 1 to 8 (protein composed of
the amino acid sequence of any one of SEQ ID NOs: 1 to 8). In
another embodiment, the reagents of the present invention include
reagents for detecting Lgr5-positive cancer cells, which preferably
contain at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 6 (protein composed of the amino acid sequence of any one
of SEQ ID NOs: 1 to 6). In still another embodiment, the reagents
of the present invention include reagents for detecting
Lgr5-negative cancer cells, which preferably contain at least one
antibody that binds to the protein of any one of SEQ ID NOs: 1 to 8
(protein composed of the amino acid sequence of any one of SEQ ID
NOs: 1 to 8).
Growth Inhibitors
[0191] In a non-limiting embodiment, preferred growth inhibitors
include DNA-damaging agents, antimitotic agents, and/or
anti-metabolites. Such a DNA-damaging agent may be an alkylating
reagent, a topoisomerase inhibitor, and/or a DNA intercalator.
Examples of preferred growth inhibitors include, but are not
limited to, carboplatin (DNA alkylating reagent), etoposide
(topoisomerase II inhibitor), doxorubicin (DNA intercalator),
docetaxel (antimitotic agent), and Gemzar (gemcitabine;
anti-metabolite).
[0192] Alkylating reagents can be at least one reagent selected
from the following. Specifically, it is possible to use at least
one alkylating reagent selected from: chlorambucil,
cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil
mustard, thiotepa, busulfan, carmustine, lomustine, streptozocin,
carboplatin, cisplatin, satraplatin, oxaliplatin, altretamine,
ET-743, XL119 (becatecarin), dacarbazine, chlormethine,
bendamustine, trofosfamide, uramustine, fotemustine, nimustine,
prednimustine, ranimustine, semustine, nedaplatin, triplatin
tetranitrate, mannosulfan, treosulfan, temozolomide, carboquone,
triaziquone, triethylene melamine, procarbazine, etc.
[0193] Topoisomerase inhibitors can be at least one inhibitor
selected from the following. Specifically, it is possible to use at
least one topoisomerase inhibitor selected from: doxorubicin
(Doxil), daunorubicin, epirubicin, idarubicin, anthracenedione
(Novantrone), mitoxantrone, mitomycin C, bleomycin, dactinomycin,
plicatomycin, irinotecan (Camptosar), camptothecin, rubitecan,
belotecan, etoposide, teniposide, topotecan (Hycamptin), etc.
[0194] At least one topoisomerase inhibitor selected from the
following can be used as a DNA intercalator: proflavin, doxorubicin
(adriamycin), daunorubicin, dactinomycin, thalidomide, etc.
[0195] Antimitotic agents can be at least one agent selected from
the following. Specifically, it is possible to use at least one
topoisomerase inhibitor selected from: paclitaxel (Abraxane)/Taxol,
docetaxel (Taxotere), BMS-275183, Xyotax, Tocosal, vinorlebine,
vincristine, vinblastine, vindesine, vinzolidine, etoposide
(VP-16), teniposide (VM-26), ixavepilone, larotaxel, ortataxel,
tesetaxel, ispinesib, etc.
[0196] Anti-metabolites can be at least one inhibitor selected from
the following. Specifically, it is possible to use at least one
topoisomerase inhibitor selected from: fluorouracil (5-FU),
floxuridine (5-FUdR), methotrexate, Xeloda, Arranon, leucovorin,
hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine,
pentostatin, fludarabine phosphate, cladribine (2-CDA),
asparaginase, gemcitabine, pemetrexed, bortezomib, aminopterin,
raltitrexed, clofarabine, enocitabine, sapacitabine, azacytidine,
etc.
[0197] Furthermore, the present invention relates to methods of
screening for anti-cancer drugs, which use cancer stem cells
isolated or induced by the above methods of the present
invention.
[0198] The present invention also relates to methods for assessing
compounds, which use cancer stem cells isolated or induced by the
above methods of the present invention.
<Method for Detecting Cancer Stem Cells>
[0199] Furthermore, the present invention provides methods for
detecting, identifying, or quantifying the presence of cancer stem
cells of the present invention. Specifically, the present invention
provides methods for detecting, identifying, or quantifying the
presence of cancer stem cells or substantially homogeneous cancer
stem cell populations of the present invention, which comprise the
steps of:
(a) preparing a sample obtained from a cancer patient; and (b)
contacting a sample with an anti-Lgr5 antibody.
[0200] In these methods, first, samples obtained from cancer
patients are prepared. In the present invention, a "sample" is not
particularly limited as long as it is preferably an organ or tissue
derived from a cancer patient. It is possible to use a frozen or
unfrozen organ or tissue. Such samples include, for example, cancer
(tumor) tissues isolated from cancer patients. In these methods, a
sample is then contacted with an anti-Lgr5 antibody.
[0201] Methods for detecting, identifying, or quantifying the
presence of above-described cancer stem cells or substantially
homogeneous cancer stem cell populations of the present invention
can be used in, for example, cancer diagnosis, selection of cancer
patients, prediction or assessment of the effectiveness of an agent
(pharmaceutical composition), treatment monitoring, and cancer
imaging.
[0202] Specifically, for example, organs or tissues are isolated
from cancer patients, and specimens are prepared. The specimens can
be used to detect, identify, or quantify the presence of cancer
stem cells. Specimens can be appropriately prepared by using known
methods, for example, the PFA-AMeX-Paraffin method (WO 09/078,386).
The samples include, for example, frozen or unfrozen organs or
tissues. First, samples from cancer patients are fixed in a PFA
solution. "PFA solution" refers to a cell fixation solution which
is an aqueous solution of 1 to 6% paraformaldehyde combined with a
buffer such as phosphate buffer. It is preferable to use 4% PFA
fixation solution (4% paraformaldehyde/0.01 M PBS (pH7.4)). For
fixation with a PFA fixation solution, organs or tissues of
interest are immersed in a PFA solution containing 1 to 6%,
preferably 4% paraformaldehyde, at 0 to 8.degree. C., preferably at
about 4.degree. C., for 2 to 40 hours, preferably for 6 to 30
hours. Then, fixed organs or tissues are washed with phosphate
buffered saline or such. Washing may be carried out after excising
portions from the observed organs or tissues.
[0203] Organs or tissues thus prepared are then embedded in
paraffin by the AMeX method. The AMeX method is a paraffin
embedding method with a series of the following steps: cold acetone
fixation, dehydration with acetone, clearing in methylbenzoate and
xylene, and paraffin embedding. Specifically, tissues are immersed
in acetone at -25 to 8.degree. C., preferably at -20 to 6.degree.
C., for 2 to 24 hours, preferably for 4 to 16 hours. Then, the
tissues in acetone are warmed to room temperature. Alternatively,
organs or tissues are transferred into acetone at room temperature.
Then, dehydration is performed for 0.5 to 5 hours, preferably 1 to
4 hours at room temperature. Subsequently, the organs or tissues
are cleared by immersion in methylbenzoate at room temperature for
0.5 to 3 hours, preferably for 0.5 to 2 hours, followed by
immersion in xylene at room temperature for 0.5 to 3 hours,
preferably 0.5 to 2 hours. Next, the organs or tissues are embedded
in paraffin by penetration at 55 to 65.degree. C., preferably at 58
to 62.degree. C. for 1 to 4 hours, preferably for 1 to 3 hours. The
paraffin blocks of organs or tissues prepared by the PFA-AMeX
method are stored at low temperature before use.
[0204] At the time of use, the paraffin blocks thus prepared are
sliced into thin sections using a microtome or the like. Then, the
thin sections are deparaffinized and rehydrated. Deparaffinization
and rehydration can be performed by known methods. For example,
deparaffinization can be performed using xylene and toluene, while
rehydration can be carried out using alcohol and acetone.
[0205] The resulting thin sections are stained, for example, by
histochemistry, immunohistochemistry, or enzyme histochemistry for
detection, identification, or quantitation.
[0206] When the prepared samples are stained by histochemistry
(special staining), it is possible to use any staining method
commonly available for paraffin-embedded sections (for example, PAS
staining, giemsa staining, and toluidine blue staining). For
staining by enzyme histochemistry, the sections may be stained by
any staining method available for sections (for example, various
staining such as with ALP, ACP, TRAP, or esterase). In addition,
histopathological tissues can be stained by the following:
hematoxylin-eosin staining for general staining; van Gieson
staining, azan staining, and Masson Trichrome staining for collagen
fiber staining; Weigert staining and Elastica van Gieson staining
for elastic fiber staining; Watanabe's silver impregnation staining
and PAM staining (periodic acid methenamine silver stain) for
reticular fibers/basal membrane staining, etc.
[0207] Staining with immunohistochemistry and enzyme histochemistry
can be performed by direct methods using primary antibodies labeled
with an enzyme or labeling substance, or indirect methods using
non-labeled primary antibodies and labeled secondary antibodies.
However, such methods are not limited thereto. Antibodies can be
labeled by conventional methods. Labeling substances include, for
example, radioisotopes, enzymes, fluorescent substances, and
biotin/avidin. The labeling substances may be those commercially
available. Radioisotopes include, for example, .sup.32P, .sup.33P,
.sup.131I, .sup.125I, .sup.3H, .sup.14C, and .sup.35S. Enzymes
include, for example, alkaline phosphatase, horse radish
peroxidase, .beta.-galactosidase, and .beta.-glucosidase.
Fluorescent substances include, for example, fluorescein
isothiocyanate (FITC) and rhodamine. These may be commercially
available. Labeling can be carried out by known methods.
[0208] Thin sections are stained, for example, by histochemistry,
immunohistochemistry, or enzyme histochemistry for detection,
identification, or quantitation.
[0209] Alternatively, detection, identification, or quantitation
can be carried out by quantifying DNA or RNA in cells in
organ/tissue samples. Assessment of the expression is not
particularly limited, and conventional expression assessment
methods can be used. RNAs include microRNAs, siRNAs, tRNAs, snRNAs,
mRNAs, and non-coding RNAs. For example, Lgr5 mRNA is extracted
according to conventional methods. Using the mRNA as a template,
the transcriptional level of each gene can be determined by the
Northern hybridization or RT-PCR method. DNA array techniques can
also be used to determine the expression level of Lgr5.
[0210] Desired tissues, cells, or such can be collected from
samples by the microdissection method, in particular, laser
microdissection (LMD) method. The LMD method can collect a group of
target cells from living tissues, and thus accurately determine
which cells express a specific gene among various cells that
constitute a tissue, and at what level the cells express the gene.
Devices used for microdissection include, for example, the AS-LMD
system (Leica Microsystems).
[0211] Furthermore, the present invention provides methods for
diagnosing cancer, detecting cancer stem cells, or selecting cancer
patients, which comprise using at least one antibody that binds to
a protein of SEQ ID NOs: 1 to 8 to detect the presence of at least
one of the proteins in a sample isolated from a cancer patient. In
order to detect the presence of cancer stem cells, it is possible
to use, instead of the anti-Lgr5 antibody described above, at least
one antibody that binds to a protein of SEQ ID NOs: 1 to 8.
[0212] In a non-limiting embodiment, the present invention provides
methods for diagnosing cancer, detecting cancer stem cells, or
selecting cancer patients, which comprise using at least one
antibody that binds to a protein of SEQ ID NOs: 1 to 6 to detect
the presence of at least one of the proteins in a sample isolated
from a cancer patient. In order to detect the presence of
Lgr5-positive cancer stem cells, it is possible to use, instead of
the anti-Lgr5 antibody described above, at least one antibody that
binds to a protein of SEQ ID NOs: 1 to 6. The presence of
Lgr5-positive cancer stem cells can be detected by detecting the
presence of the protein. However, the present invention does not
exclude detection of Lgr5 in addition to the protein.
[0213] In a non-limiting embodiment, the present invention provides
methods for diagnosing cancer, detecting cancer stem cells, or
selecting cancer patients, which comprise using at least one
antibody that binds to a protein of SEQ ID NOs: 1 to 8 to detect
the presence of at least one of the proteins in a sample isolated
from a cancer patient. In order to detect the presence of
Lgr5-negative cancer stem cells, it is possible to use, instead of
the anti-Lgr5 antibody described above, at least one antibody that
binds to a protein of SEQ ID NOs: 1 to 8. The presence of
Lgr5-negative cancer stem cells can be detected by detecting the
presence of the protein. However, the present invention does not
exclude detection of Lgr5 in addition to the protein.
[0214] Furthermore, the present invention provides methods for
assessing the effectiveness of a pharmaceutical composition
comprising at least one antibody that binds to a protein of SEQ ID
NOs: 1 to 8, which comprise detecting one or more of the proteins
of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding the proteins
in a sample isolated from a subject administered with the
pharmaceutical composition. In these methods, detection may be
carried out using at least one antibody that binds to a protein of
SEQ ID NOs: 1 to 8, or a portion of a polynucleotide encoding the
protein of any one of SEQ ID NOs: 1 to 8 and/or a complementary
strand thereof. In another non-limiting embodiment, the present
invention provides methods for assessing the effectiveness of a
cancer treatment in a test subject, which comprise comparing the
expression of at least one of the proteins of SEQ ID NOs: 1 to 8
and/or polynucleotides encoding the proteins in a first sample
obtained from a test subject before providing the subject with at
least part of the treatment, to the expression of at least one of
the proteins of SEQ ID NOs: 1 to 8 and/or polynucleotides encoding
the proteins in a second sample obtained from the subject after
providing the part of the treatment, wherein a significantly lower
level of the protein and/or polynucleotide in the second sample
than in the first sample is an indicator showing that the treatment
is effective for inhibiting cancer in the test subject.
[0215] In a non-limiting embodiment, the present invention provides
methods of monitoring in a test subject the effectiveness of a
treatment with an antibody provided by the present invention, which
comprise the steps of:
(i) collecting a pre-administration sample from the subject before
administration of the antibody; (ii) determining the expression
level of at least one marker protein selected from the proteins of
SEQ ID NOs: 1 to 8, or an mRNA or genomic DNA thereof in the
pre-administration sample; (iii) collecting one or more
post-administration sample(s) from the subject; (iv) determining
the expression or activity level of at least one marker protein
selected from the proteins of SEQ ID NOs: 1 to 8, or an mRNA or
genomic DNA thereof in the post-administration sample(s); (v)
comparing the expression or activity level of the marker protein,
mRNA, or genomic DNA in the pre-administration sample to that of
the marker protein, mRNA, or genomic DNA in the post-administration
sample(s); and (vi) modifying the antibody administration to the
test subject according to the comparison.
[0216] For example, an increased dosage of an antibody of the
present invention can be used to reduce the expression or activity
of a marker towards a level higher than the detected level (the
expression or activity level of the marker protein, mRNA, or
genomic DNA in the sample before administration), i.e., to increase
the effectiveness of the antibody.
[0217] In a different non-limiting embodiment, the present
invention provides methods of monitoring in a test subject the
effectiveness of a treatment with an antibody provided by the
present invention, which comprise the steps of:
(i) detecting an Lgr5-positive cancer stem cell in a
pre-administration sample collected from the subject before
administration of the antibody; (ii) determining the expression
level of at least one marker protein selected from the proteins of
SEQ ID NOs: 1 to 6, or mRNA or genomic DNA thereof in the
pre-administration sample; (iii) collecting one or more
post-administration sample(s) from the subject; (iv) determining
the expression or activity level of at least one marker protein
selected from the proteins of SEQ ID NOs: 1 to 6, or mRNA or
genomic DNA thereof in the post-administration sample(s); (v)
comparing the expression or activity level of the marker protein,
mRNA, or genomic DNA in the pre-administration sample to that of
the marker protein, mRNA, or genomic DNA in the post-administration
sample(s); and (vi) modifying the antibody administration to the
test subject according to the comparison.
[0218] For example, an increased dosage of an antibody of the
present invention can be used to reduce the expression or activity
of a marker towards a level higher than the detected level (the
expression or activity level of the marker protein, mRNA, or
genomic DNA in the sample before administration), i.e., to increase
the effectiveness of the antibody.
[0219] In another non-limiting embodiment, the present invention
provides methods of monitoring in a test subject the effectiveness
of a treatment with an antibody provided by the present invention,
which comprise the steps of:
(i) detecting an Lgr5-negative cancer stem cell in a
pre-administration sample collected from the subject before
administration of the antibody; (ii) determining the expression
level of at least one marker protein selected from the proteins of
SEQ ID NOs: 1 to 8, or mRNA or genomic DNA thereof in the
pre-administration sample; (iii) collecting one or more
post-administration sample(s) from the subject; (iv) determining
the expression or activity level of at least one marker protein
selected from the proteins of SEQ ID NOs: 1 to 8, or mRNA or
genomic DNA thereof in the post-administration sample(s); (v)
comparing the expression or activity level of the marker protein,
mRNA, or genomic DNA in the pre-administration sample to that of
the marker protein, mRNA, or genomic DNA in the post-administration
sample(s); and (vi) modifying the antibody administration to the
test subject according to the comparison.
[0220] For example, an increased dosage of an antibody of the
present invention can be used to reduce the expression or activity
of a marker towards a level higher than the detected level (the
expression or activity level of the marker protein, mRNA, or
genomic DNA in the sample before administration), i.e., to increase
the effectiveness of the antibody.
<Cancer Stem Cell Inhibitors>
[0221] "Cancer stem cell inhibitor" refers to, for example, an
agent having the effect of suppressing the proliferation of cancer
stem cells, suppressing the metastasis or recurrence of cancer stem
cells, killing cancer stem cells, etc. It may have the effect of
suppressing the proliferation of cancer cells, suppressing the
metastasis or recurrence of cancer cells, killing cancer cells,
etc.
[0222] When used in connection with a biological activity, whose
non-limiting examples include the proliferation or metastasis of
cancer stem cells, the terms "suppress" and "suppressing", and
synonymous expressions refer to the down-regulation of the
biological activity. This can reduce or eliminate a target function
such as protein production and phosphorylation of a molecule, etc.
In a specific embodiment, the suppression means a decrease in a
target activity by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
95%. When used in connection with a disorder or disease, the terms
refer to the prevention of development of a symptom, relief of a
symptom, or successful alleviation of a disease, condition, or
disorder.
[0223] "Metastasis" refers to a process where cancer spreads or
moves from the primary site to another site in the body, resulting
in the development of similar cancer lesions at the new site.
"Metastatic cell" or "metastasizing cell" refers to a cell that
loses the adhesive contact with adjacent cells, and leaves the
primary site of the disease and invades a nearby body structure via
blood or lymphatic circulation. "Recurrence" means that the same
malignant tumor reappears in a remaining organ after partial
resection of an organ for removing a malignant tumor from a cancer
patient, or after postoperative chemotherapy following the
resection.
<Proteins>
[0224] Proteins for use in the present invention can be easily
prepared by any method known to those skilled in the art as
follows. An expression vector containing a gene comprising a DNA
encoding a protein is constructed. The protein is produced and
accumulated by culturing transformants transformed with the
expression vector. The transformants are harvested to prepare the
protein.
[0225] Such an expression vector can be constructed according to
methods known in the art, for example, by the following:
(1) excising a DNA fragment that comprises a gene comprising a DNA
encoding a protein; and (2) ligating the DNA fragment downstream of
a promoter in an appropriate expression vector.
[0226] Such vectors used include E. coli-derived plasmids (for
example, pBR322, pBR325, pUC18, and pUC118), Bacillus
subtilis-derived plasmids (for example, pUB110, pTP5, and pC194),
yeast-derived plasmids (for example, pSH19 and pSH15),
bacteriophages such as .lamda. phage, and animal viruses such as
retroviruses, vaccinia viruses, and Baculoviruses.
[0227] Promoters for use in the present invention may be any
promoters as long as they are appropriate and compatible with a
host to be used for gene expression. For example, when the host is
E. coli, preferred promoters include the trp promoter, lac
promoter, recA promoter, .lamda.PL promoter, and lpp promoter. When
the host is Bacillus subtilis, preferred promoters include the SPO1
promoter, SPO2 promoter, and penP promoter. When the host is yeast,
preferred promoters include the PHOS promoter, PGK promoter, GAP
promoter, and ADH promoter. When the host is an animal cell,
promoters include the SR.alpha. promoter, SV40 promoter, LTR
promoter, CMV promoter, and HSV-TK promoter.
[0228] In addition to those described above, if desired, enhancers,
splicing signals, poly-A addition signals, selection markers, SV40
replication origins, or such known in the art can be added to
expression vectors. As necessary, a protein for use in the present
invention can be expressed as a fusion protein with another protein
(for example, glutathione-S-transferase or Protein A). Such a
fusion protein can be cleaved into individual proteins by using an
appropriate protease.
[0229] Host cells include, for example, bacteria of the genus
Escherichia, bacteria of the genus Bacillus, yeasts, insect cells,
insects, and animal cells.
[0230] Specific examples of bacteria of the genus Escherichia
include Escherichia coli K12 DH1 (Proc. Natl. Acad. Sci, USA, 60,
160 (1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221
(Journal of Molecular Biology, 120, 517 (1978)), and HB101 (Journal
of Molecular Biology, 41, 459 (1969)).
[0231] Bacteria of the genus Bacillus include, for example,
Bacillus subtilis MI114 (Gene, 24, 255 (1983)) and 207-21 (Journal
of Biochemistry, 95, 87 (1984)).
[0232] Yeasts include, for example, Saccharomyces cerevisiae AH22,
AH22R-, NA87-11A, DKD-5D, and 20B-12; Schizosaccaromyces pombe
NCYC1913 and NCYC2036; and Pichia pastoris.
[0233] Animal cells include, for example, monkey COS-7 cells, Vero
cells, Chinese hamster CHO cells (hereinafter abbreviated as CHO
cells), dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20
cells, mouse myeloma cells, rat GH3 cells, and human FL cells.
[0234] These host cells can be transformed according to methods
known in the art. See, for example, the following references.
Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972); Gene, Vol. 17,
107 (1982); Molecular & General Genetics, Vol. 168, 111 (1979);
Methods in Enzymology, Vol. 194, 182-187 (1991); Proc. Natl. Acad.
Sci. USA), Vol. 75, 1929 (1978); and Virology, Vol. 52, 456
(1973).
[0235] Transformants thus prepared can be cultured according to
methods known in the art.
[0236] For example, when hosts were bacteria of the genus
Escherichia, in general, they are cultured at about 15 to
43.degree. C. for about 3 to 24 hours. Aeration or stirring is
performed as necessary. When hosts are bacteria of the genus
Bacillus, in general, they are cultured at about 30 to 40.degree.
C. for about 6 to 24 hours. Aeration or stirring is performed as
necessary
[0237] When hosts are yeasts, in general, transformants are
cultured at about 20.degree. C. to 35.degree. C. for about 24 to 72
hours in a medium adjusted to about pH 5 to 8. Aeration or stirring
is performed as necessary.
[0238] When hosts are animal cells, in general, transformants are
cultured at about 30.degree. C. to 40.degree. C. for about 15 to 60
hours in a medium adjusted to about pH 6 to 8. Aeration or stirring
is performed as necessary.
[0239] To isolate and purify a protein for use in the present
invention from the above culture, for example, cells or bacteria
are harvested after culture by a known method, and this is
suspended in an appropriate buffer. After disrupting the cells or
bacteria by sonication, lysozyme, and/or freeze-thawing, a crude
protein extract is prepared by centrifugation or filtration. The
buffer may contain protein denaturants such as urea and guanidine
hydrochloride, and detergents such as Triton X-100.TM.. When the
protein is secreted to the culture medium, the supernatant is
separated from the cells or bacteria after culture by a known
method, and the supernatant is collected. A protein contained in
the resulting culture supernatant or extract can be purified by
appropriately combining known isolation/purification methods.
[0240] According to known or equivalent methods, a protein prepared
as described above can be arbitrarily modified or a polypeptide can
be partially removed from the protein by treating the protein
produced by recombinants with an appropriate protein modification
enzyme such as trypsin and chymotrypsin before or after
purification.
[0241] The presence of a protein for use in the present invention
can be assessed by various binding assays, enzyme immunoassays
using specific antibodies, etc.
<Antibodies>
[0242] Antibodies for use in the present invention are not
particularly limited as long as they bind to proteins for use in
the present invention. The antibodies may be obtained as polyclonal
or monoclonal antibodies using known methods. Particularly
preferred antibodies for use in the present invention include
monoclonal antibodies derived from mammals Monoclonal antibodies
derived from mammals include those produced by hybridomas and those
produced by hosts transformed with expression vectors carrying
antibody genes using gene engineering technologies. It is
preferable that antibodies for use in the present invention
specifically bind to proteins for use in the present invention.
[0243] Basically, hybridomas producing monoclonal antibodies can be
prepared using known techniques by the following procedure.
Specifically, immunization is carried out using as a sensitizing
antigen a protein for use in the present invention according to
conventional immunization methods. The resulting immune cells are
fused with known parental cells by conventional cell fusion
methods. Monoclonal antibody-producing cells are screened using
conventional screening methods. More specifically, monoclonal
antibodies can be prepared by the following procedure.
[0244] A gene sequence encoding the protein is inserted into a
known expression vector system, and this is transformed into
appropriate host cells. Then, the protein is purified from the host
cells or culture supernatant by known methods.
[0245] Next, the protein is used as a sensitizing antigen.
Alternatively, a partial peptide of the protein is used as a
sensitizing antigen. In this case, the partial peptide can be
prepared by chemical synthesis based on the amino acid sequence of
the protein according to common methods known to those skilled in
the art.
[0246] Such a partial polypeptide of the protein has, for example,
at least 10 or more amino acids, preferably 50 or more amino acids,
more preferably 70 or more amino acids, still more preferably 100
or more amino acids, and yet more preferably 200 or more amino
acids of the amino acid sequence constituting the protein, and has,
for example, a biological activity substantially equivalent to the
function of the protein. The C terminus of the partial peptide is
generally a carboxyl group (--COOH) or carboxylate (--COO--);
however, the C terminus may also be amide (--CONH.sub.2) or ester
(--COOR). In addition, the partial peptides include those in which
the amino group of the N-terminal methionine residue is protected
with a protecting group, those in which a glutamyl residue
resulting from in vivo N-terminal cleavage is
pyroglutamine-oxidized, those in which a substituent group in the
side chain of an amino acid in the molecule is protected with an
appropriate protecting group, and conjugated peptides such as
so-called glycopeptides linked with sugar chains
[0247] Mammals that are immunized with a sensitizing antigen are
not particularly limited, though it is preferable to take into
consideration compatibility with the parent cell used for cell
fusion. Thus, rodents such as mice, rats, or hamsters are generally
selected.
[0248] Immunization of animals with a sensitizing antigen is
performed according to known methods. For example, standard methods
of delivering sensitizing antigen to mammals involve
intraperitoneal or subcutaneous injection. More specifically, a
sensitizing antigen is diluted to be an appropriate volume with PBS
(phosphate-buffered saline), physiological saline, or the like. If
desired, this may be mixed with an appropriate amount of a typical
adjuvant, for example, Freund's complete adjuvant, made into an
emulsion, and then administered to mammals several times every 4 to
21 days. An appropriate carrier may also be used for immunization
with sensitizing antigens.
[0249] After the mammals are immunized as described above, an
increase in the level of desired antibody in the serum is
confirmed, immunocytes are collected from the mammals for cell
fusion. Immunocytes that are preferably subjected to cell fusion
are splenocytes in particular.
[0250] Regarding the other parent cell to be fused with the
above-mentioned immunocytes, mammalian myeloma cells are used. For
myeloma cells, it is preferable to use various known cell lines,
for example, P3 (P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550),
P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)
81, 1-7), NS-1 (Kohler, G. and Milstein, C. Eur. J. Immunol. (1976)
6, 511-519), MPC-11 (Margulies, D. H. et al., Cell (1976) 8,
405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270),
F0 (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35,
1-21), S194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323),
and 8210 (Galfre, G et al., Nature (1979) 277, 131-133).
[0251] In general, the above-described immunocytes and myeloma
cells can be fused according to known methods, examples of which
are described by Kohler and Milstein et al. (Kohler, G. and
Milstein, C., Methods Enzymol. (1981) 73, 3-46).
[0252] More specifically, the above-described cell fusion is
carried out, for example, in a typical nutrient culture medium in
the presence of a cell fusion promoting agent. For example,
polyethylene glycol (PEG), Sendai virus (HVJ), or such can be used
as the fusion promoting agent. If desired, adjuvants such as
dimethylsulfoxide can additionally be used to increase fusion
efficiency.
[0253] It is possible to arbitrarily determine the proportion of
immunocytes and myeloma cells used. The preferred ratio of myeloma
cells to immunocytes is, for example, from 1:1 to 1:10. The culture
medium used for the above-described cell fusion may be, for
example, RPMI1640 medium, MEM medium, which are suitable for
proliferation of the above-described myeloma cell lines, or other
kinds of culture medium commonly used for culturing such cells.
Furthermore, serum supplements such as fetal calf serum (FCS) may
be used in combination.
[0254] The cell fusion is carried out by thoroughly mixing
prescribed amounts of the above-described immunocytes and myeloma
cells in the aforementioned culture medium, adding to the medium a
PEG solution preheated to about 37.degree. C. generally at a
concentration of 30% to 60% (w/v), wherein the PEG has an average
molecular weight of about 1,000 to 6,000, for example, and mixing
them to form the desired fusion cells (hybridomas). An appropriate
culture medium is then successively added. Cell fusing agents and
such that are undesirable for the proliferation of hybridomas are
removed by repeatedly removing the supernatant by
centrifugation.
[0255] The hybridomas obtained in this manner are selected by
culturing them in a common selection culture medium, for example,
the HAT medium (a culture medium containing hypoxanthine,
aminopterin, and thymidine). Culture in the HAT medium described
above is continued for a sufficient time, usually from a few days
to a few weeks, to allow death of all cells but the target
hybridomas (the non-fused cells). The usual limiting dilution
method is then performed to screen and clone hybridomas producing
antibodies used the present invention.
[0256] In addition to methods obtaining the above-described
hybridomas by immunizing non-human animals with an antigen, desired
human antibodies having an activity of binding to the protein can
also be obtained by in vitro sensitizing human lymphocytes with the
protein and fusing the sensitized lymphocytes with human-derived
myeloma cells having permanent cell division ability (see Japanese
Patent Application Kokoku Publication No. (JP-B) H01-59878
(examined, approved Japanese patent application published for
opposition)). Furthermore, human antibodies against a protein may
be obtained from immortalized antibody-producing cells that are
prepared by administering the protein as an antigen to a transgenic
animal having a full repertoire of human antibody genes (see,
International Patent Applications WO 94/25585, WO 93/12227, WO
92/03918, and WO 94/02602).
[0257] There are known techniques for obtaining human antibodies by
panning using a human antibody library. For example, the V regions
of human antibodies can be expressed as single-chain antibodies
(scFvs) on the surface of phages using a phage display methods,
from which phages presenting scFv that binds to an antigen can be
selected. The DNA sequences encoding the V regions of human
antibodies that bind to the antigen can be determined by analyzing
the genes of selected phages. After identifying the DNA sequences
of scFvs that bind to the antigen, the V region sequences are fused
in frame with the C region sequences of a desired human antibody.
Then, the resulting DNA is inserted into an appropriate expression
vector to construct an expression vector. The expression vector is
introduced into suitable cells for expression, such as those
described above. The human antibody can be obtained by expressing
the gene encoding the human antibody. These methods are already
known (see WO 1992/001047, WO 1992/020791, WO 1993/006213, WO
1993/011236, WO 1993/019172, WO 1995/001438, and WO
1995/015388).
[0258] The hybridomas prepared in this manner that produce
monoclonal antibodies can be passaged in a common culture medium
and stored for a long time in liquid nitrogen.
[0259] Monoclonal antibodies may be obtained from the hybridomas
using common techniques; for example, the hybridomas are cultured
according to standard methods and the antibodies may be obtained
from the culture supernatants. Alternatively, the hybridomas are
administered to a compatible mammal for proliferation and then the
antibodies may be obtained from the ascites fluid. The former
method is suitable for obtaining highly pure antibodies, while the
latter method is suitable for mass production of antibodies.
[0260] Monoclonal antibodies used in the present invention may be
recombinant antibodies produced by genetic engineering techniques.
They can be produced, for example, by cloning an antibody gene from
a hybridoma, incorporating the antibody gene into an appropriate
vector, and introducing the resulting vector into a host (see, for
example, Vandamme, A. M. et al., Eur. J. Biochem., (1990) 192, p.
767-775, 1990).
[0261] Specifically, mRNAs encoding antibody variable (V) regions
are isolated from hybridomas producing the antibodies. mRNAs can be
isolated by preparing total RNAs using known methods, for example,
guanidine-ultracentrifugation method (Chirgwin, J. M. et al.,
Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. et
al., Anal. Biochem. (1987) 162, 156-159), or such. mRNAs of
interest are prepared using the mRNA Purification Kit (Pharmacia)
or such. The mRNAs can be prepared directly by using the QuickPrep
mRNA Purification Kit (Pharmacia).
[0262] The obtained mRNAs are used to synthesize cDNAs of the
antibody V regions using reverse transcriptase. cDNAs are
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Co.) or such. Alternatively, cDNA may be
synthesized and amplified following the 5'-RACE method (Frohman, M.
A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002;
Belyaysky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932)
using the 5'-Ampli FINDER RACE Kit (Clontech) and PCR, and
such.
[0263] DNA fragments of interest are purified from the resulting
PCR products, and ligated to vector DNAs. From this, a recombinant
vector is produced. The recombinant vector is then introduced into
E. coli or such, and the desired recombinant vector is prepared
from a selected colony. The nucleotide sequences of DNAs of
interest are then determined by known methods, for example, the
dideoxynucleotide chain termination method.
[0264] A DNA encoding the antibody V region of interest is
obtained, and then incorporated into an expression vector carrying
a DNA that encodes a desired antibody constant region (C
region).
[0265] To produce an antibody used in the present invention, the
antibody gene is incorporated into an expression vector so that the
gene will be expressed under the control of an expression
regulatory region, for example, an enhancer and a promoter. Then,
host cells are transformed with the resulting expression vector to
express the antibody.
[0266] When expressing antibody genes, a DNA encoding an antibody
heavy chain (H chain) or light chain (L chain) can be each
separately incorporated into an expression vector to simultaneously
transform the host cell, or alternatively DNAs encoding H and L
chains can be incorporated into a single expression vector to
transform the host cells (see, WO 94/11523).
[0267] Besides the above-described host cells, transgenic animals
can also be used to produce recombinant antibodies. For example, an
antibody gene is prepared as a fusion gene by inserting the
antibody gene into a gene encoding a protein that is specifically
produced in milk, such as goat casein. DNA fragments containing the
fusion gene to which the antibody gene has been inserted is
injected into goat embryos, which are then introduced into female
goats. The desired antibody is then obtained from the milk produced
by the transgenic goats, which are born from the goats that
received the embryos, or from their offspring. Hormones may be
suitably given to the transgenic goat to increase the production of
milk containing the antibody of interests (Ebert, K. M. et al.,
Bio/Technology (1994) 12, 699-702).
[0268] In the present invention, in addition to the antibodies
described above, artificially modified genetically-recombinant
antibodies such as chimeric, humanized, and human antibodies can be
used to reduce heterologous antigenicity against humans and such.
Such modified antibodies can be produced using known methods.
Monoclonal antibodies of the present invention include not only
those derived from animals described above but also artificially
modified genetically-recombinant antibodies such as chimeric
antibodies, humanized antibodies, and bispecific antibodies.
[0269] A chimeric antibody can be obtained by linking a DNA
encoding the antibody V region obtained as described above to a DNA
encoding the human antibody C region, incorporating this into an
expression vector, and then introducing it into a host for
production. Useful chimeric antibodies can be obtained using this
known method.
[0270] Humanized antibodies are also referred to as "reshaped human
antibodies", which are antibodies obtained by grafting the
complementarity determining regions (CDRs) of an antibody from a
non-human mammal (e.g., mouse antibody) to the complementarity
determining regions of a human antibody. General gene recombination
procedures are also known (see, European Patent Application
Publication No. EP 125023; International Patent Application
Publication No. WO 96/02576).
[0271] Specifically, a DNA sequence designed to link a mouse
antibody CDR to the framework region (FR) of a human antibody is
synthesized by PCR, using as primers several oligonucleotides
prepared to contain overlapping portions in both CDR and FR
terminal regions (see methods described in WO 98/13388).
[0272] The human antibody framework region to be linked via CDR is
selected such that complementarity determining region forms a
favorable antigen-binding site. As necessary, amino acids of the
framework region in the antibody variable region may be substituted
so that the complementarity determining region of the reshaped
human antibody forms a suitable antigen-binding site (Sato, K. et
al., 1993, Cancer Res. 53, 851-856).
[0273] Human antibody C-regions are used as the C-regions of
chimeric antibodies or humanized antibodies. For example, CH1, CH2,
CH3, and CH4 can be used for the H chain, while C.kappa. and
C.lamda. can be used for the L chain. The human antibody C-region
may be modified in order to improve stability of the antibody or
its production.
[0274] A chimeric antibody is composed of the variable region of an
antibody derived from a non-human mammal and the constant region
derived from a human antibody. On the other hand, a humanized
antibody is composed of the complementarity determining region of
an antibody derived from a non-human mammal, and the framework
region and C region derived from a human antibody. Since the
antigenicity of humanized antibodies is low in the human body, and
humanized antibodies are useful as an active ingredient in
therapeutic agents of the present invention.
[0275] Antibodies used in the present invention are not limited to
whole antibody molecules, and as long as they bind to proteins used
in the present invention, antibody fragments and modification
products thereof as well as divalent and monovalent antibodies are
also included. Antibody fragments include, for example, Fab,
F(ab')2, Fv, Fab/c having an Fab and the whole Fc, single chain Fv
(scFv) in which Fv fragments from H and L chains are ligated via an
appropriate linker, and Diabody. Specifically, antibody fragments
are prepared by treating antibodies with an enzyme, for example,
papain or pepsin. Alternatively, after genes encoding such antibody
fragments are constructed and introduced into an expression vector,
the antibody fragments are expressed in appropriate host cells
using the vector (see, for example, Co, M. S. et al., J. Immunol.
(1994) 152, 2968-2976; Better, M. & Horwitz, A. H. Methods in
Enzymology (1989) 178, 476-496, Academic Press, Inc.; Plueckthun,
A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496,
Academic Press, Inc.; Lamoyi, E., Methods in Enzymology (1989) 121,
652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121,
663-669; Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
[0276] scFv is obtained by ligating antibody H-chain V region with
an antibody L-chain V region. In this scFv, the H-chain and L-chain
V regions are ligated via a linker, preferably via a peptide linker
(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,
5879-5883). The H-chain V region and L-chain V region of an scFv
may be derived from any of the antibodies described herein. For
example, any single-chain peptides consisting of 12 to 19 amino
acid residues such as (GGGGS)n may be used as a peptide linker for
ligating the V regions.
[0277] A DNA encoding an scFv can be obtained by using, among DNAs
encoding the antibody H chain or H chain V region and the antibody
L chain or L chain V region mentioned above, all or DNA portion
encoding amino acid sequence of interest as a template, amplifying
by PCR using a primer pair that defines its two ends; and then
carrying out a subsequent amplification using a combination of a
DNA encoding the peptide linker portion, and primer pairs that
define both ends of the linker DNA to be ligated to the H chain and
L chain, respectively.
[0278] Once DNAs encoding scFvs are constructed, expression vectors
carrying the DNAs and hosts transformed with the expression vectors
can be obtained according to conventional methods. Furthermore,
scFvs can be obtained using these hosts according to conventional
methods.
[0279] Diabodies are dimers formed by linking two fragments (for
example, scFv) in which a variable region is linked to another
variable region via a linker or such, and typically have two VLs
and two VHs (P. Holliger et al., Proc. Natl. Acad. Sci. USA, 90,
6444-6448 (1993); EP 404097; WO 93/11161; Johnson et al., Method in
Enzymology, 203, 88-98, (1991); Holliger et al., Protein
Engineering, 9, 299-305 (1996); Perisic et al., Structure, 2,
1217-1226 (1994); John et al., Protein Engineering, 12(7), 597-604
(1999); Holliger et al., Proc. Natl. Acad. Sci. USA., 90, 6444-6448
(1993); Atwell et al., Mol. Immunol. 33, 1301-1312 (1996); and
such).
[0280] These antibody fragments can be produced, in a similar
manner as described above, by obtaining their genes and expressing
them in hosts. Herein, "antibody" comprises such antibody
fragments.
[0281] As modified antibodies, antibodies of the present invention
linked to various molecules such as polyethylene glycol (PEG) may
be used. Moreover, antibodies can also be linked to radioisotopes,
chemotherapeutic agents, cytotoxic substances such as
bacteria-derived toxins, or such. Herein, "antibody" includes such
modified antibodies. Modified antibodies can be obtained by
chemically modifying the prepared antibodies. Such antibody
modification methods are already established in the art.
[0282] Furthermore, antibodies used in the present invention may be
bispecific antibodies. Bispecific antibodies of the present
invention may be those having antigen-binding sites that each
recognizes different epitopes in the protein used in the present
invention or those which recognize the protein used in the present
invention and a different protein. Alternatively, bispecific
antibodies of the present invention may be those in which one
antigen-binding domain recognizes the protein used in the present
invention and the other recognizes a chemotherapeutic agent or a
cytotoxic substance such as a cell-derived toxin. In this case,
proliferation of cancer stem cells can be suppressed by allowing a
cytotoxic substance to act directly on cancer stem cells expressing
a protein used in the present invention and specifically damaging
the cancer stem cells. It is also possible to use bispecific
antibodies in which one antigen-binding domain recognizes a
molecule that constitutes the T cell receptor complex such as CD3,
expressing on cytotoxic T cells, and the other recognizes an
epitope in the protein of any one of SEQ ID NOs: 1 to 8 of the
present invention. The bispecific antibodies may be prepared by
linking pairs of H and L chains from two types of antibodies, or by
fusing hybridomas that produce different monoclonal antibodies to
yield a fusion cell producing bispecific antibodies. Furthermore,
the bispecific antibodies can be prepared using genetic engineering
techniques.
[0283] Antibody genes constructed as described above can be
expressed and obtained according to known methods. When mammalian
cells are used, antibody genes can be expressed using a DNA in
which a common useful promoter, an antibody gene to be expressed,
and a poly A signal positioned downstream of the antibody gene on
the 3' side are operably linked Promoter/enhancer includes, for
example, human cytomegalovirus immediate early
promoter/enhancer.
[0284] Furthermore, other promoter/enhancers that can be used to
express the antibody used in the present invention include viral
promoter/enhancers of retroviruses, polyoma viruses, adenoviruses,
simian virus 40 (SV40), and such; and mammalian cell-derived
promoter/enhancers such as human elongation factor 1.alpha.
(HEF1.alpha.).
[0285] When SV40 promoter/enhancer and HEF1.alpha.
promoter/enhancer is used, gene expression can be easily carried
out by the method of Mulligan et al. (Nature (1979) 277, 108) and
the method by Mizushima et al. (Nucleic Acids Res. (1990) 18,
5322), respectively.
[0286] Replication origin derived from SV40, polyoma viruses,
adenoviruses, bovine papilloma viruses (BPV), and such can be used.
Furthermore, to increase the gene copy number in a host cell
system, the expression vector may include, as a selection marker,
the aminoglycoside transferase (APH) gene, thymidine kinase (TK)
gene, E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt)
gene, dihydrofolate reductase (dhfr) gene, and such.
[0287] In the case of E. coli, the antibody gene can be expressed
by an operably linked common useful promoter, a signal sequence for
antibody secretion, and the antibody gene to be expressed. Such
promoters include, for example, the lacz promoter and araB
promoter. When the lacz promoter or araB promoter is used, the gene
can be expressed by the method of Ward et al. (Nature (1098) 341,
544-546; FASEB J. (1992) 6, 2422-2427) or the method of Better et
al. (Science (1988) 240, 1041-1043), respectively.
[0288] When an antibody is produced into the periplasm of E. coli,
the pel B signal sequence (Lei, S. P. et al., J. Bacteriol. (1987)
169, 4379) may be used as a signal sequence for antibody secretion.
After antibodies produced into the periplasm is separated, the
antibody structure is appropriately refolded and then used.
[0289] Any expression system that uses, for example, eukaryotic
cells or prokaryotic cells may be used to produce antibodies used
in the present invention. Eukaryotic cells include, for example,
animal cells such as established mammalian cell systems, insect
cell systems, cells of filamentous fungi, and yeast cells.
Prokaryotic cells include, for example, bacterial cells such as E.
coli cells. Antibodies used in the present invention are preferably
expressed in mammalian cells, for example, CHO, COS, myeloma, BHK,
Vero, and HeLa cells.
[0290] Then, transformed host cells are cultured in vitro or in
vivo to produce antibodies of interest. Host cells are cultured
according to known methods. For example, DMEM, MEM, RPMI1640, or
IMDM may be used as a culture medium, and this may also be used
with serum supplements such as fetal calf serum (FCS).
[0291] Antibodies expressed and produced as described above can be
isolated from cells or host animals and purified to be homogeneous.
Antibodies used in the present invention can be isolated/purified
by using affinity columns. For example, Protein A columns include
Hyper D, POROS, and Sepharose F. F. (Pharmacia). It is also
possible to use other common protein isolation/purification
methods. Such methods are not particularly limited. For example,
antibodies may be isolated/purified by appropriately
selecting/combining chromatography columns other than the
above-described affinity columns, filters, ultrafiltration,
salting-out, dialysis, and such (Antibodies A Laboratory Manual. Ed
Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
[0292] The antigen-binding activity (Antibodies A Laboratory
Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988)
and ligand-receptor binding-inhibitory activity (Harada, A. et al.,
International Immunology (1993) 5, 681-690) of an antibody used in
the present invention can be determined by using known methods.
[0293] Enzyme-linked immunosorbent assays (ELISAs), enzyme
immunoassays (EIAs), radioimmunoassays (RIAs), and fluorescent
antibody methods can be used to determine the antigen-binding
activity of the antibody of the present invention. For example,
when an enzyme immunoassay is used, samples containing an antibody
of the present invention such as a culture supernatant of cells
producing the antibody or the purified antibody are added to plates
coated with a protein used in the present invention. A secondary
antibody labeled with an enzyme such as alkaline phosphatase is
added, and the plates are incubated. After washing, an enzyme
substrate such as p-nitrophenyl phosphate is added and the
absorbance is measured to evaluate the antigen-binding
activity.
[0294] An antibody used in the present invention may appropriately
be linked to a cytotoxic substance described above such as a
proliferation inhibitor, toxic peptide, or radioactive chemical
substance. Such modified antibodies (hereinafter referred to as
antibody conjugates) can be obtained by chemically modifying the
obtained antibodies. Specifically, a linker molecule links a growth
inhibitor to an antibody via chemical bonding so that the antibody
and growth inhibitor or cytotoxic substance can chemically
conjugate with each other (for example, can bind covalently).
Preferred binders (linkers) are cleavable linkers. It is more
preferable that the linkers are cleaved under mild conditions
(specifically, intracellular conditions that do not affect the
activity of inhibitors). Examples of suitable cleavable linkers
include disulfide linkers, acid-labile linkers, photo-labile
linkers, peptidase-labile linkers, and esterase-labile linkers.
Disulfide-containing linkers can be cleaved via disulfide exchange,
which can occur under physiological conditions. Acid-labile linkers
can be cleaved at acid pH. For example, certain intracellular
compartments such as endosomes and lysosomes have an acidic pH (pH
4 to 5), and provide conditions suitable for cleaving acid-labile
linkers. Photo-labile linkers are useful on the body surface and in
many body cavities, which can be exposed to light. Furthermore,
infrared light can penetrate tissues. Peptidase-labile linkers can
be used to cleave certain peptides inside or outside cells (for
example, see Trouet et al., Proc. Natl. Acad. Sci. USA (1982) 79,
626-629; Umemoto et al., Int. J. Cancer (1989) 43, 677-684).
[0295] Such modified antibodies can be prepared not only by
chemical modification as described above, but also in a molecular
form such as a bispecific antibody designed to recognize a growth
inhibitor, toxic peptide, radioactive chemical substance, or the
like using genetic recombination techniques. "Antibody" of the
present invention also comprises such antibodies.
[0296] Examples of modified antibodies that are provided by the
present invention also include those modified with a toxic peptide
such as ricin, abrin, ribonuclease, onconase, DNase I,
Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,
diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin,
L-asparaginase, or PEG L-Asparaginase. In another embodiment,
antibodies may be modified by the combined use of one or more
growth inhibitors and toxic peptides. As described above, the
linkage between the antibody of the present invention that binds to
at least one protein described in SEQ ID NOs: 1 to 8 and an
above-described growth inhibitor, toxic peptide, or radioactive
chemical substance may be a covalent or non-covalent bond. Methods
for preparing modified antibodies linked to such chemotherapeutic
agents are known.
[0297] Furthermore, proteinaceous pharmaceutical agents and toxins
can be linked to an antibody by using a genetic engineering
procedure. Specifically, for example, a recombinant vector into
which a DNA encoding a toxic peptide described above and a DNA
encoding an antibody that binds to any of proteins of at least one
of SEQ ID NOs: 1 to 8 of the present invention are fused in frame
and incorporated into an expression vector is constructed.
Transformed cells obtained by introducing the vector into
appropriate host cells are cultured to express the incorporated
DNA. The modified antibody linked to the toxic peptide is obtained
as a fusion protein. When a fusion protein with an antibody is
prepared, in general, a proteinaceous pharmaceutical agent or toxin
is placed at the C terminus of the antibody. A peptide linker may
be interposed between the antibody and a proteinaceous
pharmaceutical agent or toxin.
[0298] Antibodies used in the present invention may have a
cytotoxic activity. Herein, the cytotoxic activity includes, for
example, complement-dependent cytotoxicity (CDC) and
antibody-dependent cell-mediated cytotoxicity (ADCC). Herein, CDC
refers to a cytotoxic activity mediated by the complement system,
while ADCC refers to an activity of damaging target cells, which is
caused by binding of Fc.gamma. receptor-carrying cells
(immunocytes, etc.) via Fc.gamma. receptor to the Fc portion of
specific antibody upon binding of the antibody to cell-surface
antigens on target cells.
[0299] Whether an antibody used in the present invention has ADCC
or CDC can be measured by known methods (see, for example, Current
protocols in Immunology, Chapter 7. Immunologic studies in humans,
Editor, John E, Coligan et al., John Wiley & Sons, Inc.,
(1993)).
[0300] Specifically, cytotoxicity can be measured, for example, by
the following method.
[0301] Preparation of Effector Cells
[0302] Spleen is removed from a CBA/N mouse or the like, and spleen
cells are dispersed in RPMI1640 medium (GIBCO). After washing with
the same medium containing 10% fetal bovine serum (FBS, HyClone),
effector cells with a cell concentration adjusted to
5.times.10.sup.6 cells/ml were prepared.
[0303] Preparation of Complement Solution
[0304] Baby Rabbit Complement (CEDARLANE) is diluted 10-fold with a
medium (GIBCO) containing 10% FBS to prepare a complement
solution.
[0305] Preparation of Target Cells
[0306] Cells expressing a protein used in the present invention
(cancer stem cells, etc.) are radiolabeled by incubating them with
0.2 mCi of .sup.51Cr-sodium chromate (Amersham Pharmacia Biotech)
in DMEM medium containing 10% FBS for one hour at 37.degree. C.
After radiolabeled, the cells are washed three times with RPMI1640
medium containing 10% FBS, and the target cells with a cell
concentration adjusted to 2.times.10.sup.5 cells/ml were
prepared.
[0307] ADCC Measurement
[0308] 50 .mu.l the target cells and 50 .mu.l of the antibody used
in the present invention are each added to a 96-well U-bottom plate
(Becton Dickinson), and reacted for 15 minutes on ice. Thereafter,
100 .mu.l of effector cells are added and incubated in a carbon
dioxide incubator for four hours. The final antibody concentration
is adjusted to 0 or 10 .mu.g/ml. After incubation, 100 .mu.l of the
supernatant is collected and the radioactivity is measured with a
gamma counter (COBRAIIAUTO-GMMA, MODEL D5005, Packard Instrument
Company). The cytotoxic activity (%) can be calculated according
to:
(A-C)/(B-C).times.100.
A represents the radioactivity (cpm) of each sample, B represents
the radioactivity (cpm) of a sample where 1% NP-40 (nacalai tesque)
has been added, and C represents the radioactivity (cpm) of a
sample containing the target cells alone.
[0309] CDC Measurement
[0310] 50 .mu.l of the target cells and 50 .mu.l of the antibody
used in the present invention are each added to a 96-well
flat-bottom plate (Becton Dickinson), and reacted for 15 minutes on
ice. Thereafter, 100 .mu.l of the complement solution is added, and
incubated in a carbon dioxide incubator for four hours. The
antibody final concentration is adjusted to 0 or 3 .mu.g/ml. After
incubation, 100 .mu.l of the supernatant is collected to measure
the radioactivity with a gamma counter. The cytotoxic activity can
be calculated by the similar way as in the ADCC determination.
[0311] Antibodies with modified sugar chains can appropriately be
used in the antibodies provided by the present invention. It is
known that cytotoxic activity of antibodies can be increased by
modifying its sugar chains. Known antibodies with modified sugar
chains include, for example:
[0312] glycosylated antibodies (WO 1999/054342 and such);
[0313] antibodies with defucosylated sugar chain (WO 2000/061739,
WO 2002/031140, and such); and
[0314] antibodies having a sugar chain with bisecting GlcNAc
(bisecting N-acetylglucosamine) (WO 2002/079255).
[0315] Antibodies of the present invention preferably include
antibodies with modified sugar chains whose sugar chain composition
has been altered to increase the ratio of defucosylated antibody or
to increase the ratio of antibody attached with bisecting
N-acetylglucosamine.
[0316] Antibodies having a neutralizing activity can also be used
appropriately in the present invention. In general, "neutralizing
activity" refers to the activity of a foreign molecule such as a
toxin or virus, or an internal molecule such as a hormone or
cytokine to inhibit a ligand's biological activity on cells.
Specifically, substance having a neutralizing activity refers to a
substance that binds to a ligand or a receptor to which the ligand
binds, thereby inhibiting the ligand-receptor binding. The receptor
whose ligand binding is inhibited by the neutralizing activity
cannot exert their receptor-mediated biological activity. When the
antigen-binding molecule is an antibody, in general, such an
antibody with a neutralizing activity is called a neutralizing
antibody. The neutralizing activity of a test substance can be
assessed by comparing biological activities in the presence of a
ligand, in the condition of when the test substance is present or
not present.
[0317] EREG, which is a target of EP27 antibody described later on
in the Examples, is exemplified below. EGF receptor, which is
believed to be a main receptor for the EREG represented by SEQ ID
NO: 3, dimerizes upon ligand binding and activates its own
cytoplasmic tyrosine kinase domain. The activated tyrosine kinase
causes a peptide having phosphotyrosine by autophosphorylation,
which allows association of various signal transduction accessory
molecules. The molecules are mainly phospholipase C.gamma.
(PLC.gamma.), Shc, Grb2, and such. Of these accessory molecules,
the former two are further phosphorylated by the tyrosine kinase of
the EGF receptor. The main signaling pathway from the EGF receptor
is the one in which phosphorylation occurs in order of Shc, Grb2,
Sos, Ras, and Raf/MAPK kinase/MAP kinase. It is believed that there
is also an alternative pathway from PLC.gamma. to PKC. Since such
intracellular signal cascades vary depending on cell type, a target
molecule can appropriately be selected for each target cell type of
interest and is not limited to the factors described above. It is
possible to use an appropriate in vivo signal activation assay kit
available on the market (for example, protein kinase C activation
measurement system (GE Healthcare Bioscience, etc.)).
[0318] Alternatively, the in vivo signal activation can be detected
by using as an indicator the transcriptional induction of a target
gene downstream in the in vivo signal cascade. Changes in the
transcriptional activity can be detected based on the principle of
reporter assay. Specifically, a reporter gene such as GFP (Green
Fluorescence Protein) or luciferase is placed downstream of the
transcriptional factor or promoter region of the target gene. A
change in the transcriptional activity can be determined as
reporter activity by measuring the reporter activity.
[0319] In addition, the EGF receptor typically functions to promote
cell proliferation, and thus the activation of in vivo signal
transduction can be assessed by measuring the proliferative
activity of the target cell. In the present invention, the
neutralizing activities of neutralizing antibodies of the present
invention are assessed by measuring the cell proliferative
activity. However, methods are not limited thereto, and methods
described above can suitably be used to assess the activity
depending on the type of selected target cells.
[0320] Specifically, the neutralizing activity of an anti-EREG
antibody can be assessed or determined by measuring the cell
proliferative activity, for example, using the method described
below. For example, the method where the incorporation of
[.sup.3H]-labeled thymidine, which is added to a culture medium,
into viable cells is measured as an indicator for the DNA
replication ability is used.
[0321] Simpler methods include the MTT method and dye exclusion
tests in which the ability of cells to exclude dyes such as Trypan
Blue outside is measured under a microscope. The MTT method
utilizes the ability of viable cells to convert MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), a
tetrazolium salt, into a blue formazan product. More specifically,
the MTT method is performed as follows: a test antibody is added to
a culture medium containing test cells; after a certain period, an
MTT solution is added to the culture medium; the mixture is allowed
to stand for a certain period so that MMT is incorporated into the
cells. As a result, a yellow compound MTT is converted into a blue
compound by succinate dehydrogenase in cytoplasmic mitochondria.
After the blue product is dissolved for color development, the
absorbance is measured as an indicator for the viable cell
count.
[0322] In addition to MTT, other commercially available reagents
such as MTS, XTT, WST-1, and WST-8 (nacalai tesque, etc.) can
preferably be used. There are also known methods for assessing the
cell proliferative activity using as an indicator an intracellular
ATP content or impedance of cell culture. When assessing the
activity, a control antibody that is of the same isotype as the
anti-EREG antibody of interest but does not have the neutralizing
activity is used in the same manner as for the anti-EREG antibody.
The activity can be assessed whether the anti-EREG antibody
exhibits the neutralizing activity greater than that of the control
antibody.
[0323] Cells whose proliferation is inhibited by the anti-EREG
antibody are not particularly limited, as long as they express EREG
protein. Examples of preferred EREG-expressing cells include cancer
cells. Specifically, cells derived from colorectal cancer, lung
adenocarcinoma, pancreatic cancer, stomach cancer, or kidney cancer
are preferable EREG-expressing cells in the present invention. A
cell proliferation-inhibitory effect against both primary and
metastatic lesions of all these cancers can be achieved by the
present invention. More preferred cancer cells include those of
primary colorectal cancer, metastatic colorectal cancer, lung
adenocarcinoma, pancreatic cancer, stomach cancer, and kidney
cancer. Thus, anti-EREG antibodies can be used to treat/prevent
diseases caused by cell proliferation, for example, colorectal
cancer, lung adenocarcinoma, pancreatic cancer, stomach cancer, and
kidney cancer. These cancers can be targets of treatment or
prevention, regardless of primary or metastatic lesions. More
preferably, anti-EREG antibodies are used to treat and/or prevent
primary colorectal cancer, metastatic colorectal cancer, and
pancreatic cancer. Furthermore, among these cancers, those which
grow in an EREG-dependent manner are preferred as a target of
treatment and/or prevention in the present invention.
[0324] In the description and Tables herein, when nucleotides and
amino acids are represented by abbreviations, these abbreviations
are based on the abbreviations by IUPAC-IUB Commission on
Biochemical Nomenclature, or the conventional abbreviations in the
art. Regarding amino acids, when an optical isomer exists, it
represents L form, unless otherwise specified.
<Cancer Stem Cell Inhibitors of the Present Invention>
[0325] The effective dosage of cancer stem cell inhibitors of the
present invention is selected within the range of 0.001 to 1,000
mg/kg weight for each administration. Alternatively, the dosage may
be selected within the range of 0.01 to 100,000 mg/body for each
patient. However, the dosage of the inhibitors of the present
invention is not limited to these doses. Meanwhile, with respect to
the timing of administration, an inhibitor of the present invention
may be administered before or after manifestation of clinical
symptoms of diseases. The inhibitors of the present invention can
be formulated according to conventional methods (Remington's
Pharmaceutical Science, latest edition, Mark Publishing Company,
Easton, US), and may contain both pharmaceutically acceptable
carriers and additives. Such carriers and medical additives
include, for example, water, pharmaceutically acceptable organic
solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone,
carboxyvinyl polymer, carboxymethylcellulose sodium, sodium
polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl
starch sodium, pectin, methyl cellulose, ethyl cellulose, xanthan
gum, gum arabic, casein, agar, polyethylene glycol, diglycerin,
glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol,
stearic acid, human serum albumin (HSA), mannitol, sorbitol,
lactose, and surfactants that are acceptable as a medical additive.
In practice, additives are selected alone or in appropriate
combination from those listed above depending on the dosage form of
an inhibitor of the present invention; but are not limited thereto.
For example, when used as a preparation for injection, it can be
used in a form in which the inhibitor is dissolved in a medium such
as physiological saline, buffer, or glucose solution and an
adsorption inhibitor such as Tween80, Tween20, gelatin, or human
serum albumin is added; alternatively, the inhibitor of the preset
invention may be in a lyophilized form for dissolution and
reconstitution before use. As excipients for lyophilization, for
example, sugar alcohols and saccharides such as mannitol and
glucose can be used. The inhibitors of the present invention are
generally administered by a parenteral route, for example, via
injection (subcutaneous, intravenous, intramuscular,
intraperitoneal, etc.), transdermal, transmucosal, intranasal, or
pulmonary administration; however, the inhibitor can be
administered orally.
[0326] Herein, "combined use" of a cancer stem cell inhibitor and
an anti-cancer agent means that these agents may be administered at
the same time or in succession; alternatively, one is administered
at an interval after administration of the other.
[0327] Herein, cancer stem cell inhibitors can be used as various
embodiments such as, for example, prevention of cancer recurrence,
suppression of cancer recurrence, prevention of cancer metastasis,
suppression of cancer metastasis, and adjuvant therapy for
preventing postoperative recurrence for application. When used in
the above-described embodiments, any cancer stem cell inhibitor can
be used as a cancer stem cell inhibitor of the present invention.
However, non-limiting preferred examples include agents for
inhibiting cancer stem cell proliferation or agents for disrupting
cancer stem cell. As long as agents for inhibiting cancer stem cell
proliferation that are provided by the present invention can
suppress the proliferation of target cancer stem cells, mechanism
of suppressing cancer stem cell proliferation is not relevant. As a
non-limiting example, such agents for inhibiting cancer stem cell
proliferation include those comprising as an active ingredient an
antibody having neutralizing activity against cancer stem cell
proliferation or growth or an antibody having cytotoxicity against
cancer stem cells. Similarly, as long as agents for disrupting
cancer stem cells that are provided by the present invention can
destroy target cancer stem cells, mechanism of disrupting cancer
stem cells is not relevant. As a non-limiting example, such agents
for disrupting cancer stem cells include agents for inhibiting
cancer stem cell proliferation comprising as an active ingredient
an antibody having cytotoxicity or apoptotic activity against
cancer stem cells. Those skilled in the art can determine whether a
test agent for disrupting cancer stem cells has apoptotic activity
by using known methods including terminal deoxynucleotidyl
transferase biotin-dUTP nick end labeling (TUNEL) assay, caspase
activity (in particular, caspase-3) assay, fas ligand assay, and
Annexin V assay as apoptotic activity assay methods. Non-limiting
preferred examples include cancer stem cell differentiation
enhancers. Non-limiting examples of differentiation enhancers
include BMP4, i.e., the polypeptide of SEQ ID NO: 9, or polypeptide
equivalents having one or several amino acid addition(s),
deletion(s), and/or substitution(s) among amino acids of the
polypeptide. Such polypeptide equivalents preferably have a CSC
differentiation-inducing activity equivalent to that of the
polypeptide of SEQ ID NO: 9. The equivalency of the
differentiation-inducing activity can be defined, for example, by
whether the CK20-inducing activity for CSCs is 10%, preferably 20%,
more preferably 30%, even more preferably 40%, and still more
preferably 50% of that of the polypeptide of SEQ ID NO: 9. In
another non-limiting embodiment, the equivalency of the
differentiation-inducing activity can be defined, for example, by
whether the CK20-inducing activity for CSCs is 60%, preferably 70%,
more preferably 80%, even preferably 90%, and still preferably 95%
of that of the polypeptide of SEQ ID NO: 9.
[0328] Anti-cancer agents that are used in combination with a
cancer stem cell inhibitor of the present invention include
alkylating agents, metabolic antagonists, natural products,
platinum complexes, and other pharmaceutical agents. Alkylating
agents include nitrogen mustards, ethylenimines, methylmelamines,
alkyl sulfonates, nitrosoureas, and triazens. Nitrogen mustards
include, for example, mechlorethamine, cyclophosphamide,
ifosfamide, melphalan, and chlorambucil. Ethylenimines and
methylmelamines include, for example, hexamethylmelamine and
thiotepa. Alkyl Sulfonates include busulfan. Nitrosoureas include,
for example, carmustine (BCNU), lomustine (CCNU), semustine
(methyl-CCNU), and streptozocin. Triazens include dacarbazine
(DTIC). Metabolic antagonists include folic acid analogs,
pyrimidine analogs, and purine analogs. Folic acid analogs include
methotrexate. Pyrimidine analogs include, for example, fluorouracil
(5-FU), doxifluridine (5'-DFUR; trade name: FURTULON), capecitabine
(trade name: Xeloda), floxuridine (FudR), and cytarabine. Purine
analogs include, for example, mercaptopurine (6-MP), thioguanine
(TG), and pentostatin. Natural products include vinca alkaloids,
epipodophyllotoxins, and antibiotics. Vinca alkaloids include, for
example, vinblastine (VLB) and vincristine (VCR).
Epipodophyllotoxins include, for example, etoposide and teniposide.
Antibiotics include, for example, dactinomycin (actinomycin D),
daunorubicin, doxorubicin, bleomycin, plicamycin, and mitomycin.
Platinum complex refers to platinum coordination complex, and
includes, for example, cisplatin (CDDP) and carboplatin. Other
pharmaceutical agents include: topoisomerase inhibitors such as
irinotecan and camptothecin; taxols, for example, paclitaxel,
docetaxel; anthracenediones, for example, mitoxantrone;
urea-substituted derivatives, for example, hydroxyurea; methyl
hydrazines, for example, procarbazine hydrochloride (trade name:
Natulan), vitamin A metabolites, for example, tretinoin (trade
name: VESANOID), as well as include rituximab, alemtuzumab,
trastuzumab, bevacizumab, cetuximab, panitumumab, trastuzumab, and
gemutuzumab.
[0329] All prior-art documents cited herein are incorporated herein
by reference.
EXAMPLES
[0330] Herein below, the present invention will be specifically
described with reference to examples, however, it is not to be
construed as being limited thereto.
Establishment of Human Colorectal Cancer Cell Lines with
Immunodeficient NOG Mice
[0331] Colorectal cancer specimens were obtained from patients with
their consent under approval of the ethical committees of
PharmaLogicals Research (Singapore) and Parkway Laboratory Services
(Singapore). Tumor blocks were cut into small pieces with a razor
blade, and grafted in the lateral region of NOG mice. Human
colorectal cancer xenografts were maintained by passaging them in
NOG mice provided by the Central Institute for Experimental Animals
(Japan). Mice used in this experiment were treated in accordance
with the animal experiment guidelines of PharmaLogicals Research.
For histopathological examination, small blocks of xenograft tumors
and surgical human tissue samples were fixed with 4%
paraformaldehyde at 4.degree. C. for 16 to 24 hours, and embedded
in paraffin by the AMeX method (Sato Y, et al., (1986) Am J Pathol,
125: 431-435; Sato Y, et al., (1992) Am J Pathol, 140: 775-779;
Suzuki M, et al. (2002) J Toxicol Sci, 27: 165-172). The thin
sections were stained with eosin and hematoxylin and assessed by
microscopic observation.
Isolation and In Vitro Culture of Large Intestine CSCs
[0332] Tissues of xenograft were cut with a razor blade in order to
prepare single cancer cell suspensions. After the suspensions were
incubated at 37.degree. C. for 3 hours in DPBS containing
collagenase/dispase (Roche) and DNaseI (Roche), the suspensions
were filtered through 40-.mu.m Cell Strainers (BD Biosciences). The
cells were suspended in the lysis buffer (BD Biosciences) to remove
erythrocytes. The prepared xenograft-derived cells (such cells are
referred to as primary cells) were cultured under 5% CO.sub.2
atmosphere at 37.degree. C. in DMEM/F12 (Invitrogen) containing N-2
Supplement (Invitrogen), 20 ng/ml human EGF (Invitrogen), 10 ng/ml
human basic fibroblast growth factor (Sigma), 4 .mu.g/ml heparin
(Sigma), 4 mg/ml BSA (Invitrogen), 20 .mu.g/ml human insulin zinc
solution (Invitrogen), and 2.9 mg/ml glucose (Sigma) (Todaro M, et
al. (2007) Cell Stem Cell 1: 389-402). Adherent and floating CSCs
were cultured using conventional polystyrene-treated cell culture
flasks (BD Biosciences) and Ultra-low attachment cell culture
flasks (Corning), respectively.
In Vivo Tumor Formation Analysis
[0333] Cell suspensions were prepared by serial dilution. 100 .mu.l
of cancer cell suspensions in Hanks' balanced salt solution
(Invitrogen) were subcutaneously inoculated into lateral region of
mice using 50% matrigel (BD Bioscience). The tumor development was
monitored over seven weeks. In order to inoculate single cell,
cells were labeled using an FITC-labeled mouse anti-human CD326
(EpCAM) antibody (Miltenyi Biotec), and plated in a terasaki plate
(Termo Fisher Scientific). Cell singularity was confirmed under a
microscope. Single cells were inoculated into lateral region of
mice using 50 .mu.l of 50% matrigel. The tumor development was
monitored over 10 weeks.
Establishment of Cells Expressing Full-Length Human Lgr4, Lgr5, and
Lgr6
[0334] Full-length human Lgr4, Lgr5, and Lgr6 cDNAs were cloned by
PCR based on the sequences of NM.sub.--018490 (Lgr4),
NM.sub.--001017403 (Lgr6), and NM.sub.--003667 (Lgr5). The cloned
genes were expressed with or without adding HA tag to their N
termini Cells of Chinese hamster ovary cell line CHO DG44
(Invitrogen) were transfected with expression plasmids using Gene
Pulser (BioRad). Stable cell lines HA-Lgr4/DG, HA-Lgr5/DG, and
HA-Lgr6/DG were selected using G418.
Preparation of Soluble Lgr5-Fc Protein
[0335] Soluble Lgr5 protein (amino acids 1 to 555) was expressed as
a fusion protein with the Fc domain of mouse IgG2a in CHO DG44.
Transfectants were screened by sandwich ELISA using a goat
anti-mouse IgG2a (Bethyl laboratories) and HRP-rat anti-mouse IgG2a
mAb (Serotec). A clone that produces sLgr5-Fc at the highest level
was named 2D3. A culture supernatant of 2D3 was collected, and
Lgr5-Fc protein was affinity-purified using a Protein A-Sepharose
column (Pharmacia). Lgr5-Fc was used as an antigen in protein
immunization and ELISA screening.
Generation of Anti-Lgr5 Monoclonal Antibody by Immunization with
Lgr5-Fc Protein
[0336] Balb/c mice (Charles River Japan) were immunized
subcutaneously with 50 .mu.g of Lgr5-Fc emulsified in Freund's
complete adjuvant. After two weeks, the mice were injected with the
same amount of Lgr5-Fc in Freund's incomplete adjuvant once a week
over two weeks. Three days before cell fusion, 25 .mu.g of Lgr5Fc
was intravenously injected to the mice. Spleen lymphocytes derived
from the immunized mice were fused with cells of mouse myeloma line
P3-X63Ag8U1 (ATCC) using a conventional method (Kremer L and
Marquez G (2004) Methods Mol. Biol., 239: 243-260). Hybridoma
culture supernatants were screened for antibodies reactive to
sLgr5-Fc by ELISA to establish Lgr5-specific mouse mAb 2T15E-2 and
2U2E-2.
Flow Cytometry Analysis
[0337] Large intestine CSCs were incubated with labeled antibodies
and analyzed using the EPICS ALTRA (Beckman Coulter) and
FACSCalibur (Becton Dickinson)). Antibodies used were: PE-labeled
mouse anti-human CD133 antibody (Miltenyi Biotec), PE-labeled mouse
anti-human CD44 antibody (BD Pharmingen), FITC-labeled mouse
anti-human CD326 (EpCAM) antibody (Miltenyi Biotec), PE-labeled
mouse anti-human CD166 antibody (R&D Systems), PE-labeled mouse
anti-human CD24 antibody (BD Pharmingen), PE-labeled mouse
anti-human CD26 antibody (BD Pharmingen), and PE-labeled mouse
anti-human CD29 antibody (BD Pharmingen).
[0338] Large intestine CSCs were incubated with mouse anti-human
Lgr5 antibody (2T15E-2) and then with PR-labeled rat anti-mouse IgG
antibody (Invitrogen) to stain Lgr5. The aldehyde dehydrogenase
activity was measured using the AldeFluor Kit (Stemcell
Technologies). Mouse cells were discriminated from human large
intestine CSCs by staining with anti-mouse MHC class I antibody
(Abcam), and PE- or APC-labeled goat anti-human IgG2a antibody
(BioLegend). Dead cells were also removed using the 7-AAD Viability
Dye (Beckman Coulter).
Western Blot Analysis
[0339] Protein was extracted using RIPA buffer (Sigma) supplemented
with the Complete Mini Protease Inhibitor Cocktail (Roche).
Proteins were fractionated by the NuPAGE Gel (Invitrogen) and
transferred onto PVDF membrane. After blocking with PBS containing
1% skimmed milk, the membrane was probed with rabbit anti-human
.beta.-catenin antibody (Sigma), rabbit anti-human phospho-c-JUN
antibody (Sigma), rabbit anti-human TCF1 antibody (Cell Signaling),
rabbit anti-human TCF3 antibody (Cell Signaling), rabbit anti-human
TCF4 antibody (Cell Signaling), rabbit anti-human Lgr5 antibody
(Abcam), mouse anti-human E-cadherin antibody (Abcam), rabbit
anti-human Snail antibody (Abcam), and mouse anti-human GAPDH
antibody (Santa Cruz). Reactive bands were detected using BCIP/NBT
substrate (KPL).
Quantitative Real-Time Polymerase Chain Reaction
[0340] Total RNAs were isolated using the RNeasy Mini Kit including
DNAase treatment (Qiagen). cDNAs were synthesized using the
First-Strand cDNA Synthesis Kit (SABiosciences). Quantitative
real-time PCR (QRT-PCR) analysis was performed with the SYBR
Green/Rox qPCR(SABiosciences) using the Mx3005P Real-Time PCR
System (Stratagene). The fold induction value was calculated
according to the 2-.DELTA..DELTA.Ct method. GAPDH and ACTB were
used as a reference. All experiments were performed in
triplicate.
Primers for Quantitative Real-Time PCR Analysis
[0341] The following primers were used to amplify reactive
transcripts.
TABLE-US-00001 Lgr5: forward primer: (SEQ ID NO: 10)
5'-AGTTTATCCTTCTGGTGGTAGTCC-3'; reverse primer: (SEQ ID NO: 11)
5'-CAAGATGTAGAGAAGGGGATTGA-3'; GAPDH: forward primer: (SEQ ID NO:
12) 5'-CTCTGCTCCTCCTGTTCGAC-3'; reverse primer: (SEQ ID NO: 13)
5'-ACGACCAAATCCGTTGACTC-3'; ACTB: forward primer: (SEQ ID NO: 14)
5'-AAGTCCCTTGCCATCCTAAAA-3'; reverse primer: (SEQ ID NO: 15)
5'-ATGCTATCACCTCCCCTGTG-3'
Cell Proliferation Assay
[0342] Floating and adherent CSCs were plated at about 100 and
1.times.10.sup.4 cells/well in 96-well plates, respectively. On
days 0 and 3, viable cell counts were determined by the Cell
Counting Kit-8 Assay (Doujindo) according to the manufacturer's
protocol. Average absorbance on day 0 was taken as 100%. For
chemosensitivity assay, floating and adherent CSCs were plated at
about 100 and 1.times.10.sup.4 cells/well in 96-well plates,
respectively. After 24 hours of incubation, 10 .mu.g/ml 5-FU
(Hospira), 10 .mu.g/ml irinotecan (Hospira), 50 mM TCF inhibitor
FH535 (Merck), or 50 mM .beta.-catenin inhibitor Cardamonin (Merck)
were added to the plates. After three days of culture in the
presence of the agents, the Cell Counting Kit-8 was added to the
cells. The average absorbance of cells exposed to DMSO or medium
alone was taken as 100%. All experiments were performed in
triplicate.
Immunofluorescent Staining of Cultured Cells and Xenograft
Tissues
[0343] For immunofluorescent cytochemistry, cells were fixed with
4% paraformaldehyde and methanol, and incubated with a mouse
anti-human E-cadherin antibody (Abcam), rabbit anti-human Snail
antibody (Abcam), or rabbit anti-human .beta.-catenin antibody
(Sigma)). Then, the cells were visualized using the AlexaFluor
488-labeled goat anti-mouse IgG antibody or goat anti-rabbit IgG
antibody. For immunofluorescent cytochemistry, thin sections
derived from paraffin blocks of xenograft tumors described above
were incubated with a mouse anti-human Lgr5 antibody (2U2E-2) or
rabbit anti-human Snail antibody (Abcam). After incubation with a
primary antibody, Lgr5 protein was detected with a goat anti-mouse
antibody conjugated with the polymer-HRP (DAKO) and visualized with
the AlexaFluor 488-labeled tyramide (Invitrogen), while Snail
protein was detected with biotinylated goat anti-rabbit antibody
(VECTOR) and visualized with the AlexaFluor 568-labeled
streptavidin (Invitrogen). These cells and samples were also
stained with DAPI (Invitrogen).
Example 1
Establishment of Colorectal Cancer Xenografts
[0344] As described in a previous report (Fujii E. et al., (2008)
Establishment and characterization of in vivo human tumor models in
the NOD/SCID/gamma(c)(null) mouse. Pathol Int 58: 559-567), the
present inventors established 11 types of human colorectal cancer
xenografts using NOD/Shi-scid, IL-2R.gamma.null (NOG) mice (Table
1; the number of human colorectal cancer cell lines established
with immunodeficient NOG mice).
TABLE-US-00002 TABLE 1 Adenocarcinoma G1 G2 G3 Total Number of
cases 4 46 3 53 Established 0 10 1 11 Impracticable* 0 6 0 6 EBV
lymphoma 2 16 1 19 Aggravated animal condition .dagger. 1 12 1 14
No viable cancer 1 2 0 3
[0345] In the above Table 1, asterisk indicates cases established
but unsuitable for experiment, and dagger indicates cases with
infection and such.
[0346] As shown in Table 1, 17 types of colorectal cancer
xenografts were established from samples of 53 human colorectal
cancer patients. Except for the 17 types of xenografts, associated
EBV-infected lymphoma cells occurred in 19 cases (which aggravated
the condition of NOG mice); other infections were found in 14
cases; and no tumor growth was observed in three cases. Of the 17
types, 11 xenografts survived even after freeze-thawing, and had
the capacity to reconstitute tumor, and showed similar
histopathological features as those of the original tumors. Of the
11 types, 10 xenografts were derived from grade-2
moderately-differentiated adenocarcinomas, and the remaining one
was derived from a grade-3 poorly-differentiated
adenocarcinoma.
[0347] Of the 11 types, 10 xenografts were derived from
moderately-differentiated colorectal cancer (MDCC), and the
remaining one was derived from poorly-differentiated colorectal
cancer (PDCC) (Table 2; histopathological classification of the
original human colorectal cancers that were used to establish the
11 xenografts).
TABLE-US-00003 TABLE 2 Histopathological classification of original
human tumor AJCC Dukes' Line No. Type Grade TNM stage stage PLR30
Adenocarcinoma G2 pT3N0MX II B PLR59 Adenocarcinoma G2 pT3N2MX III
C2 PLR123 Adenocarcinoma G2 pT4N1MX III C1 PLR168 Adenocarcinoma G2
pT3N0MX II B PLR215 Adenocarcinoma G2 pT3N0MX II B PLR241
Adenocarcinoma G2 pT4N3M1 IV D PLR254 Adenocarcinoma G2 pT4N2MX III
C2 PLR261 Adenocarcinoma G2 pT3N0MX II B PLR325 Adenocarcinoma G3
pT4N1M1 III C1 PLR379 Adenocarcinoma G2 pT4N2MX IIIC C1 PLR423
Adenocarcinoma G2 pT3N0M1 IV D
[0348] Both MDCC and PDCC xenografts reconstituted
histopathological morphologies almost equivalent to those of the
original tumors. MDCC xenografts formed specific epithelial ducts
which contained goblet cells, and small budding clusters (may
undergo epithelial-mesenchymal transition (EMT)). In contrast, PDCC
xenografts did not form such specific epithelial duct structures
(FIGS. 1 and 16).
Example 2
Isolation of Large Intestine CSCs
[0349] In order to isolate large intestine CSCs, the present
inventors used two types of MDCC xenografts, i.e., PLR59 and
PLR123. These xenografts were chosen by the present inventors
because they grew rapidly even after 10 passages in NOG mice while
maintaining the capacity to reconstitute tumors with epithelial
ducts and small budding clusters (FIG. 1). Thus, the present
inventors predicted that stable CSCs can be obtained from the
xenografts.
[0350] Flow cytometry analysis of primary cells passaged in NOG
mice derived from PLR59 and PLR123 showed that signal level of
CD44, ALDH, CD26, and Lgr5 were lower than that of CD133, EpCAM,
CD166, CD24, and CD29. This suggests the existence of a small
population of CSCs (FIG. 2). Indeed, when primary cells derived
from PLR59 and PLR123 were grafted subcutaneously to NOG mice at
100 cells/injection site, tumors were formed at half of the
injection sites (five of 12 injection sites; Table 3), and the
histopathological morphology of tumors is highly similar to that of
original tumors in that they had hierarchical organization (FIG.
3). However, primary cells derived from PLR59 and PLR23 were
injected subcutaneously at 10 cells/site, any tumor was not formed
in NOG mice (Table 3). Table 3 shows the cancer formation ability
49 days after inoculation.
TABLE-US-00004 TABLE 3 Number of cells/inoculation site Cell line*
Specimen .dagger. 1,000 100 10 PLR59 Primary
12.sup.+/12.sup..dagger-dbl. 5/12 0/12 (100) (42) (0) Non-adherent
6/6 6/6 1/6 (Lgr5-) (100) (100) (17) Adherent (Lgr5+) 6/6 6/6 6/6
(100) (100) (100) PLR123 Primary 12/12 5/12 0/12 (100) (42) (0)
Non-adherent 6/6 5/6 2/6 (Lgr5-) (100) (83) (33) Adherent (Lgr5+)
6/6 6/6 6/6 (100) (100) (100)
[0351] In Table 3 shown above, asterisk indicates tumor xenografts
established with NOG mice, and dagger indicates cell preparations.
Primary indicates cells (primary cells) prepared by harvesting
xenograft tumor tissues grown in NOG mice and removing erythrocytes
and mouse cells. Floating indicates cells prepared by in vitro
culturing primary cells under a non-adherent culture condition.
Adherent indicates cells prepared by in vitro culturing primary
cells under adherent culture conditions. Plus symbol (single)
indicates the number of formed tumors, while plus symbol (double)
indicates the total number of inoculation sites. Parenthesis
indicates percent tumor (cancer) formation. Lgr5.sup.+ represents
Lgr5 positive, and Lgr5.sup.- represents Lgr5 negative.
[0352] Primary cells derived from PLR59 and PLR123 were cultured in
serum-free media supplemented with EGF and FGF. This yielded
adherent and non-adherent cells. The present inventors harvested
the adherent and non-adherent cells and cultured them separately.
The adherent cells were grown with a doubling time of about 2.5
days and exhibited mesenchymal cell-like morphology. The
non-adherent cells, on the other hand, formed spheroid-like cell
clusters but did not proliferate significantly (FIGS. 4, 5, 18, and
19). After one-week or longer culture, the cells were assessed for
large intestine CSC markers. This demonstrated that both adherent
and non-adherent cells were highly homogeneous. The adherent cells
were of Lgr5.sup.+, ALDH.sup.+, CD133.sup.+, CD44.sup.+,
EpCAM.sup.+, CD 166.sup.+, CD24.sup.+, CD26.sup.+, and CD29.sup.+.
Meanwhile, the non-adherent cells were of Lgr5.sup.- and
ALDH.sup.-, and thus were different from the adherent cells (FIGS.
6 and 20).
[0353] Lgr5 mRNA was detected at a significant level in the
adherent cells, while it was undetectable in non-adherent cells
(FIG. 27).
Example 3
Analysis of Lgr5 Protein Expression
[0354] In order to assess the expression of Lgr5 protein, the
present inventors prepared two types of Lgr5-specific monoclonal
antibodies (2L36, 2T15E-2, and 2U2E-2) respectively for
immunohistochemistry and flow cytometry analysis. The antibodies
produced by the present inventors were highly specific to Lgr5
without any cross-reactivity to Lgr4 or Lgr6, both of which are
highly homologous to Lgr5 (FIGS. 28 and 29). Using the antibodies,
the present inventors demonstrated the expression of Lgr5 in the
adherent CSCs.
[0355] Lgr5-positive cells were detected in tumor tissues that were
the origins of PLR59 and PLR123 as well as in xenograft cancer
tissues therefrom through all passages (FIG. 39). The frequency of
Lgr5-positive cells was low in the original tumor tissues (0.01%
and 0.04% for PLR59 and PLR123, respectively). Regarding the
xenograft cancer tissues, the frequency of Lgr5-positive cells was
increased as passage number increased; however, there was no
further change after tenth generation (FIG. 39). On the other hand,
the tumor-reconstituting capacity of primary cells from PLR123
xenograft model was also potentiated as passage number increased.
The ratio of CSCs in the primary cells, which was estimated based
on the capacity to reconstitute tumor, was about 0.1% after 5
passages, and was increased to about 0.4% after 14 passages.
Example 4
Tumor-Reconstituting Capacities of Lgr5-Positive and Lgr5-Negative
Colorectal CSCs
[0356] If a group of colorectal cancer stem cells is characterized
by Wnt signaling, Lgr5-positive adherent cells alone can form
tumors in vivo. To confirm whether this prediction is true, the
present inventors assessed the tumor-forming capacities of
Lgr5-positive adherent cells and Lgr5-negative non-adherent
cells.
[0357] The result showed that Lgr5-positive adherent cells were
more potent than Lgr5-negative non-adherent cells in the capacity
to form tumors. However, both Lgr5-positive and Lgr5-negative cells
had the capacity to form tumors in NOG mice. Subcutaneous injection
of ten Lgr5-positive cells caused tumor formation at every
injection site (six of six sites), while Lgr5-negative cells formed
tumors at two of six injection sites (PLR123-derived cells) or at a
single site (PLR59-derived cells) (Table 3). Lgr5-positive cells,
even when injected at only one cell per inoculation site,
reconstituted tumors at two of 12 injection sites (PLR123-derived
cells) or at a single site (PLR59-derived cells) (FIG. 7). The
histopathological morphologies of tumors derived from the
Lgr5-positive and Lgr5-negative cells were almost the same as those
of the original tumors (FIGS. 17 and 40). Furthermore, there was no
change in the expression of cell surface markers and
tumor-initiating activity of the Lgr5-positive CSCs even after one
month of passage culture (FIGS. 30 and 31).
[0358] Under adherent culture conditions, the Lgr5-positive cells
underwent symmetric cell division (FIG. 41). Meanwhile, in the
presence of matrigel and serum, Lgr5 protein was distributed to one
of two daughter cells under the same culture conditions (FIGS. 42C
and 42D), demonstrating that the Lgr5-positive cells undergo
asymmetrical cell division. One of CSC's properties is symmetrical
cell division for self-renewal, and another characteristic property
is asymmetrical cell division. The Lgr5-positive adherent cells
divided symmetrically under adherent culture conditions (FIG. 41),
whereas, in the presence of matrigel and FBS, as seen from the fact
that Lgr5 protein was distributed to one of daughter cells (FIG.
42), the Lgr5-positive cells underwent asymmetrical cell division,
which resulted in two distinct progenies.
[0359] The results described above demonstrate that Lgr5-positive
and Lgr5-negative cells derived from PLR59 and PLR123 are highly
pure large intestine CSCs, and that the Lgr5-positive and
Lgr5-negative cells correspond to two independent CSC states in
colorectal cancer.
Example 5
Effect of TCF and .beta.-Catenin
[0360] In the Lgr5-positive cells, the levels of .beta.-catenin,
TCF1, TCF3, and TCF4 proteins were upregulated in accordance with
the expression of Lgr5. This was not observed in the Lgr5-negative
cells (FIGS. 7 and 21).
[0361] On the other hand, the N-terminal phosphorylation of c-Jun
was undetectable in the Lgr5-positive CSCs as compared to the
Lgr5-negative CSCs (FIGS. 7 and 21).
[0362] To answer the question of whether Wnt signaling promotes the
proliferation of large intestine CSCs, the present inventors
assessed the effects of .beta.-catenin/TCF inhibitor FH535 and
Wnt/.beta.-catenin inhibitor cardamonin (which induces
.beta.-catenin degradation) on the proliferation of large intestine
CSCs.
[0363] The result showed that 50 .mu.M FH535 significantly reduced
the proliferation of Lgr5-positive large intestine CSCs but had no
effect on the proliferation of Lgr5-negative large intestine CSCs
(FIGS. 8 and 22). Meanwhile, 50 .mu.M cardamonin reduced the viable
cell count to 70% for the Lgr5-positive large intestine CSCs and to
about 50% for the Lgr5-negative large intestine CSCs (FIGS. 8 and
22).
[0364] This result suggests that TCF mediates the proliferation of
Lgr5-positive cells and that .beta.-catenin is involved in the
survival of large intestine CSCs. Interestingly, the Lgr5-positive
cells proliferated even without supplement of EGF and FGF (FIGS. 9
and 23). This finding shows that large intestine CSCs have an
intrinsic/autocrine mechanism for activating the Wnt signaling for
their proliferation.
Example 6
Ability of Large Intestine CSCs to Convert from Lgr5-Positive to
Lgr5-Negative State
[0365] One of CSC's properties is resistance to chemotherapeutic
agents. Thus, the present inventors tested large intestine CSCs for
the sensitivity to 5-FU and irinotecan. As described above, the
Lgr5-positive cells proliferated with a doubling time of about 2.5
days. Meanwhile, the Lgr5-negative CSCs were in a quiescent state
in terms of growth. Both 5-FU (10 .mu.g/ml) and irinotecan (10
.mu.g/ml) treatments significantly inhibited the proliferation of
Lgr5-positive large intestine CSCs, while they did not affect the
proliferation and survival of Lgr5-negative large intestine CSCs
(FIGS. 10 and 24). Three-day exposure of the Lgr5-positive large
intestine CSCs to 5-FU (10 .mu.g/ml) or irinotecan (10 .mu.g/ml)
caused the appearance of cells resistant to the chemotherapeutic
agents. Surprisingly, the drug-resistant cells were negative for
Lgr5 and had changed in morphology (FIGS. 11, 32, and 25). This
finding demonstrates the transition from the Lgr5-positive to
Lgr5-negative state.
[0366] HLA-DMA, TMEM173, ZMAT3, and GPR110 were chosen as markers
for use in specific detection of such CSCs stably negative for
Lgr5. Immunostaining performed using specific antibodies against
the above molecules yielded a specific staining pattern with colon
CSCs that converted to negative for Lgr5 after three days of
irinotecan exposure (FIG. 43). Furthermore, this immunostaining
method was demonstrated to be applicable to tissue sections
prepared from paraffin blocks, which are used commonly (FIG. 43).
These findings suggest that HLA-DMA, TMEM173, ZMAT3, GPR110 can
serve as specific markers for CSCs that converted to negative for
Lgr5.
[0367] The fluorescence representing Lgr5 positivity (FIG. 44A),
which had been observed before irinotecan treatment, disappeared
after the treatment (FIG. 44B). From Lgr5-negative cells again
inoculated and cultured in the absence of irinotecan, Lgr5-positive
cells appeared four days after the inoculation (FIG. 44C), and
expanded by eight days after the inoculation (FIG. 44D). All the
Lgr5-negative drug-resistant cells are negative for Lgr5 (FIGS. 44
and 45) and remained negative for CK20 (FIG. 46). This suggests
that the transition of colon CSCs from the actively proliferating
state to a quiescent state is correlated with the disappearance of
Lgr5 molecule. The correlation was also verified by in vitro growth
inhibitor-resistance assay (FIG. 47). In addition, the ALDH
activity was reduced, while there was no alteration in other CSC
markers (FIG. 48).
[0368] The Lgr5-negative cells prepared via irinotecan treatment
were assessed for the tumor-forming activity. Subcutaneous
injection of ten cells derived from PLR59 and PLR123 resulted in
formation of tumors in two and one NOG mice (Table 4),
respectively. Table 4 shows the tumor-forming activity of
Lgr5-negative CSCs 49 hours after inoculation. In Table 4 shown
below, asterisk indicates tumor xenografts established in NOG mice;
plus symbol (single) indicates the number of animals bearing
tumors, and plus symbol (double) indicates the total number of
animals.
TABLE-US-00005 TABLE 4 Number of cells/inoculation site Cell line*
1,000 100 10 PLR59 6.sup..dagger./6.sup..dagger-dbl. 6/6 2/6 PLR123
6/6 6/6 1/6
[0369] In addition, to assess whether Lgr5-negative large intestine
CSCs undergo a transition into an Lgr5-positive state, the present
inventors cultured adherently Lgr5-negative large intestine CSCs
prepared via irinotecan treatment again in a serum-free stem cell
culture medium. The cells became positive for Lgr5 and exhibited
mesenchymal cell-like morphology (FIGS. 12 and 33), and started to
proliferate. On the other hand, when Lgr5-positive adherent large
intestine CSCs were cultured in an ultra low adherent plate, the
present inventors observed that some of the cells halted their
growth and formed a spheroid-like structure and that the Lgr5 mRNA
level was very low (FIGS. 12 and 33). The transition from the
Lgr5-positive to Lgr5-negative state (and the reverse) was also
confirmed by observations using a single cell in culture. When
single Lgr5-positive cells were individually cultured in a
multi-well plate, the cells underwent a transition into the
Lgr5-negative state within three days after irinotecan treatment.
When single Lgr5-negative cells prepared via irinotecan treatment
were individually cultured in a multi-well plate in the absence of
irinotecan, 19 to 43% of the cells underwent a transition into the
Lgr5-positive state within four days (FIG. 49 and Table 5).
TABLE-US-00006 TABLE 5 Number of cells Transition of state Cell
line Lgr5 positive Lgr5 negative Total Lgr5 positive to negative
PLR59 0 132 132 (0%) (100%) PLR123 1 173 174 (1%) (99%) Lgr5
negative to positive PLR59 18 78 96 (19%) (81%) PLR123 29 39 68
(43%) (57%)
[0370] Table 5 shows cell count ratios of Lgr5-positive and
-negative cells stained by immunocytochemistry using an anti-Lgr5
antibody (antibody 2L36). Number in parenthesis represents the
ratio of Lgr5-positive or -negative cell count.
[0371] Thus, the present inventors concluded that large intestine
CSCs underwent interconversion between the Lgr5-positive and
-negative states and the transition does not require any exogenous
factor and/or niche environment.
Example 7
In Vitro and In Vivo EMT of Lgr5-Positive Large Intestine CSCs
[0372] Mesenchymal-like cells expressing nuclear .beta.-catenin are
considered migratory CSCs and metastatic CSCs that undergo EMT
(Brabletz T, Jung A, Spaderna S, Hlubek F, Kirchner T (2005)
Opinion: migrating cancer stem cells--an integrated concept of
malignant tumor progression. Nat Rev Cancer 5:744-749). Since the
morphology of Lgr5-positive large intestine CSCs was similar to
that of mesenchymal cells, the present inventors tested whether
Lgr5-positive large intestine CSCs correspond to migratory CSCs.
Western blot analysis revealed low level expression of cell-surface
E-cadherin, high level expression of Snail, and nuclear
.beta.-catenin (which is characteristic of EMT) in the
Lgr5-positive large intestine CSCs (FIGS. 13, and 14, and 26). In
contrast, the Lgr5-negative colorectal CSCs did not show any
evidence of EMT. Specifically, cell-surface E-cadherin was
expressed at a high level; Snail was expressed at a low level, and
there was no nuclear localization of .beta.-catenin. Furthermore,
concomitant expression of Snail and Lgr5 was observed in cells that
underwent EMT in budding areas of xenograft tumor tissues (FIG.
15). This finding supports the view that the Lgr5-positive large
intestine CSCs correspond to migratory stem cells.
[0373] In addition, the present inventors demonstrated that the
Lgr5-positive large intestine CSCs formed tumors in multiple
tissues including lung, liver, lymph node, and subcutaneous
tissues. Interestingly, in the liver, lymph node, and subcutaneous
tissues, tumors with epithelial ductal structures were
reconstituted by at least 40 days after intravenous injection of
tumor cells, whereas such structures were not reconstituted in the
lung (FIGS. 34 and 35).
[0374] Next, the present inventors examined whether Lgr5-negative
CSCs directly form the hierarchical organization of cancer or first
undergo transition to Lgr5-positive cells in vivo. To find markers
for detecting Lgr5-negative CSCs, gene expression profiling was
carried out using Lgr5-positive cells, Lgr5-negative cells, and
primary cells from xenograft tumors. As a result, HLA-DMA was
selected from molecules whose expression can be detected at high
level in the Lgr5-negative CSCs as compared to the Lgr5-positive
CSC and primary cells (FIG. 50). By immunohistochemistry using
anti-Lgr5 antibody, anti-HLA-DMA antibody, and anti-EREG antibody,
HLA-DMA was demonstrated to be specifically expressed in the
Lgr5-negative CSCs (FIG. 51). HLA-DMA is also expressed in
macrophages. Then, to rule out the possibility that cells stained
by immunohistochemistry using the anti-HLA-DMA antibody are
macrophages, the present inventors tested not only HLA-DMA but also
other markers expressed in CSCs. Immunohistochemistry using an
antibody against EREG expressed in both Lgr5-positive and -negative
CSCs (FIG. 50) confirmed that EREG was expressed in both of
Lgr5-positive and Lgr5-negative CSCs (FIG. 51). It was demonstrated
that Lgr5-negative CSCs could be identified as cells that are
positive for both HLA-DMA and EREG by detection using both markers
in combination. After injection of a homogeneous population of
Lgr-negative CSCs to NOG mice, cells expressing Lgr5 only weakly
for one day after the injection, which however remained positive
for HLA-DMA and EREG, appeared. Then, cells that are negative for
HLA-DMA but remain positive for Lgr5 and EREG appeared by five days
after the injection (FIG. 52). Tumors derived from Lgr5-negative
CSCs had specific ductal structures and included Lgr5-positive
cells (FIG. 53).
[0375] To probe the possibility of in vivo transition to a growth
inhibitor-resistant state, irinotecan was administered at the
maximum tolerated dose (MTD) (120 mg/kg) into the peritoneal
cavities of NOG mice bearing tumors derived from Lgr5-positive
CSCs. Tumor growth was inhibited almost completely (FIG. 55), and
the ductal structures were collapsed extensively (FIG. 54). This
condition resulted in a dramatic decrease of Lgr5-positive cells
(FIGS. 54 and 56). The number of Lgr5-negative and HLA-DMA-positive
cells increased significantly after irinotecan treatment. By
contrast, in vehicle-treated control mice, about one third of
cancer cells were positive for Lgr5 in both ductal and budding
areas (FIG. 54). Both Lgr5-positive cells and HLA-DMA-positive and
Lgr5-negative cells were positive for EREG, and were identified to
be CSCs (FIG. 54). After irinotecan treatment, Lgr5-positive cells
appeared again (FIG. 54). The results described above, when
considered together, suggest that Lgr5-negative CSCs can be the
origin of colorectal cancer after growth inhibitor treatment and
reconstitute cancer hierarchy via transition to Lgr5-positive
cells.
Example 8
Identification of Molecules Specifically Expressed in Cancer Stem
Cells
1. Preparation of Lgr5-Negative Adherent Cells by Irinotecan
Treatment
[0376] Using a stem cell medium, Lgr5-positive adherent cells were
seeded at 3.times.10.sup.5 cells/well in a 6-well plate (BD, Cat.
No. 353046). On the following day, irinotecan (Hospira,
61703-349-09) was added to cells at a final concentration of 10
.mu.g/ml. After three-day culture, irinotecan-resistant cells were
detected. The cells were harvested using Accutase, and suspended in
FACS buffer. Then, the cells were incubated at 4.degree. C. for 30
minutes with 7-AAD Viability Dye as dead cell staining and each of
the following antibodies as cancer stem cell markers:
[0377] FITC-labeled mouse mAb to human CD326 (EpCAM), PE-labeled
mouse mAb to human CD133/1 (AC133), PE-labeled mouse mAb to human
CD44, PE-labeled mouse mAb to human CD166, PE-labeled mouse mAb to
human CD24, PE-labeled mouse mAb to human CD26, or PE-labeled mouse
mAb to human CD29. To detect Lgr5, the cells were incubated with
the mouse mAb to human Lgr5 at 4.degree. C. for 30 minutes. After
washing once with FACS buffer, the cells were incubated with the
PE-labeled goat Ab to mouse IgG2a at 4.degree. C. for 30 minutes.
Then, after washing once with FACS buffer, the cells were subjected
to flow cytometry analysis. The ALDH activity was detected using
AideFluor Kit according to the procedure recommended by the
manufacturer. Flow cytometry analysis was performed using EPICS
ALTRA. Cells negative for 7-AAD Viability Dye were analyzed for
cancer stem cell markers. The irinotecan-resistant cells were
demonstrated to change from positive to negative for Lgr5.
2. Identification of Molecules Specifically Expressed in Cancer
Stem Cells
[0378] Primary cells from PLR59 and PLR123, high proliferative
Lgr5-positive cancer stem cells prepared by adherent culture of
primary cells, and low proliferative Lgr5-negative cancer stem
cells prepared by irinotecan treatment of the cells as described
above were homogenized mechanically with QIAshredder (Qiagen, Cat.
No. 79654), and RNAs were extracted from them using RNeasy Mini Kit
(Qiagen, Cat. No. 74104) and RNase-Free DNase Set (Qiagen, Cat. No.
79254) according to the procedure recommended by the manufacturer.
The extracted RNAs were analyzed for the purity and quality using
Agilent 2100 Bioanalyzer. Following cRNA synthesis, gene expression
information was obtained using GeneChip (HG-U133 plus2) of
Affymetrix. Data analysis was performed with Microsoft Excel and
Statistics software R. The three types of cells (primary cells,
Lgr5-positive cells, and Lgr5-negative cells) were compared to each
other to make a list of genes whose expression levels are
significantly increased in each cell type. Specifically, raw data
from GeneChip were normalized and log.sub.2 transformed by GCRMA to
calculate differences in the expression level between distinct
sample types (three types: primary cells and Lgr5-positive cells,
Lgr5-positive cells and Lgr5-negative cells, and Lgr5-negative
cells and primary cells). The criteria used for selecting
differently expressed genes were:
(1) genes showing a twofold or more change in Lgr5-positive cells
as compared to primary cells and a twofold or more change in
Lgr5-negative cells as compared to primary cells (expressed at high
levels in both Lgr5-positive and -negative cancer stem cells)
(Table 6-1 to 6-10) (partial amino acid sequences of proteins
encoded by the genes are shown in SEQ ID NOs: 1 to 6, and 9); (2)
genes showing a twofold or more change in Lgr5-positive cells as
compared to primary cells and a less than twofold change in
Lgr5-negative cells as compared to primary cells and (expressed at
a high level in Lgr5-positive cancer stem cells alone) (Table 7-1
to 7-5); (3) genes showing a less than twofold change in
Lgr5-positive cells as compared to primary cells and a twofold or
more change in Lgr5-negative cells as compared to primary cells
(expressed at a high level in Lgr5-negative cancer stem cells
alone) (Table 8-1 and 8-2) (partial amino acid sequences of
proteins encoded by the genes are shown in SEQ ID NOs: 7 and
8).
[0379] Furthermore, to identify genes encoding proteins that are
presented specifically on cell membrane of cancer stem cells, genes
of GO:0005886 [plasma membrane] were extracted from GeneOntology
(GO). Then, the present inventors extracted genes with GO:0005576
[extracellular region], GO:0009986 [cell surface], and GO:0016020
[membrane], or genes which are predicted to have a transmembrane
region by membrane protein prediction software TMHMM and to have a
signal peptide by signal peptide prediction software SignalP, and
which do not have GO:0031090 [organelle membrane]. Furthermore,
with the aid of GeneChip data from normal colorectal tissues, the
present inventors exclude genes whose expression levels are
relatively high in normal tissues or primary cells as well as gene
only showing a small fold-change in Lgr5-positive or Lgr5-negative
cells.
TABLE-US-00007 TABLE 6-1 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary- ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO. NP_036438.2 FAIM2 Fas apoptotic
inhibitory molecule 2 8.4 7.0 5.4 4.1 -- NP_116265.1 JUB jub, ajuba
homolog (Xenopus laevis) 5.8 5.7 6.2 5.5 -- NP_116281.2 FRMD5 FERM
domain containing 5 4.0 6.7 2.5 4.8 -- NP_071731.1 EDAR
ectodysplasin A receptor 7.0 7.6 5.3 4.6 1 NP_001240622.1 MCOLN3
mucolipin 3 3.7 5.1 0.8 2.5 -- NP_001007098.1 NTRK2 neurotrophic
tyrosine kinase, receptor, type 2 3.6 5.2 1.0 2.6 -- NP_001243.1
CD70 CD70 molecule 4.0 4.9 4.1 4.7 2 NP_062818.1 SLCO1B3 solute
carrier organic anion transporter family, 4.9 4.4 3.0 3.9 -- member
1B3 NP_003606.3 SLC4A7 solute carrier family 4, sodium bicarbonate
2.9 4.0 1.6 3.2 -- cotransporter, member 7 NP_005836.2 ABCC4
ATP-binding cassette, sub-family C 4.0 4.0 1.8 2.4 -- (CFTR/MRP),
member 4 NP_003920.1 RAB7L1 RAB7, member RAS oncogene family-like 1
5.5 5.3 5.6 6.0 -- NP_009162.1 SLC6A14 solute carrier family 6
(amino acid 1.9 3.7 4.7 6.3 -- transporter), member 14 NP_872631.1
EFNA4 ephrin-A4 2.6 3.6 1.4 2.5 -- NP_001423.1 EREG epiregulin 2.5
3.3 2.6 3.1 3 NP_001127839.1 SLC6A6 solute carrier family 6
(neurotransmitter 3.3 3.3 0.8 1.6 -- transporter, taurine), member
6 NP_003497.2 FZD6 frizzled homolog 6 (Drosophila) 3.7 3.2 4.6 4.0
-- NP_003264.2 TM7SF2 transmembrane 7 superfamily member 2 4.0 4.2
6.8 6.2 -- NP_001172024.1 AIF1L allograft inflammatory factor
1-like 3.5 2.9 2.3 2.0 -- NP_060033.3 IL17RD interleukin 17
receptor D 3.1 2.8 2.4 3.0 -- NP_000013.2 ADA adenosine deaminase
2.1 2.7 2.8 2.9 -- NP_004834.1 IL27RA interleukin 27 receptor,
alpha 3.6 2.6 2.2 1.8 --
[0380] Table 6-2 is a continuation of Table 6-1.
TABLE-US-00008 TABLE 6-2 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_001731.2 CALB2 calbindin 2 3.8 2.5
2.4 0.5 -- NP_065209.2 ANK1 ankyrin 1, erythrocytic 5.8 2.5 4.9 2.4
-- NP_001157412.1 PYGL phosphorylase, glycogen, liver 1.2 2.5 1.7
2.1 -- NP_001097.2 ACVR2B activin A receptor, type IIB 2.4 2.5 1.5
1.2 -- NP_001129141.1 XPR1 xenotropic and polytropic retrovirus
receptor 1 3.4 2.5 3.5 2.4 -- NP_001523.2 SLC29A2 solute carrier
family 29 (nucleoside transporter), 1.6 2.4 0.5 1.7 -- member 2
NP_563615.3 DCBLD2 discoidin, CUB and LCCL domain containing 2 2.9
5.3 5.2 4.9 -- NP_689522.2 PIK3AP1 phosphoinositide-3-kinase
adaptor protein 1 3.5 2.4 1.3 1.0 -- NP_054772.1 FLVCR1 feline
leukemia virus subgroup C cellular receptor 1 3.5 2.4 1.7 1.2 --
NP_006849.1 TMED1 transmembrane emp24 protein transport domain 2.6
2.3 2.4 1.9 -- containing 1 NP_116254.4 TNS4 tensin 4 1.9 2.3 1.9
1.5 -- NP_001193874.1 CSPG5 chondroitin sulfate proteoglycan 5
(neuroglycan C) 3.9 2.2 1.5 0.5 -- NP_000667.1 ADORA2B adenosine
A2b receptor 1.5 2.1 0.7 1.5 -- NP_064423.2 ACCN2
amiloride-sensitive cation channel 2, neuronal 1.8 1.9 1.7 1.3 --
NP_001018000.1 KAZ kazrin 2.5 1.8 2.0 1.8 -- NP_004773.1 SNAP29
synaptosomal-associated protein, 29 kDa 0.3 1.7 0.8 1.9 --
NP_066292.2 KCNJ12 potassium inwardly-rectifying channel, subfamily
J, 3.4 3.5 4.0 3.4 -- member 12 NP_938205.1 FLRT3 fibronectin
leucine rich transmembrane protein 3 2.8 1.5 7.1 5.0 -- NP_115899.1
PARD6G par-6 partitioning defective 6 homolog gamma 1.8 1.0 2.7 1.9
-- (C. elegans) NP_066924.1 CLDN1 claudin 1 1.4 0.7 3.4 3.5
NP_066939.1 ADCY1 adenylate cyclase 1 (brain) 2.9 2.2 2.1 1.4
--
[0381] Table 6-3 is a continuation of Table 6-2.
TABLE-US-00009 TABLE 6-3 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_001127807.1 AIMP2 aminoacyl tRNA
synthetase complex-interacting 1.0 0.6 1.2 0.7 -- multifunctional
protein 2 NP_619539.1 AKAP7 A kinase (PRKA) anchor protein 7 2.0
1.3 1.4 1.2 -- NP_064715.1 ANKMY2 ankyrin repeat and MYND domain
containing 2 2.7 2.5 3.2 3.4 -- NP_112591.2 APH1B anterior pharynx
defective 1 homolog B (C. elegans) 0.8 1.6 1.1 3.1 -- NP_658985.2
APOA1BP apolipoprotein A-1 binding protein 1.8 1.3 2.2 1.6 --
NP_940852.3 APOOL apolipoprotein O-like 1.6 1.6 2.3 2.3 --
NP_647537.1 ATRN attractin 1.5 2.1 1.1 1.8 -- NP_001193.2 BMP4 bone
morphogenetic protein 4 2.9 4.2 1.9 2.8 9 NP_001720.1 BTC
betacellulin 1.4 2.5 1.7 3.2 -- NP_001224.1 CAV2 caveolin 2 1.9 2.9
3.7 4.2 -- NP_001788.2 CDH11 cadherin 11, type 2, OB-cadherin
(osteoblast) 6.2 2.4 3.8 0.6 -- NP_857592.1 CKLF chemokine-like
factor 0.9 1.6 -0.8 1.1 -- NP_612419.1 CMTM7 CKLF-like MARVEL
transmembrane domain 2.8 4.2 2.3 3.2 -- containing 7 NP_054860.1
CNTNAP2 contactin associated protein-like 2 4.6 4.3 3.8 3.7 --
NP_004738.3 DLG5 discs, large homolog 5 (Drosophila) 1.5 1.0 1.5
1.2 -- NP_001926.2 DPP4 dipeptidyl-peptidase 4 0.1 1.3 2.0 3.8 --
NP_690611.1 FAS Fas (TNF receptor superfamily, member 6) 1.4 0.1
3.4 2.1 -- NP_001099043.1 FBXO45 F-box protein 45 1.0 1.3 0.3 1.2
-- NP_001138390.1 FGFR2 fibroblast growth factor receptor 2 3.9 1.7
3.0 1.0 -- NP_001457.1 FZD2 frizzled homolog 2 (Drosophila) 5.2 5.4
2.6 2.9 -- NP_031379.2 GNA12 guanine nucleotide binding protein (G
protein) alpha 12 1.2 0.5 1.1 0.3 --
[0382] Table 6-4 is a continuation of Table 6-3.
TABLE-US-00010 TABLE 6-4 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_001243343.1 GNAI1 guanine nucleotide
binding protein (G protein), alpha 4.0 4.6 7.6 8.2 -- inhibiting
activity polypeptide 1 NP_002766.1 HTRA1 HtrA serine peptidase 1
5.1 7.0 5.0 6.3 -- NP_001543.2 IGFBP4 insulin-like growth factor
binding protein 4 3.4 2.7 0.5 1.1 -- NP_001002915.2 IGFL2 IGF-like
family member 2 3.0 3.3 2.3 2.5 -- NP_001034659.2 KREMEN1 kringle
containing transmembrane protein 1 3.0 3.7 1.3 2.3 -- NP_005597.3
LGMN legumain 1.1 1.8 0.5 1.4 -- NP_002303.2 LIG4 ligase IV, DNA,
ATP-dependent 3.7 3.7 5.6 5.4 -- NP_006024.1 LIPG lipase,
endothelial 2.1 2.6 0.1 1.1 -- NR_001392.2 LPAR1 lysophosphatidic
acid receptor 1 2.9 3.4 1.3 2.4 -- NP_036284.1 LPAR3
lysophosphatidic acid receptor 3 6.7 4.9 3.7 2.9 -- NP_002327.2
LRP6 low density lipoprotein receptor-related protein 6 2.8 1.8 1.5
0.6 -- NP_055414.2 MAGED2 melanoma antigen family D, 2 1.1 1.8 2.7
2.9 -- NP_005922.2 MICB MHC class I polypeptide-related sequence B
2.6 3.6 2.3 3.7 -- NP_001182555.1 MLLT10 myeloid/lymphoid or
mixed-lineage leukemia 2.5 2.0 1.0 1.3 -- (trithorax homolog,
Drosophila); translocated to, 10 NP_002435.1 MSN moesin 3.0 1.7 2.2
0.0 -- NP_001018169.1 NAE1 NEDD8 activating enzyme E1 subunit 1 1.8
1.7 1.2 1.8 -- NP_056146.1 NCSTN nicastrin 1.5 0.4 2.3 0.8 --
NP_060562.3 NETO2 neuropilin (NRP) and tolloid (TLL)-like 2 7.3 6.1
8.0 6.9 -- NP_009014.2 NUDT6 nudix (nucleoside diphosphate linked
moiety X)-type 3.2 1.9 4.2 2.9 -- motif 6 NP_689501.1 ORAI3 ORAI
calcium release-activated calcium modulator 3 1.5 1.1 4.3 2.2 --
NP_002605.2 PDZK1 PDZ domain containing 1 8.1 8.3 4.9 3.6 --
[0383] Table 6-5 is a continuation of Table 6-4.
TABLE-US-00011 TABLE 6-5 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_055824.1 PDZRN3 PDZ, domain
containing ring finger 3 2.6 2.5 1.4 0.6 -- NP_077734.1 PLA2G4A
phospholipase A2, group IVA (cytosolic, calcium- 7.7 8.2 5.0 5.9 --
dependent) NP_001123508.1 PLEKHB1 pleckstrin homology domain
containing, family B 4.0 5.3 5.1 6.3 -- (evectins) member 1
NP_079501.2 PNPLA3 patatin-like phospholipase domain containing 3
4.4 4.9 1.7 3.5 -- NP_004641.1 PNPLA4 patatin-like phospholipase
domain containing 4 2.3 1.9 2.8 2.2 -- NP_006395.2 PROCR protein C
receptor, endothelial 1.3 1.5 4.2 3.6 4 NP_001159449.1 PROM2
prominin 2 5.4 4.6 10.5 10.4 5 NP_077748.3 PSTPIP2
proline-serine-threonine phosphatase interacting protein 2.2 2.7
5.1 4.9 -- 2 NP_002834.3 PTPRJ protein tyrosine phosphatase,
receptor type, J 2.9 3.0 1.5 2.2 -- NP_002861.1 RAB13 RAB13, member
RAS oncogene family 1.3 0.9 2.0 1.4 -- NP_066361.1 RAP2A RAP2A,
member or RAS oncogene family 2.0 1.7 2.4 1.7 -- NP_001094058.1
RC3H2 ring finger and CCCH-type zinc finger domains 2 1.2 0.3 2.0
0.0 -- NP_002897.1 RDX radixin 4.7 5.3 4.5 4.6 -- NP_006502.1
RSC1A1 regulatory solute carrier protein, family 1, member 1 1.1
0.0 1.2 0.4 -- (DDI2) (DNA-damage inducible 1 homolog 2)
NP_004162.2 SLC1A2 solute carrier family 1 (glial high affinity
glutamate 7.4 7.3 2.1 1.9 -- transporter), member 2 NP_006349.1
SLC25A17 solute carrier family 25 (mitochondrial carrier; 2.4 1.9
2.4 1.5 -- peroxisomal membrane protein, 34 kDa), member 17
NP_075053.2 SLC30A5 solute carrier family 30 (zinc transporter),
member 5 1.3 0.9 1.2 0.8 -- NP_001070253.1 SLC7A6 solute carrier
family 7 (cationic amino acid transporter, 1.0 0.2 1.5 0.8 -- y+
system), member 6 NP_071420.1 SMOC1 SPARC related modular calcium
binding 1 2.6 5.4 -0.1 2.4 -- NP_001159884.1 SMOC2 SPARC related
modular calcium binding 2 7.2 8.4 -0.9 2.2 -- NP_054730.1 SOCS5
suppressor of cytokine signaling 5 2.3 1.7 2.5 1.9 --
[0384] Table 6-6 is a continuation of Table 6-5.
TABLE-US-00012 TABLE 6-6 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_001030127.1 SORBS1 sorbin and SH3
domain containing 1 6.4 4.3 1.1 -0.2 -- NP_003095.2 SORD sorbitol
dehydrogenase 1.5 1.8 1.0 2.1 -- NP_003705.1 STC2 stanniocalcin 2
3.6 3.1 1.2 2.3 -- NP_005810.1 STX6 syntaxin 6 3.4 2.5 4.4 3.2 --
NP_003229.1 TGFB2 transforming growth factor, beta 2 5.3 5.1 4.2
3.6 -- NP_001124388.1 TGFBR1 transforming growth factor, beta
receptor 1 1.9 1.8 1.5 1.0 -- NP_057635.1 TM7SF3 transmembrane 7
superfamily member 3 1.9 1.6 2.7 2.2 -- NP_653233.3 TMEM182
transmembrane protein 182 3.3 3.4 4.6 4.6 -- NP_003802.1 TNFSF9
tumor necrosis factor (ligand) superfamily, member 9 4.5 4.4 5.2
4.9 6 NP_005714.2 TSPAN5 tetraspanin 5 1.8 2.9 0.0 1.5 --
NP_068835.1 UTS2 urotensin 2 2.4 1.7 2.5 2.3 --
[0385] Table 6-7 is a continuation of Table 6-6.
TABLE-US-00013 TABLE 6-7 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID No. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_056222.2 ABHDI4A abhydrolase domain
containing 14A 1.3 0.9 2.7 1.9 -- NP_004449.1 ACSL4 acyl-CoA
synthetase long-chain family member 4 4.5 4.5 3.6 4.2 --
NP_665812.1 AIFM1 apoptosis-inducing factor,
mitochondrion-associated, 1 1.9 1.6 2.0 1.9 -- NP_001344.2 AKR1C1
aido-keto reductase family 1, member Cl (dihydrodiol 9.4 7.8 8.1
2.7 -- dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid
dehydrogenase) NP_940683.1 ANKRD46 ankyrin repeat domain 46 2.6 1.8
3.5 2.2 -- NP_077027.1 APOO apolipoprotein O 1.4 1.5 1.2 1.0 --
NP_004766.2 B4GALT6 UDP-Gal: betaGlcNAc beta
1,4-galactosyltransferase, 2.0 1.5 0.3 1.1 -- polypeptide 6
NP_004326.1 BST2 bone marrow stromal cell antigen 2 -3.9 4.4 -3.9
5.9 -- NP_001185983.1 C16orf5 chromosome 16 open reading frame 5
3.1 2.6 6.9 5.4 -- NP_115700.1 C1orf57 chromosome 1 open reading
frame 57 1.5 1.3 3.0 3.0 -- NP_653181.1 C1orf85 chromosome 1 open
reading flame 85 1.1 0.8 2.9 2.4 -- NP_001074293.1 C2orf89
chromosome 2 open reading frame 89 1.9 1.7 0.8 1.1 -- NP_775823.1
C3orf58 chromosome 3 open reading frame 58 2.0 0.3 1.6 -0.4 --
NP_439896.1 C6orf192 chromosome 6 open reading frame 192 4.0 2.5
4.5 3.3 -- NP_001135942.1 C6orf203 chromosome 6 open reading frame
203 1.2 1.0 2.0 1.8 -- NP_620140.1 C6orf72 chromosome 6 open
reading frame 72 1.3 1.8 1.2 1.7 -- NP_001243894.1 CCDC51
coiled-coil domain containing 51 1.1 0.9 3.6 3.1 -- NP_ 001157882.1
CDK5 cyclin-dependent kinase 5 1.9 2.0 3.5 3.3 -- NP_055061.1
CELSR1 cadherin, EGF LAG seven-pass G-type receptor 1 0.5 1.5 0.6
1.1 -- (flamingo homolog, Drosophila) NP_004077.1 COCH coagulation
factor C homolog, cochlin (Limulus 2.1 2.3 0.5 2.2 -- polyphemus)
NP_001896.2 CTPS CTP synthase 2.0 1.0 1.4 0.7 -- NP_055191.2 CYFIP2
cytoplasmic FMR1 interacting protein 2 2.6 3.9 5.0 5.3 --
NP_004393.1 DFFB DNA fragmentation factor, 40 kDa, beta polypeptide
2.3 1.8 1.7 1.5 -- (caspase-activated DNase) NP_001077058.1 E2F5
E2F transcription factor 5, p130-binding 2.8 2.2 2.7 2.2 --
[0386] Table 6-8 is a continuation of Table 6-7.
TABLE-US-00014 TABLE 6-8 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_056067 2 EHBP1 EH domain binding
protein 1 2.3 1.9 1.1 0.4 -- NP_060682.2 ENAH enabled homolog
(Drosophila) 4.4 3.5 1.5 1.2 -- NP_001180289.1 EXD2 exonuclease
3'-5' domain containing 2 2.3 1.2 2.7 1.9 -- NP_660323.3 FAM119A
family with sequence similarity 119, member A 5.4 5.1 3.0 3.7 --
NP_620775.2 FAM175A family with sequence similarity 175, member A
3.4 2.8 3.8 2.6 -- NP_937995.1 FAM189B family with sequence
similarity 189, member B 1.7 1.7 1.3 1.1 -- NP_055679.1 FAM20B
family with sequence similarity 20, member B 1.6 0.8 1.5 0.8 --
NP_942600.1 FIBP fibroblast growth factor (acidic) intracellular
binding 1.0 1.2 2.0 1.7 -- protein NP_000139.1 FUT1
fucosyltransferase 1 (galactoside 2-alpha-L- 7.0 5.6 3.1 4.0 --
fucosyltransferase, H blood group) NP_000135.2 FXN frataxin 1.7 1.1
2.0 1.6 -- NP_000393.4 G6PD glucose-6-phosphate dehydrogenase 0.8
1.0 2.0 1.4 -- NP_000143.2 GAA glucosidase, alpha; acid 1.2 1.4 3.3
2.2 -- NP_000160.1 GLA galactosidase, alpha 2.0 1.1 1.5 0.9 --
NP_002072.2 GPC1 glypican 1 1.0 1.2 2.6 0.5 -- NP_001008398.2 GPX8
glutathione peroxidase 8 (putative) 5.0 5.5 7.1 7.0 -- NP_005329.3
HIP1 huntingtin interacting protein 1 2.6 2.7 2.6 2.5 --
NP_254274.1 IL33 interleukin 33 -0.5 1.2 3.6 3.4 -- NP_002262.3
IPO5 importin 5 3.6 2.3 1.9 0.4 -- NP_060573.2 LRRC8D leucine rich
repeat containing 8 family, member D 2.0 1.0 1.9 1.2 -- NP_067679.6
MFAP3L microfibrillar-associated protein 3-like 1.5 0.2 3.3 1.2 --
NP_612440.1 MFSD3 major facilitator superfamily domain containing 3
0.9 1.3 2.0 2.4 -- NP_066014.1 MOV10 Mov10, Moloney leukemia virus
10, homolog (mouse) 0.5 1.4 1.3 2.0 -- NP_036351.3 MRAS muscle RAS
oncogene homolog 1.1 0.6 4.3 1.3 -- NP_057034.2 MRPL2 mitochondrial
ribosomal protein L2 1.3 0.8 0.8 0.0 -- NP_065972.3 NIN ninein
(GSK3B interacting protein) 2.0 3.1 2.7 3.0 --
[0387] Table 6-9 is a continuation of Table 6-8.
TABLE-US-00015 TABLE 6-9 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_065181.1 NIPAL3 NIPA-like domain
containing 3 1.7 1.3 3.0 2.3 -- NP_002504.2 NME3 non-metastatic
cells 3, protein expressed in 1.1 2.1 1.5 1.9 -- NP_057672.1 NRN1
neuritin 1 8.5 5.7 7.7 6.3 -- NP_689643.2 OR51E1 olfactory
receptor, family 51, subfamily E, member 1 6.9 9.8 0.3 3.0 --
NP_071748.2 OSGEPL1 O-sialoglycoprotein endopeptidase-like 1 4.1
4.7 3.1 4.4 -- NP_079431.1 PAAF1 proteasomel ATPase-associated
factor 1 1.9 2.1 1.5 1.9 -- NP_000523.2 PCCB propionyl CoA
carboxylase, beta polypeptide 1.4 1.6 1.4 2.0 -- NP_061757.1
PCDHB14 protocadherin beta 14 1.4 0.5 1.4 0.1 -- NP_002622.2 PGD
phosphogluconate dehydrogenase 1.2 1.4 1.2 1.2 -- NP_003550.1
PIP4K2B phosphatidylinositol-5-phosphate 4-kinase, type II, beta
1.8 1.3 1.8 1.1 -- NP_056530.2 PLA2G3 phospholipase A2, group III
2.6 4.3 0.1 3.2 -- proteasome (prosome, macropain) 26S subunit,
non- NP_005038.1 PSMD5 ATPase, 5 1.5 0.7 1.7 0.7 -- NP_006255.1
PTPN13 protein tyrosine phosphatase, non-receptor type 13 0.1 1.1
0.7 2.2 -- (APO-1/CD95 (Fas)-associated phosphatase) NP_057161.1
PTRH2 peptidyl-tRNA hydrolase 2 1.4 1.4 1.5 1.8 -- NP_037390.2
PYCARD PYD and CARD domain containing 0.2 1.3 1.1 2.1 --
NP_055113.2 QPRT quinolinate phosphoribosyltransferase 2.5 3.6 3.1
4.5 -- NP_060233.3 RNF43 ring finger protein 43 2.7 2.7 1.2 1.5 --
NP_060616.1 RNMTL1 RNA methyltransferase like 1 1.5 0.9 1.7 1.1 --
NP_003698.1 RUVBL1 RuvB-like 1 (E. coli) 1.9 2.2 1.3 2.2 --
NP_002949.2 RYK RYK receptor-like tyrosine kinase 1.5 1.0 1.2 1.2
-- NP_116250.3 SERAC1 serine active site containing 1 1.8 0.8 2.7
1.5 -- NP_005016.1 SERPINI1 serpin peptidase inhibitor, clade I
(neuroserpin), 5.5 8.7 2.1 4.2 -- member 1 NP_008927.1 SLC19A2
solute carrier family 19 (thiamine transporter), member 2 3.8 2.7
2.0 1.2 -- NP_065075.1 SLC39A10 solute carrier family 39 (zinc
transporter), member 10 2.9 3.1 2.6 2.7 -- NP_060306.3 SLC41A3
solute carrier family 41, member 3 1.9 1.5 1.3 0.8 --
[0388] Table 6-10 is a continuation of Table 6-9.
TABLE-US-00016 TABLE 6-10 PLR59 PLR123 PLR59 PLP123 SEQ DB
accession primary primary primary primary ID NO. Abbreviation
Molecule name Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_005692.1 SNUPN
snurportin 1 2.2 2.4 2.3 2.5 -- NP_055563.1 SNX17 sorting nexin 17
0.8 1.1 1.0 1.0 -- NP_003156.1 STXBP1 syntaxin binding protein 1
4.3 5.7 4.9 5.3 -- NP_003192.1 TFAM transcription factor A,
mitochondrial 2.1 1.6 1.1 1.6 -- NP_444283.2 THEM4 thioesterase
superfamily member 4 1.6 -0.5 2.8 0.4 -- NP_612472.1 TLCD1 TLC
domain containing 1 2.7 2.1 3.9 4.2 -- NP_057548.1 TMEM138
transmembrane protein 138 1.8 2.2 2.0 2.7 -- NP_085054.2 TMEM177
transmembrane protein 177 3.1 3.2 3.5 4.1 -- NP_056236.2 TMEM186
transmembrane protein 186 2.4 2.0 2.0 2.3 -- NP_078863.2 TMEM53
transmembrane protein 53 1.2 1.3 1.2 1.0 -- NP_001008495.2 TMEM64
transmembrane protein 64 6.7 5.7 8.3 8.0 -- NP_689630.1 TMBM68
transmembrane protein 68 1.8 1.8 3.4 2.9 -- NP_861448.2 TMTC3
transmembrane and tetratricopeptide repeat 2.5 1.8 3.8 2.3 --
containing 3 NP_775107.1 TRIM59 tripartite motif-containing 59 3.7
4.3 3.0 4.0 -- NP_003293.2 TRIP6 thyroid hormone receptor
interactor 6 2.7 1.3 5.1 3.3 -- NP_009215.1 TWF2 twinfilin,
actin-binding protein, homolog 2 (Drosophila) 0.6 1.5 1.4 1.8 --
NP_079094.1 UBA5 ubiquitin-like modifier activating enzyme 5 1.8
2.1 2.4 2.4 -- NP_060769.4 UBE2W ubiquitin-conjugating enzyme E2W
(putative) 1.3 1.7 2.4 2.4 -- NP_001017980.1 VMA21 VMA21 vacuolar H
+- ATPase homolog (S. cerevisiae) 3.0 2.6 2.9 2.6 -- NP_660295.2
ZG16B zymogen granule protein 16 homolog B (rat) 1.1 2.0 1.4 1.9 --
NP_115549.2 ZNRF3 zinc and ring finger 3 4.7 4.1 2.2 2.6 --
(The values in Tables 6-1 to 6-10 shown above represent the
expression difference (log 2 ratio).)
TABLE-US-00017 TABLE 7-1 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lar5+ Lgr5+ Lgr5- Lgr5- NO NP_003658.1 LGR5 leucine-rich
repeat-containing G protein-coupled 3.0 3.8 -1.5 0.4 -- receptor 5
NP_066301.1 SNTB1 syntrophin, beta 1 (dystrophin-associated protein
A1, 3.1 3.1 0.0 0.4 -- 59 kDa, basic component 1) NP_001123575.1
COL13A1 collagen, type XIII, alpha 1 2.0 2.9 -0.2 0.3 --
NP_001167538.1 FGFR1 fibroblast growth factor receptor 1 1.3 2.3
-0.7 0.6 -- NP_004432.1 EPHB1 EPH receptor B1 1.0 2.3 -0.3 0.7 --
NP_002002.3 FGFR4 fibroblast growth factor receptor 4 2.0 2.2 -1.9
-0.5 -- NP_004622.2 LRP8 low density lipoprotein receptor-related
protein 8, 2.3 2.1 0.5 0.7 -- apolipoprotein e receptor NP_002832.3
PTPRG protein tyrosine phosphatase, receptor type, G 1.8 2.1 -5.0
-5.4 -- NP_004727.2 XPR1 xenotropic and polytropic retrovirus
receptor 1 1.5 2.1 0.7 0.9 -- NP_859052.3 QSOX2 quiescin Q6
suithydryl oxidase 2 1.8 2.0 0.7 0.8 -- NP_003876.1 CDK5R1
cyclin-dependent kinase 5, regulatory subunit 1 (p35) 3.1 3.3 0.3
2.0 -- NP_002821.1 PTPN4 protein tyrosine phosphatase, non-receptor
type 4 1.7 1.9 0.3 0.7 -- (megakaryocyte) NP_001457.1 FZD2 frizzled
homolog 2 (Drosophila) 5.2 5.4 2.6 2.9 -- NP_000674.2 ADRA2C
adrenergic, alpha-2C-, receptor 1.8 1.6 0.1 -0.1 -- NP_000334.1
SLC5A1 solute carrier family 5 (sodium/glucose cotransporter), 2.2
1.6 0.2 0.6 -- member 1 NP_005901.2 MAPT microtubule-associated
protein tau 1.9 1.5 0.4 0.6 -- NP_598328.1 SYN2 synapsin II 0.3 1.7
0.2 0.5 -- NP_005496.4 SCARB1 scavenger receptor class B, member 1
1.1 1.2 -0.3 -0.3 -- NP_004434.2 EPHB3 EPH receptor B3 0.7 1.0 -0.5
-0.4 -- NP_000259.1 NF2 neurofibromin 2 (merlin) 1 2 0.9 0.6 0.1 --
NP_ 003477.4 SLC7A5 solute carrier family 7 (cationic amino acid
2.0 0.8 -0.4 -0.7 -- transporter, y+ system), member 5 NP_054740.3
SSX2IP synovial sarcoma, X breakpoint 2 interacting protein 1.8 0.7
0.9 -0.3 -- NP_055736 2 LPHN1 latrophilin 1 1.4 0.6 0.9 0.3 --
NP_004435.3 EPHB4 EPH receptor B4 1.3 0.5 0.7 -0.1 --
[0389] Table 7-2 is a continuation of Table 7-1.
TABLE-US-00018 TABLE 7-2 DB PLR59 PLR123 PLR59 PLR123 SEQ accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_116197.4 LINGO1 leucine rich repeat
and Ig domain containing 1 1.2 0.0 0.6 -1.3 -- NP_004987.2 ABCC1
ATP-binding cassette, sub-family C (CPTR/MRP), 1.1 0.9 0.0 -0.2 --
member 1 -- NP_003174.3 ADAM17 ADAM metallopeptidase domain 17 1.0
0.3 -0.3 -0.4 -- NP_620686.1 ADAMTS15 ADAM metallopeptidase with
thrombospondin type 1 6.1 4.6 -3.6 -4.2 -- motif, 15 -- NP_005091.2
AKAP12 A kinase (PRKA) anchor protein 12 2.4 3.7 -1.2 0.3 --
NP_001618.2 ALCAM activated leukocyte cell adhesion molecule 1.3
0.9 0.3 0.6 -- NP_001648.1 AREGB amphiregulin B 1.7 0.4 0.0 -0.5 --
NP_001164.2 ARHGAP5 Rho GTPase activating protein 5 1.2 0.4 0.8 0.3
-- NP_542172.2 B3GALT6 UDP-Gal: betaGal beta
1,3-galactosyltransferase 1.1 0.6 0.6 0.3 -- polypeptide 6
NP_001711.2 BMP8B bone morphogenetic protein 8b 0.9 1.3 0.4 0.3 --
NP_006560.3 CGREF1 cell growth regulator with EF-hand domain 1 0.9
1.0 0.3 0.8 -- NP_058647.1 CKLF chemokine-like factor 0.9 1.5 -0.5
0.9 -- NP_849199.2 CMTM8 CKLF-like MARVEL transmembrane domain
containing 1.3 1.4 0.6 0.7 -- 8 NP_001422.1 EPB41L2 erythrocyte
membrane protein band 4. 1-like 2 1.3 0.9 -0.4 -0.4 -- NP_004433.2
EPHB2 EPH receptor B2 1.0 0.5 -3.1 -2.8 -- NP_000496.2 F12
coagulation factor XII (Hageman factor) 1.2 1.6 0.2 1.0 --
NP_057133.2 FAM158A family with sequence similarity 158, member A
0.8 1.2 0.1 0.4 -- NP_001990.2 FBN2 fibrillin 2 2.6 2.5 -0.4 0.1 --
NP_068656.2| FGG fibrinogen gamma chain 1.3 0.6 0.6 0.5 -- (OSMR)
(oncostatin M receptor) NP_001439.2 GPC4 glypican 4 2.0 2.1 -0.9
0.0 -- NP_065857.1 GPHN gephyrin 3.2 2.9 -3.4 -2.6 -- NP_057399.1
GULP1 GULP, engulfment adaptor PTB domain containing 1 1.4 2.0 0.4
0.9 -- NP_036616.2 HMMR hyaluronan-mediated motility receptor
(RHAMM) 5.0 4.5 -1.7 -1.5 -- NP_000866.1 IGF1R insulin-like growth
factor 1 receptor 0.7 1.1 -1.9 -1.2 --
[0390] Table 7-3 is a continuation of Table 7-2.
TABLE-US-00019 TABLE 7-3 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_000588.2 IGFBP2 insulin-like growth
factor binding protein 2, 36 kDa 1.4 0.7 0.3 -0.4 -- NP_061195.2
IL17RB interleukin 17 receptor B 1.0 1.2 -2.7 -1.9 -- NP_002326.2
LRP5 low density lipoprotein receptor-related protein 5 1.2 1.7
-0.8 -0.2 -- NP_006491.2| MCAM melanoma cell adhesion molecule 2.1
1.5 -0.2 -0.4 -- NP_055456.2 MDC1 mediator of DNA-damage checkpoint
1 2.6 2.2 -1.3 -1.7 -- NP_055606.1 MELK maternal embryonic leucine
zipper kinase 1.5 1.6 -2.0 -1.1 -- NP_000236.2 MET met
proto-oncogene (hepatocyte growth factor receptor) 1.2 1.2 0.6 0.4
-- NP_065825.1 MIB1 mindbomb homolog a (Drosophila) 1.3 1.3 0.7 0.5
-- NP_005952.2 MUC6 mucin 6, oligomeric mucus/gel-forming 1.1 3.0
-0.1 1.0 -- NP_777596.3 PCSK9 proprotein convertase
subtilisin/kexin type 9 1.5 1.8 -1.6 -0.4 -- NP_003619.2 PKP4
plakophilin 4 1.5 1.4 0.0 -0.1 -- NP_003042.3 SLC16A1 solute
carrier family 16, member 1 (monocarboxylic acid 1.7 0.6 0.3 0.3 --
transporter 1) NP_057438.3 SLCO4A1 solute carrier organic anion
transporter family, member 0.5 1.2 -1.4 -0.2 -- 4A1 NP_003093.2
SOD3 superoxide dismutase 3, extracellular 2.2 1.7 0.6 -1.2 --
NP_006425.2 SORBS1 sorbin and SH3 domain containing 1 3.2 3.4 -0.9
0.2 -- NP_003095.2 SORD sorbitol dehydrogenase 1.1 0.7 0.9 0.8 --
NP_000342.2 STS steroid sulfatase (microsomal), isozyme S 1.8 2.2
-0.5 -0.7 -- NP_003234.2 TGFBR3 transforming growth factor, beta
receptor III 1.4 1.8 -0.2 -0.2 -- NP_055388.2 TMEM97 transmembrane
protein 97 2.6 2.1 1.0 -0.3 -- CAA26435.1 TRAC T cell receptor
alpha constant 0.0 1.1 0.0 0.0 --
[0391] Table 7-4 is a continuation of Table 7-3.
TABLE-US-00020 TABLE 7-4 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_075417.2 AACS acetoacetyl-CoA
synthetase 1.1 1.6 0.2 0.6 -- NP_653191.2 ANKRD22 ankyrin repeat
domain 22 2.0 1.5 -1.8 -1.7 -- NP_056020.2 ATP11A ATPase, class VI,
type 11A 1.3 1.0 0.7 0.6 -- NP_001159.2 BIRC5 baculoviral IAP
repeat-containing 5 2.7 2.4 -4.8 -4.4 -- NP_061189.2 CDCA7L cell
division cycle associated 7-like 2.1 1.8 -0.6 0.4 -- NP_005183.2
CDKN3 cyclin-dependent kinase inhibitor 3 4 8 5.3 -3.1 -1.8 --
NP_004077.1 COCH coagulation factor C homolog, cochlin (Limulus 1.2
1.2 -0.4 0.7 -- polyphemus) NP_005302.3 GRB10 growth factor
receptor-bound protein 10 1.1 0.4 -1.2 -1.5 -- NP_671704.3 HS6ST2
heparan sulfate 6-O-sulfotransferase 2 1.8 1.6 -1.1 -0.2 --
NP_002262.3 IPO5 importin 5 2.6 1.7 0.5 -0.3 -- NP_114428.1 ITFG3
integrin alpha FG-GAP repeat containing 3 1.0 0.9 0.0 -1.1 --
NP_002241.1 KCNN4 potassium intermediate/small conductance calcium-
1.2 0.9 -0.7 -0.7 -- activated channel, subfamily N, member 4
NP_061159.1 KIAA1199 KIAA1199 1.1 1.1 -1.5 -2.4 -- NP_115940.2
KISS1R KISS1 receptor 1.1 1.8 -3.0 0.2 -- NP_057034.2 MRPL2
mitochondrial ribosomal protein L2 1.3 0.8 0.8 0.0 -- NP_ 000242.1
MSH2 mutS homolog 2, colon cancer, nonpolyposis type 1 (E. 2.5 2.8
-0.8 -0.4 -- coli) NP_055452.3 MTFR1 mitochondrial fission
regulator 1 1.6 0.9 0.2 -0.1 -- NP_005947.3 MTHFD1
methylenetetrahydrofolate dehydrogenase (NADP+ 1.7 1.6 0.3 0.1 --
dependent) 1, methenyltetrahydrofolate cyclohydrolase,
formyltetrahydrofolate synthetase NP_005366.2 MYB v-myb
myeloblastosis viral oncogene homolog (avian) 2.7 2.3 -4.4 0.2 --
NP_078938.2 NAT10 N-acetyltransferase 10 (GCN5-related) 1.9 1.3 0.1
-0.1 -- NP_777549.1 NDUFAF2 NADH dehydrogenase (ubiquinone) 1 alpha
1.0 1.4 -0.9 0.1 -- subcomplex, assembly factor 2 NP_004280.5
NFE2L3 nuclear factor (erythroid-derived 2)-like 3 1.2 1.2 -0.9
-0.1 -- NP_006672.1 NMU neuromedin U 0.2 1.6 -4.7 0.2 --
NP_055950.1 NUP205 nucleoporin 205 kDa 1.9 1.7 -0.3 -0.2 --
[0392] Table 7-5 is a continuation of Table 7-4.
TABLE-US-00021 TABLE 7-5 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_942590.1 NUP43 nucleoporin 43 kDa 1.3
0.9 0.0 0.0 -- NP_055484.3 NUP93 nucleoporin 93 kDa 2.1 2.4 0.1 0.8
-- NP_006614 2 PHGDH phosphoglycerate dehydrogenase 3.0 3.4 -2.0
-0.4 -- NP_060904.2 RNF130 ring finger protein 130 0.9 1.0 0.4 0.3
-- NP_060259.4 SEMA4C sema domain, immunoglobulin domain (Ig), 1.9
2.3 0.2 0.5 -- transmembrane domain (TM) and short cytoplasmic
domain, (semaphorin) 4C NP_005857.1 SIGMAR1 sigma non-opioid
intracellular receptor 1 1.0 1.1 0.8 0.9 -- NP_055413.1 SOCS7
suppressor of cytokine signaling 7 1.0 0.6 0.7 0.1 -- NP_005554.1
STMN1 stathmin 1 1 9 2.1 -3.1 -1.4 -- NP_054897.4 STXBP6 syntaxin
binding protein 6 (amisyn) 2.1 2.1 -3.4 -0.1 -- NP_001070884.1
TMEM231 transmembrane protein 231 0.5 1.5 -2.2 -0.8 -- NP_002537.3
TNFRSF11B tumor necrosis factor receptor superfamily, member 11b
1.7 6.3 -2.9 -0.9 -- NP_443195.1 TOP1MT topoisomerase (DNA) I,
mitochondrial 1.1 0.4 0.5 -0.1 -- NP_001058.2 TOP2A topoisomerase
(DNA) II alpha 170 kDa 2.2 2.7 -6.1 -3.1 -- NP_006364.2 VAT1
vesicle amine transport protein 1 homolog (T. 1.0 1.5 0.5 1.0 --
californica) NP_612471.1 ZMYND19 zinc finger, MYND-type containing
19 1.2 0.7 0.7 0.3 --
(The values in Tables 7-1 to 7-5 shown above represent the
expression difference (log 2 ratio).)
TABLE-US-00022 TABLE 8-1 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_076404.3 GPR87 G protein-coupled
receptor 87 0.0 0.0 7.1 7.4 -- NP_005109.2 TNFSF15 tumor necrosis
factor (ligand) superfamily, member 15 -2.4 -2.3 3.5 3.3 --
NP_001926.2 DPP4 dipeptidyl-peptidase 4 0.1 1.3 2.0 3.8 --
NP_149017.2 BBS4 Bardet-Biedl syndrome 4 0.2 0.6 1.7 2.6 --
NP_542386.1 C9orf30 chromosome 9 open reading frame 30 0.2 0.2 1.5
1.7 -- NP_001447.2 FLNA filamin A, alpha -0.4 0.8 0.7 1.7 --
NP_000425.1 NEU1 sialidase 1 (lysosomal sialidase) 0.3 0.4 1.9 1.7
-- NP_002125.3 HMOX2 heme oxygenase (decycling) 2 0.3 0.4 1.5 1.6
-- NP_001078.2 AAMP angio-associated, migratory cell protein 0.1
0.3 1.2 1.6 -- NP_061985.2 ABCA7 ATP-binding cassette, sub-family A
(ABC1), member 7 -1.3 0.4 2.1 1.6 -- NP_000401.1 HFE
hemochromatosis 2.2 2.0 1.2 2.8 -- NP_001142.2 SLC25A4 solute
carrier family 25 (mitochondrial carrier; adenine 0.6 0.8 1.3 1.5
-- nucleotide translocator), member 4 NP_006569.1 GNB5 guanine
nucleotide binding protein (G protein), beta 5 0.2 0.8 0.9 1.4 --
NP_005846.1 RAMP1 receptor (G protein-coupled) activity modifying
protein 1 -0.5 -0.5 1.1 1.4 -- NP_598378.3 RHOV ras homolog gene
family, member V 0.1 -0.2 0.5 1.4 -- NP_112178.2 PVRL4 poliovirus
receptor-related 4 -0.3 -2.8 6.1 1.8 7 NP_003686.1 LY6D lymphocyte
antigen 6 complex, locus D -3.8 -1.8 -0.5 2.7 -- NP_775876.1 KCNRG
potassium channel regulator 1.0 0.2 2.6 1.9 -- NP_005063.1 SLC12A4
solute carrier family 12 (potassium/chloride -0.2 -0.1 1.2 1.2 --
transporters), member 4 NP_005292.2 GPR35 G protein-coupled
receptor 35 -0.8 -1.4 1.1 -0.8 -- NP_001607.1 ACVR2A activin A
receptor. type IIA 0.2 -0.5 1.4 1.0 -- NP_001687.1 ATP6V1E1 ATPase,
H+ transporting, lysosomal 31 kDa, V1 subunit 0.1 0.3 1.1 1.2 -- E1
NP_036474.1 BAMBI BMP and activin membrane-bound inhibitor homolog
-0.8 0.7 1.3 2.4 -- (Xenopus laevis) NP_005177.2 CAPN1 calpain 1,
(mu/l) large subunit -1.3 -1.3 1.3 -0.2 --
[0393] Table 8-2 is a continuation of Table 8-1.
TABLE-US-00023 TABLE 8-2 PLR59 PLR123 PLR59 PLR123 SEQ DB accession
primary primary primary primary ID NO. Abbreviation Molecule name
Lgr5+ Lgr5+ Lgr5- Lgr5- NO NP_001798.2 CEL carboxyl ester lipase
(bile salt-stimulated lipase) 0.0 0.0 7.4 5.4 -- NP_056034.2 EXOC7
exocyst complex component 7 -0.2 -0.2 1.9 1.2 -- NP_000034.1 FAS
Fas (TNF receptor superfamily, member 6) 0.8 0.4 2.8 2.3 8
NP_005802.1 GDF11 growth differentiation factor 11 0.7 0.9 1.3 1.9
-- NP_060456.3 GPR172B G protein-coupled receptor 172B 0.0 0.0 5.6
4.3 -- NP_665895.1 KLK10 kallikrein-related peptidase 10 -0.9 0.4
1.2 1.6 -- NP_055414.2 MAGED2 melanoma antigen family D, 2 0.0 0.3
2.1 3.0 -- NP_000519.2 MAN2B1 mannosidase, alpha, class 2B, member
1 -0.2 -0.2 2.3 1.5 -- NP_002434.1 MSMB microseminoprotein, beta-
0.0 0.0 5.6 4.7 -- NP_009014.2 NUDT6 nudix (nucleoside diphosphate
linked moiety X)-type 0.0 0.0 1.3 0.8 -- motif 6 NP_036528.1 PHLDA3
pleckstrin homology-like domain, family A, member 3 -0.3 -0.2 2.9
1.0 -- NP_002629.1 PI3 peptidase inhibitor 3, skin-derived -7.6
-7.9 1.4 -0.6 -- NP_597998.1 SAT2 spermidine/spermine
N1-acetyltransferase family 0.7 0.9 1.7 1.2 -- member 2 NP_006207.1
SERPINE2 serpin peptidase inhibitor, clade E (pexin, plasminogen
3.2 -2.5 1.3 0.7 -- activator inhibitor type 1), member 2
NP_036303.1 TSPAN17 tetraspanin 17 0.6 0.4 1.7 1.3 --
(The values in Tables 8-1 and 8-2 shown above represent the
expression difference (log 2 ratio).)
[0394] Furthermore, genes that meet a criterion described below and
have GO:0005886 [plasma membrane] from GeneOntology (GO) (Tables 9
and 10) were extracted in order to obtain genes encoding proteins
that are specifically presented on cell membrane of cancer stem
cells.
[0395] Markers Common for Both Proliferating and Quiescent
CSCs:
genes whose expression levels are in average greater than 64 in
Lgr5-negative and Lgr5-positive cells; which show a greater than
four-fold change in both Lgr5-negative and Lgr5-positive cells
relative to primary cells; and which show a significant difference
by t-test (Table 9).
TABLE-US-00024 TABLE 9 PLR59 PLR123 PLR59 PLR123 PLR59 PLR123 SEQ
DB accession primary primary primary primary Lgr5+ Lgr 5+ ID NO.
Abbreviation Molecule name Lgr5+ Lgr5+ LgrS- Lgr5- Lgr5- Lgr5- NO
NP_001423.1 EREG epiregulin 2.2 3.4 2.5 3.1 0.3 -0.3 3 NP_001986.2
ACSL1 acyl-CoA synthetase long-chain family member 1 3.0 3.9 2.9
3.7 -0.2 -0.2 -- NP_005922.2 MICB MHC class I polypeptide-related
sequence B 2.3 3.8 2.2 3.7 -0.1 -0.1 -- NP_000296.2 PON2
paraoxonase 2 2.3 4.6 2.9 4.6 0.7 0.0 -- NP_00 3458.1 CXCR4
chemokine (C--X--C motif) receptor 4 10.2 9.5 8.5 7.8 -1.7 -1.7 --
NP_062818.1 SLCO1B3 solute carrier organic anion transporter
family, 4.4 5.1 3.0 4.1 -1.4 -1.0 -- member 1b3 NP_000280.1 PFKM
phosphofructokinase, muscle 3.3 2.3 3.3 2.1 0.0 -0.2 -- NP_057672.1
NRN1 neuritin 1 8.8 8.5 7.8 6.5 -1.0 -2.0 -- NP_060369.3 TESC
tescalcin 3.3 5.0 2.0 3.6 -1.3 -1.3 -- NP_000139.1 FUT1
fucosyltransferase 1 (galactoside 2-alpha-L- 6.9 5.9 3.1 4.3 -3.9
-1.6 -- fucosyltransferase, H blood group) NP_003802.1 TNFSF9 tumor
necrosis factor (ligand) superfamily member 9 4.5 4.4 5.4 5.1 0.9
0.7 -- NP_059108.1 FZD3 frizzled homolog 3 (Drosophila) 2.8 2.6 2.6
3.2 -0.2 0.6 -- NP_005810.1 STX6 syntaxin 6 2.8 2.7 4.2 3.5 1.4 0.9
-- NP_057635.1 TM7SF3 transmembrane 7 superfamily member 3 3.0 2.5
3.9 3.0 0.9 0.5 -- NP_000013.2 ADA adenosine deaminase 2.0 2.7 2.8
2.9 0.8 0.2 -- NP_071731.1 EDAR ectodysplasin A receptor 6.2 6.8
5.0 3.7 -1.2 -3.1 -- NR_003264.2 TM7SF2 transmembrane 7 superfamily
member 2 3.6 4.3 6.9 6.7 3.2 2.4 -- NP_116265.1 JUB jub, ajuba
homolog (Xenopus laevis) 3.7 4.3 4.7 3.8 1.0 -0.5 -- NP_689740.2
SLC16A14 solute carrier family 16, member 14 5.4 6.6 5.8 6.1 0.3 -0
4 -- (monocarboxylic acid transporter 14) NP_065209.2 ANK1 ankyrin
1, erythrocytic 6.6 3.0 5.3 2.7 -1.3 -0.3 --
[0396] Quiescent CSC-specific markers: genes whose expression
levels are in average greater than 64 in Lgr5-negative cells and
are in average less than 64 in both primary cells and Lgr5-positive
cells; which show a greater than 20 fold change in Lgr5-negative
cells relative to Lgr5-positive cells; and which show a significant
difference by t-test (Table 10).
TABLE-US-00025 TABLE 10 PLR59 PLR123 PLR59 PLR123 PLR59 PLR123 SEQ
DB accession primary primary primary primary Lgr5+ Lgr5+ ID NO.
Abbreviation Molecule name Lgr5+ Lgr5+ Lgr5- Lgr5- Igr5- Lgr5- NO
NP_006111.2 HLA-DMA major histocompatibility complex, class II, DM
alpha 0.0 0.2 8.0 8.7 8.0 8.5 -- NP_862830.1 AMIGO2 adhesion
molecule with Ig-like domain 2 0.0 0.0 7.0 7.5 7.0 7.5 --
NP_001159449.1 PROM2 prominin 2 3.3 2.9 10.1 10.5 6.9 7.6 5
NP_076404.3 GPR87 G protein-coupled receptor 87 0.0 0.0 7.0 7.4 7.0
7.4 -- NP_722582.2 GPR110 G protein-coupled receptor 110 0.0 0.0
6.0 7.0 6.0 7.0 -- NP_112178.2 PVRL4 poliovirus receptor-related 4
-0.6 -2.7 5.8 1.7 6.4 4.4 7 NP_938205.1 FLRT3 fibronectin leucine
rich transmembrane protein 3 1.3 0.2 6.8 5.4 5.5 5.2 --
3. Expression Analysis by Flow Cytometry Analysis
3.1. Flow Cytometry Analysis of NOG-Established Cancer Cell
Lines
[0397] After suspending in FACS buffer, cells of NOG-established
cancer lines collected from mice were incubated at 4.degree. C. for
30 minutes with rat mAb to mouse MHC I (Abcam; ab15680) and mAb to
human EREG (EP27; WO2008/047723). Then, following washing once with
FACS buffer, the cells were incubated at 4.degree. C. for 30
minutes with 7-AAD Viability Dye (Beckman Coulter; A07704) as dead
cell staining and secondary antibodies: PE-labeled goat F(ab')2
fragment to mouse IgG (H+L) (Beckman Coulter; IM0855) and
APC-labeled goat Ab to rat IgG (BioLegend; 405406). After washing
once with FACS buffer, the cells were subjected to flow cytometry
analysis. Flow cytometry analysis was performed using EPICS ALTRA.
Cells negative for 7-AAD Viability Dye and mouse MHC were analyzed
for EREG expression.
3.2. Flow Cytometry Analysis of In Vitro Cultured Cancer Cell
Lines
[0398] Lgr5-positive adherent cells and Lgr5-negative adherent
cells resulting from induction by irinotecan treatment were
harvested using Accutase. The cells were suspended in FACS buffer,
and then incubated at 4.degree. C. for 30 minutes with mouse mAb to
human EREG. After the cells were washed once with FACS buffer,
7-AAD Viability Dye as dead cell staining and a PE-labeled goat
F(ab')2 fragment to mouse IgG (H+L) as a secondary antibody were
added thereto. The cells were incubated at 4.degree. C. for 30
minutes. Then, the cells were washed once with FACS buffer, and
subjected to flow cytometry analysis. Flow cytometry analysis was
performed using EPICS ALTRA. Cells negative for 7-AAD Viability Dye
were analyzed for EREG expression. The result showed that the
corresponding protein was expressed at a high level on cell
membrane surface.
[0399] The result obtained by EREG flow cytometry analysis of
primary cells from PLR59 and PLR123, and Lgr5.sup.+ and Lgr5.sup.-
cancer stem cells is shown in FIG. 37. The cells were stained using
an antibody against EREG and analyzed by flow cytometry. It was
demonstrated that primary cells were negative for EREG while
Lgr5.sup.+ and Lgr5.sup.- cancer stem cells were homogeneous
EREG-positive cell populations. Gray indicates fluorescence
intensity after cell staining with an indicated antibody; and white
indicates fluorescence intensity after cell staining with a control
isotype antibody.
4. In Vitro Assessment of Drug Efficacy by ADCC Activity
Measurement
4.1. Preparation of Effector Cell Suspension
[0400] A mononuclear cell fraction collected from human peripheral
blood was used as human effector cells. Fifty ml of peripheral
blood was collected from a healthy volunteer (adult male) of the
inventors' company using a syringe loaded in advance with 200 .mu.l
of 1000 units/ml heparin solution (Novo-Heparin 5,000 units/5 ml
for Injection; Novo Nordisk). The peripheral blood was diluted
twofold with PBS(-), and then introduced into a Leucosep lymphocyte
separation tube (Greiner bio-one) in advance loaded with
Ficoll-Paque PLUS and subjected to centrifugation. After
centrifugation (2150 rpm, room temperature, 10 minutes), the
monocyte fraction layer was collected from the tube. The cells were
washed once with 10% FBS/D-MEM, and then suspended at a cell
density of 5.times.10.sup.6/ml in 10% FBS/D-MEM. The suspension was
used as an effector cell suspension.
4.2. Preparation of Target Cell Suspensions
[0401] Target cell suspensions were prepared at the time of use.
One.times.10.sup.6 cells of cancer lines were centrifuged (1200
rpm, room temperature, 5 minutes). The cell pellets were suspended
in 200 .mu.l of 0.2 mg/ml calcein-AM (Nacalai Tesque)/DMEM (10%
FBS) medium. Cell suspensions in calcein-AM solution were incubated
for two hours in a CO.sub.2 incubator set to 37.degree. C. and to a
CO.sub.2 concentration of 5%. After washing once with 10%
FBS/D-MEM, the cells were adjusted to a cell density of
2.times.10.sup.5/ml with 10% FBS/D-MEM to prepare target cell
suspensions.
4.3. ADCC Activity Measurement
[0402] Anti-EREG antibody was prepared at a concentration of 0.5
mg/ml, which was further diluted with 10% FBS/D-MEM to give
antibody solutions. The final concentration was adjusted to 0.4, 4,
and 40 .mu.g/ml. The antibody solutions of respective
concentrations or 10% FBS/D-MEM were each added at 50 .mu.l/well to
a 96-well round-bottomed plate. Then, the target cell suspensions
were added at 50 .mu.l/well to every well. The plate was incubated
at room temperature for 15 minutes. Next, 100 .mu.l of the effector
cell suspension was added to each well containing target cell
suspension, and antibody solution or 10% FBS/D-MEM. One-hundred
.mu.l of 10% FBS/D-MEM or 2% NP-40 solution (NP-40 substitute; Wako
Pure Chemical Industries) was added to each of other wells
containing 10% FBS/D-MEM and target cell suspension. The plate was
centrifuged (1200 rpm, room temperature, 5 minutes) and incubated
for 4 hours in a CO.sub.2 incubator set to 37.degree. C. and to a
CO.sub.2 concentration of 5%. The plate was centrifuged (1200 rpm,
room temperature, 5 minutes), and a 100-.mu.l aliquot of
supernatant was collected from each well. The fluorescence
intensity (.lamda..sub.ex=490 nm, .lamda..sub.em=515 nm) was
determined using a spectrofluorimeter. The specific calcein release
rate (cytotoxicity (%)) was determined according to the following
formula.
cytotoxicity(%)=(A-C).times.100/(B-C) Formula 1:
where A represents the fluorescence intensity in each well; B
represents the mean value of fluorescence intensity in a well where
50 .mu.l of target cell suspension and 100 .mu.l of NP-40 solution
were added to 50 .mu.l of 10% FBS/D-MEM; and C represents the mean
value of fluorescence intensity in a well where 50 .mu.l of target
cell suspension and 100 .mu.l of 10% FBS/D-MEM were added to 50
.mu.l of 10% FBS/D-MEM. This assay was carried out in triplicate,
and the cytotoxicity (%) at each antibody concentration was
determined using Microsoft Office Excel 2007.
[0403] The anti-EREG antibody-mediated ADCC activities against
Lgr5-positive and -negative cells derived from PLR59 cells, and
those against Lgr5-positive and -negative cells derived from PLR123
cells are shown in FIG. 38. The result showed that the anti-EREG
antibody exerted cytotoxic activity against both Lgr5-positive and
-negative cancer stem cells from PRL59 or PLR123 in a dose
dependent manner whereas the control antibody had no cytotoxic
activity.
[0404] To assess in vivo EREG expression, the Lgr5-positive cells
were administered into the peritoneal cavities of NOG mice. In the
early stage of tumor generation, EREG was expressed at a high
level. In the late stage where the tumor formed specific ductal
structures, EREG expression was somewhat localized to the budding
clusters rather than ductal structures. EREG-positive cells were
detected even after irinotecan administration to tumor-bearing mice
(FIG. 54). The anti-EREG antibody was assessed for anti-tumor
activity after irinotecan treatment. Effector cells are essential
for the anti-EREG antibody to mediate ADCC activity. Thus, SCID
mice were used as a model to assess the pharmacological efficacy of
the anti-EREG antibody. Tumor growth was suppressed when the
antibody was administered at the time points of days 4 and 11 after
the final irinotecan administration (FIG. 57).
[0405] As a first step to assess the pharmacological efficacy based
on the metastasis model, it was tested whether EREG is expressed in
the metastasis model. When Lgr5-positive cells were intravenously
injected into NOG mice, tumors were formed in multiple tissues
including lung. Cells of the tumors formed in lungs are mostly
positive for EREG (FIG. 58A). The pharmacological efficacy of the
anti-EREG antibody was assessed using SCID-Beige mice where
macrophages and mononuclear cells can serve as effector cells to
mediate ADCC. The anti-EREG antibody was administered to mice once
a week for a total of five times starting at three days after the
injection of Lgr5-positive cells. The number of tumor cells in
distal locations was demonstrated to be markedly reduced as
compared to that in control mice (FIG. 58B). In addition, the size
of each tumor was also shown to be remarkably reduced in mice
administered with the antibody (FIGS. 58C and 58D).
Example 9
Presence of Lgr5-Negative and -Positive CSCs in Clinical Tumor
Specimens
[0406] Proliferating and quiescent CSCs were identified by
immunohistochemistry using anti-Lgr5 antibody (2U2E-2),
anti-HLA-DMA antibody, and anti-EREG antibody (FIG. 59 and Table
11). Proliferative CSC represents Lgr5-positive cell, while
quiescent CSC represents HLA-DMA-positive and EREG-positive cell
(Table 11). Lgr5-positive cells which are positive for both HLA-DMA
and EREG, and Lgr5-negative cells which are positive for both
HLA-DMA and EREG were present in a very small number in primary and
metastatic colorectal cancer specimens isolated from colorectal
cancer patients (FIG. 59). Both Lgr5-positive and -negative cells
were detected in eight of 12 specimens of human colorectal cancer
tissues. Meanwhile, either Lgr5-positive or Lgr5-negative cells
were observed in the remaining four specimens. Throughout all
specimens, Lgr5-positive cells accounted for 0.003 to 1.864%, and
Lgr5-negative cells accounted for 0.001 to 10.243% (Table 11).
TABLE-US-00026 TABLE 11 Property of Case number CSC 1 2 3 4 5 6 7 8
9 10 11 12 Duct Proliferative P.sup..dagger. P P P P P P P P N P N
Quiescent N P N P P P P P P P N P Budding Proliferative P N N P P N
N P P P N N area Quiescent N P N P N P N P N N N P Frequency
Proliferative 1.864 0.786 0.136 0.121 0.119 0.095 0.063 0.054 0.018
0.010 0.003 0.000 Quiescent 0.000 0.243 0.000 0.187 0.001 0.228
0.045 0.065 0.003 0.003 0.000 0.073 (P.sup..dagger. indicates that
proliferating or quiescent CSCs were detected; N indicates that
proliferating or quiescent CSCs were undetectable) (Frequency
indicates cell percentage)
[0407] Both Lgr5-positive and -negative CSCs were detected in the
ductal and budding areas (FIG. 59). Furthermore, in ducts,
Lgr5-positive and -negative CSCs were not limited to particular
areas but distributed at random over the entire ducts.
Example 10
Anti-Tumor Effect of Various Antibodies Used in Combination with
Mab-ZAP and Rat-ZAP
[0408] The present inventors tested whether an anti-tumor effect
can be expected with target therapy using a membrane protein
expressed at a high level as the target in irinotecan-treated or
non-treated PLR59 and PLR123. Commercially available antibodies
shown in Table 12 were assessed by flow cytometry (FCM) for the
binding activity to antigens expressed on the cell surface of
irinotecan-treated or non-treated PLR59 or PLR123. The result is
summarized in Table 13.
TABLE-US-00027 TABLE 12 Antigen name Subtype Manufacturer CD70
mIgG3 BD Pharmingen EDAR mIgG1 MBL FAS mIgG1 BD Pharmingen PROM2
mIgG2b R & D Systems PVRL4 migG2b R & D Systems TNFSF9
mIgG1 BioLegend PROCR ratIgG1 BD Pharmingen EPCAM mIgG2a ABGENT
TABLE-US-00028 TABLE 13 Irinotecan non-treated Irinotecan-treated
CSC PLR59 PLR123 PLR59 PLR123 Antibody +/- - + - + - + - + CD70 4
1177 4 1843 NT NT NT NT EDAR 7 37 7 61 NT NT NT NT FAS 4 519 4 230
22 1871 25 1480 PROM2 NT NT NT NT 29 391 19 298 PVRL4 4 8 4 19 22
109 25 129 TNFSF9 4 86 4 33 22 183 25 104 PROCR 6 19 5 13 22 519 25
127 EPCAM 7 7563 7 7701 NT NT NT NT (NT indicates not tested.)
[0409] Using Mab-ZAP and Rat-ZAP, various antibodies that had been
demonstrated to have binding activity were assessed for the
activity of internalization (into cells). Mab-ZAP and Rat-ZAP are
anti-mouse IgG antibody and anti-rat IgG antibody, respectively,
conjugated with saporin, a toxin that inhibits protein synthesis
(Advanced Targeting Systems). To assess the activity of
internalization into irinotecan-non-treated cells, PLR59 and PLR123
cells were seeded at a cell density of 30000 cells/80 .mu.l/well to
respective wells of a 96-well plate. Following day, each antibody
solution was added at a final concentration of 0.01, 0.1, or 1
.mu.g/ml to the respective wells. Then, Mab-ZAP or Rat-ZAP was
added at a final concentration 1 .mu.g/ml to the respective wells,
and the plate was incubated at 37.degree. C. for 72 hours in a
CO.sub.2 incubator. To assess the activity of internalization into
irinotecan-treated cells, PLR59 and PLR123 cells were seeded in a
96-well plate, and irinotecan was added at a final concentration of
15 .mu.M to each well. The plate was incubated at 37.degree. C. for
72 hours in a CO.sub.2 incubator. Various antibodies were assessed
for the activity of internalization to cells cultured as described
above in the presence or absence of irinotecan. For assay, the
various antibodies were each assessed for the internalization
activity into cells contained in each well where the medium was
replaced with the same irinotecan-free medium as used for
irinotecan-non-treated cells. Seventy-two hours after addition of
antibodies and Mab-ZAP or Rat-ZAP, 10 .mu.l of 3% SDS (Nacalai
Tesque) was added to each well of the plate. The cells in the plate
were lysed thoroughly by stirring the plate using a plate mixer.
Then, 100 .mu.l of CellTiter-Glo.TM. Luminescent Cell Viability
Assay (Promega) was added to each well. The mixture in each well
was assayed to determine its luminescent signal. The determined
anti-tumor activity is shown in Table 14 and FIGS. 60 to 72. In
FIGS. 60 to 72, the percent suppression of cell proliferation
indicated by the vertical axis represents a relative value for the
difference in the luminescence signal value between the mixtures in
wells, one of which contained a test antibody alone (without
Mab-ZAP and Rat-ZAP) and the other contained a test antibody, and
Mab-ZAP or Rat-ZAP, when taking as 100% the difference in the
luminescence signal value between the mixtures in wells, one of
which contained a test antibody alone (without Mab-ZAP and Rat-ZAP)
and the other did not contain any cells. In Table 14, symbols, -,
+, ++, and +++, represent relative values for the internalization
activity when a test antibody was assayed at a concentration of 1
.mu.g/.mu.l. The relative value refers to a relative value for the
difference in the luminescence signal value between the mixtures in
wells, one of which contained a test antibody alone (without
Mab-ZAP and Rat-ZAP) and the other contained a test antibody, and
Mab-ZAP or Rat-ZAP, when taking as 100% the difference in the
luminescence signal value between the mixtures in wells, one of
which contained a test antibody alone (without Mab-ZAP and Rat-ZAP)
and the other did not contain any cells. Symbols, -, +, ++, and
+++, indicate that the relative value is less than 5%, 5% or more
and less than 15%, 15% or more and less than 25%, and 25% or more,
respectively.
TABLE-US-00029 TABLE 14 PLR59 PLR123 Antigen Irinotecan Irinotecan-
Irinotecan Irinotecan- name non-treated treated non-treated treated
CD7O +++ NT +++ NT EDAR ++ NT - NT FAS +++ +++ +++ +++ PROM2 NT +
NT + PVRL4 + +++ - + TNFSF9 - +++ - +++ PROCR + ++ - + EPCAM +++
+++ +++ +++
[0410] As shown in FIG. 66, regarding irinotecan-non-treated PLR59
and PLR123, anti-CD70 antibody and anti-FAS antibody showed the
internalization activity level of 25% or more under a condition
where the anti-EPCAM antibody used as a positive control had been
demonstrated to exhibit a sufficient anti-tumor activity (FIGS. 60
and 62). Furthermore, regarding PLR59, anti-EDAR antibody showed
the internalization activity level of 15 to 25%, and anti-PVRL4
antibody and anti-PROCR antibody exhibited the internalization
activity level of 5 to 15% (FIGS. 61, 63, and 65). Meanwhile,
regarding irinotecan-treated PLR59 and PLR123, anti-FAS antibody
and anti-TNFRSF9 antibody showed the internalization activity level
of 25% or more, and anti-PROM2 antibody exhibited the
internalization activity level of 5 to 15% (FIGS. 67, 70, and 68).
Both anti-PVRL4 antibody and anti-PROCR antibody showed the
internalization activity for irinotecan-treated PLR59 and PLR123,
and the activity for PLR59 was greater than that for PLR123 (FIGS.
69 and 71). The result described above demonstrates that all
antibodies tested have an anti-tumor effect against PLR59 and
PLR123.
[0411] BMP4 was assessed for the differentiation-enhancing effect
on irinotecan-non-treated and irinotecan-treated PLR59 and PLR123.
To assess the differentiation-enhancing effect on
irinotecan-non-treated cells, PLR59 and PLR123 cells suspended in
the media where BMP4 (R&D Systems; a final concentration of 20
nM) or a control buffer was added to culture media were seeded at a
cell density of 5.times.10.sup.5 cells/1.5 ml/well to each well of
a 12-well plate. The cells were passaged while changing the culture
media with the same type of medium 2, 4, and 7 days after seeding.
To assess the differentiation-enhancing effect on
irinotecan-treated cells, PLR59 or PLR123 was seeded at a cell
density of 17.times.10.sup.5 cells/5 ml/flask to a 12.5-ml culture
flask. Following day, irinotecan was added at a final concentration
of 15 .mu.M. The flask was incubated at 37.degree. C. for 72 hours
in a CO.sub.2 incubator. Then, the medium in the flask was changed
with a medium containing BMP4 or a control buffer. The medium was
further changed with the same type of medium 2, 4, and 7 days after
the initial medium change. From cells isolated 4 and 9 days after
the initial medium change, RNAs were extracted using RNeasy Plus
Mini Kit and RNase-Free DNase Set (QIAGEN). cDNAs were synthesized
with ThermoScript RT-PCR System (Invitrogen) using the extracted
RNAs as a template.
[0412] Quantitative real-time PCR was carried out using the cDNAs
isolated as described above. As shown in FIG. 73, elevated CK20
levels were observed in PLR59 and PLR123 cells cultured in the
presence of BMP4.
INDUSTRIAL APPLICABILITY
[0413] The present inventors identified cell surface molecules that
are expressed specifically on cancer stem cells. The present
invention provides novel anti-cancer drugs and reagents for
detecting cancer stem cells, which use antibodies against the cell
surface molecules.
Sequence CWU 1
1
151448PRTHomo sapiens 1Met Ala His Val Gly Asp Cys Thr Gln Thr Pro
Trp Leu Pro Val Leu 1 5 10 15 Val Val Ser Leu Met Cys Ser Ala Arg
Ala Glu Tyr Ser Asn Cys Gly 20 25 30 Glu Asn Glu Tyr Tyr Asn Gln
Thr Thr Gly Leu Cys Gln Glu Cys Pro 35 40 45 Pro Cys Gly Pro Gly
Glu Glu Pro Tyr Leu Ser Cys Gly Tyr Gly Thr 50 55 60 Lys Asp Glu
Asp Tyr Gly Cys Val Pro Cys Pro Ala Glu Lys Phe Ser 65 70 75 80 Lys
Gly Gly Tyr Gln Ile Cys Arg Arg His Lys Asp Cys Glu Gly Phe 85 90
95 Phe Arg Ala Thr Val Leu Thr Pro Gly Asp Met Glu Asn Asp Ala Glu
100 105 110 Cys Gly Pro Cys Leu Pro Gly Tyr Tyr Met Leu Glu Asn Arg
Pro Arg 115 120 125 Asn Ile Tyr Gly Met Val Cys Tyr Ser Cys Leu Leu
Ala Pro Pro Asn 130 135 140 Thr Lys Glu Cys Val Gly Ala Thr Ser Gly
Ala Ser Ala Asn Phe Pro 145 150 155 160 Gly Thr Ser Gly Ser Ser Thr
Leu Ser Pro Phe Gln His Ala His Lys 165 170 175 Glu Leu Ser Gly Gln
Gly His Leu Ala Thr Ala Leu Ile Ile Ala Met 180 185 190 Ser Thr Ile
Phe Ile Met Ala Ile Ala Ile Val Leu Ile Ile Met Phe 195 200 205 Tyr
Ile Leu Lys Thr Lys Pro Ser Ala Pro Ala Cys Cys Thr Ser His 210 215
220 Pro Gly Lys Ser Val Glu Ala Gln Val Ser Lys Asp Glu Glu Lys Lys
225 230 235 240 Glu Ala Pro Asp Asn Val Val Met Phe Ser Glu Lys Asp
Glu Phe Glu 245 250 255 Lys Leu Thr Ala Thr Pro Ala Lys Pro Thr Lys
Ser Glu Asn Asp Ala 260 265 270 Ser Ser Glu Asn Glu Gln Leu Leu Ser
Arg Ser Val Asp Ser Asp Glu 275 280 285 Glu Pro Ala Pro Asp Lys Gln
Gly Ser Pro Glu Leu Cys Leu Leu Ser 290 295 300 Leu Val His Leu Ala
Arg Glu Lys Ser Ala Thr Ser Asn Lys Ser Ala 305 310 315 320 Gly Ile
Gln Ser Arg Arg Lys Lys Ile Leu Asp Val Tyr Ala Asn Val 325 330 335
Cys Gly Val Val Glu Gly Leu Ser Pro Thr Glu Leu Pro Phe Asp Cys 340
345 350 Leu Glu Lys Thr Ser Arg Met Leu Ser Ser Thr Tyr Asn Ser Glu
Lys 355 360 365 Ala Val Val Lys Thr Trp Arg His Leu Ala Glu Ser Phe
Gly Leu Lys 370 375 380 Arg Asp Glu Ile Gly Gly Met Thr Asp Gly Met
Gln Leu Phe Asp Arg 385 390 395 400 Ile Ser Thr Ala Gly Tyr Ser Ile
Pro Glu Leu Leu Thr Lys Leu Val 405 410 415 Gln Ile Glu Arg Leu Asp
Ala Val Glu Ser Leu Cys Ala Asp Ile Leu 420 425 430 Glu Trp Ala Gly
Val Val Pro Pro Ala Ser Gln Pro His Ala Ala Ser 435 440 445
2193PRTHomo sapiens 2Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg
Arg Arg Pro Tyr Gly 1 5 10 15 Cys Val Leu Arg Ala Ala Leu Val Pro
Leu Val Ala Gly Leu Val Ile 20 25 30 Cys Leu Val Val Cys Ile Gln
Arg Phe Ala Gln Ala Gln Gln Gln Leu 35 40 45 Pro Leu Glu Ser Leu
Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His 50 55 60 Thr Gly Pro
Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala 65 70 75 80 Leu
Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90
95 Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu
100 105 110 Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr
Thr Leu 115 120 125 Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile
Ser Leu Leu Arg 130 135 140 Leu Ser Phe His Gln Gly Cys Thr Ile Ala
Ser Gln Arg Leu Thr Pro 145 150 155 160 Leu Ala Arg Gly Asp Thr Leu
Cys Thr Asn Leu Thr Gly Thr Leu Leu 165 170 175 Pro Ser Arg Asn Thr
Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg 180 185 190 Pro
3169PRTHomo sapiens 3Met Thr Ala Gly Arg Arg Met Glu Met Leu Cys
Ala Gly Arg Val Pro 1 5 10 15 Ala Leu Leu Leu Cys Leu Gly Phe His
Leu Leu Gln Ala Val Leu Ser 20 25 30 Thr Thr Val Ile Pro Ser Cys
Ile Pro Gly Glu Ser Ser Asp Asn Cys 35 40 45 Thr Ala Leu Val Gln
Thr Glu Asp Asn Pro Arg Val Ala Gln Val Ser 50 55 60 Ile Thr Lys
Cys Ser Ser Asp Met Asn Gly Tyr Cys Leu His Gly Gln 65 70 75 80 Cys
Ile Tyr Leu Val Asp Met Ser Gln Asn Tyr Cys Arg Cys Glu Val 85 90
95 Gly Tyr Thr Gly Val Arg Cys Glu His Phe Phe Leu Thr Val His Gln
100 105 110 Pro Leu Ser Lys Glu Tyr Val Ala Leu Thr Val Ile Leu Ile
Ile Leu 115 120 125 Phe Leu Ile Thr Val Val Gly Ser Thr Tyr Tyr Phe
Cys Arg Trp Tyr 130 135 140 Arg Asn Arg Lys Ser Lys Glu Pro Lys Lys
Glu Tyr Glu Arg Val Thr 145 150 155 160 Ser Gly Asp Pro Glu Leu Pro
Gln Val 165 4408PRTHomo sapiens 4Met Ile Pro Gly Asn Arg Met Leu
Met Val Val Leu Leu Cys Gln Val 1 5 10 15 Leu Leu Gly Gly Ala Ser
His Ala Ser Leu Ile Pro Glu Thr Gly Lys 20 25 30 Lys Lys Val Ala
Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly 35 40 45 Gln Ser
His Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 50 55 60
Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro 65
70 75 80 Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu
Glu Glu 85 90 95 Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu
Arg Pro Ala Ser 100 105 110 Arg Ala Asn Thr Val Arg Ser Phe His His
Glu Glu His Leu Glu Asn 115 120 125 Ile Pro Gly Thr Ser Glu Asn Ser
Ala Phe Arg Phe Leu Phe Asn Leu 130 135 140 Ser Ser Ile Pro Glu Asn
Glu Val Ile Ser Ser Ala Glu Leu Arg Leu 145 150 155 160 Phe Arg Glu
Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His 165 170 175 Arg
Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro 180 185
190 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg Leu Val His His Asn
195 200 205 Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val Leu
Arg Trp 210 215 220 Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Ala Ile
Glu Val Thr His 225 230 235 240 Leu His Gln Thr Arg Thr His Gln Gly
Gln His Val Arg Ile Ser Arg 245 250 255 Ser Leu Pro Gln Gly Ser Gly
Asn Trp Ala Gln Leu Arg Pro Leu Leu 260 265 270 Val Thr Phe Gly His
Asp Gly Arg Gly His Ala Leu Thr Arg Arg Arg 275 280 285 Arg Ala Lys
Arg Ser Pro Lys His His Ser Gln Arg Ala Arg Lys Lys 290 295 300 Asn
Lys Asn Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val 305 310
315 320 Gly Trp Asn Asp Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe
Tyr 325 330 335 Cys His Gly Asp Cys Pro Phe Pro Leu Ala Asp His Leu
Asn Ser Thr 340 345 350 Asn His Ala Ile Val Gln Thr Leu Val Asn Ser
Val Asn Ser Ser Ile 355 360 365 Pro Lys Ala Cys Cys Val Pro Thr Glu
Leu Ser Ala Ile Ser Met Leu 370 375 380 Tyr Leu Asp Glu Tyr Asp Lys
Val Val Leu Lys Asn Tyr Gln Glu Met 385 390 395 400 Val Val Glu Gly
Cys Gly Cys Arg 405 5238PRTHomo sapiens 5Met Leu Thr Thr Leu Leu
Pro Ile Leu Leu Leu Ser Gly Trp Ala Phe 1 5 10 15 Cys Ser Gln Asp
Ala Ser Asp Gly Leu Gln Arg Leu His Met Leu Gln 20 25 30 Ile Ser
Tyr Phe Arg Asp Pro Tyr His Val Trp Tyr Gln Gly Asn Ala 35 40 45
Ser Leu Gly Gly His Leu Thr His Val Leu Glu Gly Pro Asp Thr Asn 50
55 60 Thr Thr Ile Ile Gln Leu Gln Pro Leu Gln Glu Pro Glu Ser Trp
Ala 65 70 75 80 Arg Thr Gln Ser Gly Leu Gln Ser Tyr Leu Leu Gln Phe
His Gly Leu 85 90 95 Val Arg Leu Val His Gln Glu Arg Thr Leu Ala
Phe Pro Leu Thr Ile 100 105 110 Arg Cys Phe Leu Gly Cys Glu Leu Pro
Pro Glu Gly Ser Arg Ala His 115 120 125 Val Phe Phe Glu Val Ala Val
Asn Gly Ser Ser Phe Val Ser Phe Arg 130 135 140 Pro Glu Arg Ala Leu
Trp Gln Ala Asp Thr Gln Val Thr Ser Gly Val 145 150 155 160 Val Thr
Phe Thr Leu Gln Gln Leu Asn Ala Tyr Asn Arg Thr Arg Tyr 165 170 175
Glu Leu Arg Glu Phe Leu Glu Asp Thr Cys Val Gln Tyr Val Gln Lys 180
185 190 His Ile Ser Ala Glu Asn Thr Lys Gly Ser Gln Thr Ser Arg Ser
Tyr 195 200 205 Thr Ser Leu Val Leu Gly Val Leu Val Gly Ser Phe Ile
Ile Ala Gly 210 215 220 Val Ala Val Gly Ile Phe Leu Cys Thr Gly Gly
Arg Arg Cys 225 230 235 6834PRTHomo sapiens 6Met Lys His Thr Leu
Ala Leu Leu Ala Pro Leu Leu Gly Leu Gly Leu 1 5 10 15 Gly Leu Ala
Leu Ser Gln Leu Ala Ala Gly Ala Thr Asp Cys Lys Phe 20 25 30 Leu
Gly Pro Ala Glu His Leu Thr Phe Thr Pro Ala Ala Arg Ala Arg 35 40
45 Trp Leu Ala Pro Arg Val Arg Ala Pro Gly Leu Leu Asp Ser Leu Tyr
50 55 60 Gly Thr Val Arg Arg Phe Leu Ser Val Val Gln Leu Asn Pro
Phe Pro 65 70 75 80 Ser Glu Leu Val Lys Ala Leu Leu Asn Glu Leu Ala
Ser Val Lys Val 85 90 95 Asn Glu Val Val Arg Tyr Glu Ala Gly Tyr
Val Val Cys Ala Val Ile 100 105 110 Ala Gly Leu Tyr Leu Leu Leu Val
Pro Thr Ala Gly Leu Cys Phe Cys 115 120 125 Cys Cys Arg Cys His Arg
Arg Cys Gly Gly Arg Val Lys Thr Glu His 130 135 140 Lys Ala Leu Ala
Cys Glu Arg Ala Ala Leu Met Val Phe Leu Leu Leu 145 150 155 160 Thr
Thr Leu Leu Leu Leu Ile Gly Val Val Cys Ala Phe Val Thr Asn 165 170
175 Gln Arg Thr His Glu Gln Met Gly Pro Ser Ile Glu Ala Met Pro Glu
180 185 190 Thr Leu Leu Ser Leu Trp Gly Leu Val Ser Asp Val Pro Gln
Glu Leu 195 200 205 Gln Ala Val Ala Gln Gln Phe Ser Leu Pro Gln Glu
Gln Val Ser Glu 210 215 220 Glu Leu Asp Gly Val Gly Val Ser Ile Gly
Ser Ala Ile His Thr Gln 225 230 235 240 Leu Arg Ser Ser Val Tyr Pro
Leu Leu Ala Ala Val Gly Ser Leu Gly 245 250 255 Gln Val Leu Gln Val
Ser Val His His Leu Gln Thr Leu Asn Ala Thr 260 265 270 Val Val Glu
Leu Gln Ala Gly Gln Gln Asp Leu Glu Pro Ala Ile Arg 275 280 285 Glu
His Arg Asp Arg Leu Leu Glu Leu Leu Gln Glu Ala Arg Cys Gln 290 295
300 Gly Asp Cys Ala Gly Ala Leu Ser Trp Ala Arg Thr Leu Glu Leu Gly
305 310 315 320 Ala Asp Phe Ser Gln Val Pro Ser Val Asp His Val Leu
His Gln Leu 325 330 335 Lys Gly Val Pro Glu Ala Asn Phe Ser Ser Met
Val Gln Glu Glu Asn 340 345 350 Ser Thr Phe Asn Ala Leu Pro Ala Leu
Ala Ala Met Gln Thr Ser Ser 355 360 365 Val Val Gln Glu Leu Lys Lys
Ala Val Ala Gln Gln Pro Glu Gly Val 370 375 380 Arg Thr Leu Ala Glu
Gly Phe Pro Gly Leu Glu Ala Ala Ser Arg Trp 385 390 395 400 Ala Gln
Ala Leu Gln Glu Val Glu Glu Ser Ser Arg Pro Tyr Leu Gln 405 410 415
Glu Val Gln Arg Tyr Glu Thr Tyr Arg Trp Ile Val Gly Cys Val Leu 420
425 430 Cys Ser Val Val Leu Phe Val Val Leu Cys Asn Leu Leu Gly Leu
Asn 435 440 445 Leu Gly Ile Trp Gly Leu Ser Ala Arg Asp Asp Pro Ser
His Pro Glu 450 455 460 Ala Lys Gly Glu Ala Gly Ala Arg Phe Leu Met
Ala Gly Val Gly Leu 465 470 475 480 Ser Phe Leu Phe Ala Ala Pro Leu
Ile Leu Leu Val Phe Ala Thr Phe 485 490 495 Leu Val Gly Gly Asn Val
Gln Thr Leu Val Cys Gln Ser Trp Glu Asn 500 505 510 Gly Glu Leu Phe
Glu Phe Ala Asp Thr Pro Gly Asn Leu Pro Pro Ser 515 520 525 Met Asn
Leu Ser Gln Leu Leu Gly Leu Arg Lys Asn Ile Ser Ile His 530 535 540
Gln Ala Tyr Gln Gln Cys Lys Glu Gly Ala Ala Leu Trp Thr Val Leu 545
550 555 560 Gln Leu Asn Asp Ser Tyr Asp Leu Glu Glu His Leu Asp Ile
Asn Gln 565 570 575 Tyr Thr Asn Lys Leu Arg Gln Glu Leu Gln Ser Leu
Lys Val Asp Thr 580 585 590 Gln Ser Leu Asp Leu Leu Ser Ser Ala Ala
Arg Arg Asp Leu Glu Ala 595 600 605 Leu Gln Ser Ser Gly Leu Gln Arg
Ile His Tyr Pro Asp Phe Leu Val 610 615 620 Gln Ile Gln Arg Pro Val
Val Lys Thr Ser Met Glu Gln Leu Ala Gln 625 630 635 640 Glu Leu Gln
Gly Leu Ala Gln Ala Gln Asp Asn Ser Val Leu Gly Gln 645 650 655 Arg
Leu Gln Glu Glu Ala Gln Gly Leu Arg Asn Leu His Gln Glu Lys 660 665
670 Val Val Pro Gln Gln Ser Leu Val Ala Lys Leu Asn Leu Ser Val Arg
675 680 685 Ala Leu Glu Ser Ser Ala Pro Asn Leu Gln Leu Glu Thr Ser
Asp Val 690 695 700 Leu Ala Asn Val Thr Tyr Leu Lys Gly Glu Leu Pro
Ala Trp Ala Ala 705 710 715 720 Arg Ile Leu Arg Asn Val Ser Glu Cys
Phe Leu Ala Arg Glu Met Gly 725 730 735 Tyr Phe Ser Gln Tyr Val Ala
Trp Val Arg Glu Glu Val Thr Gln Arg 740 745 750 Ile Ala Thr Cys Gln
Pro Leu Ser Gly Ala Leu Asp Asn Ser Arg Val 755 760 765 Ile Leu Cys
Asp Met Met Ala Asp Pro Trp Asn Ala Phe Trp Phe Cys 770 775 780 Leu
Ala Trp Cys Thr Phe Phe Leu Ile Pro Ser Ile Ile Phe Ala Val 785 790
795 800 Lys Thr Ser Lys Tyr Phe Arg Pro Ile Arg Lys Arg Leu Ser Ser
Thr 805 810 815 Ser Ser Glu Glu Thr Gln Leu Phe His Ile Pro Arg Val
Thr Ser Leu 820 825 830 Lys Leu
7254PRTHomo sapiens 7Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro
Glu Ala Pro Trp Pro 1 5 10 15 Pro Ala Pro Arg Ala Arg Ala Cys Arg
Val Leu Pro Trp Ala Leu Val 20 25 30 Ala Gly Leu Leu Leu Leu Leu
Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45 Leu Ala Cys Pro Trp
Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 55 60 Ala Ala Ser
Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 65 70 75 80 Pro
Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val 85 90
95 Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
100 105 110 Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr
Lys Glu 115 120 125 Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val
Tyr Tyr Val Phe 130 135 140 Phe Gln Leu Glu Leu Arg Arg Val Val Ala
Gly Glu Gly Ser Gly Ser 145 150 155 160 Val Ser Leu Ala Leu His Leu
Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175 Ala Ala Leu Ala Leu
Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190 Arg Asn Ser
Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200 205 Gly
Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His 210 215
220 Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val
225 230 235 240 Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser
Glu 245 250 8510PRTHomo sapiens 8Met Pro Leu Ser Leu Gly Ala Glu
Met Trp Gly Pro Glu Ala Trp Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu
Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly 20 25 30 Glu Leu Glu Thr
Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala 35 40 45 Lys Leu
Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln 50 55 60
Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala 65
70 75 80 Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr
Glu Gly 85 90 95 Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu
Asp Gly Ser Val 100 105 110 Leu Leu Arg Asn Ala Val Gln Ala Asp Glu
Gly Glu Tyr Glu Cys Arg 115 120 125 Val Ser Thr Phe Pro Ala Gly Ser
Phe Gln Ala Arg Leu Arg Leu Arg 130 135 140 Val Leu Val Pro Pro Leu
Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu 145 150 155 160 Glu Gly Gln
Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser 165 170 175 Pro
Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser 180 185
190 Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe
195 200 205 His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr
Cys Val 210 215 220 Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile
Thr His Ile Leu 225 230 235 240 His Val Ser Phe Leu Ala Glu Ala Ser
Val Arg Gly Leu Glu Asp Gln 245 250 255 Asn Leu Trp His Ile Gly Arg
Glu Gly Ala Met Leu Lys Cys Leu Ser 260 265 270 Glu Gly Gln Pro Pro
Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro 275 280 285 Leu Pro Ser
Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro 290 295 300 Leu
Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu 305 310
315 320 Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro
Gln 325 330 335 Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser
Val Val Val 340 345 350 Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu
Leu Val Val Val Val 355 360 365 Val Leu Met Ser Arg Tyr His Arg Arg
Lys Ala Gln Gln Met Thr Gln 370 375 380 Lys Tyr Glu Glu Glu Leu Thr
Leu Thr Arg Glu Asn Ser Ile Arg Arg 385 390 395 400 Leu His Ser His
His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val 405 410 415 Gly Leu
Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser 420 425 430
Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu 435
440 445 Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro
Gly 450 455 460 Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly
Ile Lys Gln 465 470 475 480 Ala Met Asn His Phe Val Gln Glu Asn Gly
Thr Leu Arg Ala Lys Pro 485 490 495 Thr Gly Asn Gly Ile Tyr Ile Asn
Gly Arg Gly His Leu Val 500 505 510 9335PRTHomo sapiens 9Met Leu
Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 1 5 10 15
Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20
25 30 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln
Asn 35 40 45 Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys
Pro Cys Pro 50 55 60 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val
Asn Gly Asp Glu Pro 65 70 75 80 Asp Cys Val Pro Cys Gln Glu Gly Lys
Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser Lys Cys Arg Arg
Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110 Leu Glu Val Glu Ile
Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125 Cys Lys Pro
Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140 Pro
Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150
155 160 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly
Trp 165 170 175 Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp
Val Lys Arg 180 185 190 Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg
Lys Glu Asn Gln Gly 195 200 205 Ser His Glu Ser Pro Thr Leu Asn Pro
Glu Thr Val Ala Ile Asn Leu 210 215 220 Ser Asp Val Asp Leu Ser Lys
Tyr Ile Thr Thr Ile Ala Gly Val Met 225 230 235 240 Thr Leu Ser Gln
Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu 245 250 255 Ala Lys
Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu 260 265 270
Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys 275
280 285 Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu
Cys 290 295 300 Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp
Ile Thr Ser 305 310 315 320 Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu
Ile Gln Ser Leu Val 325 330 335 1024DNAArtificial Sequencean
artificially synthesized primer sequence 10agtttatcct tctggtggta
gtcc 241123DNAArtificial Sequencean artificially synthesized primer
sequence 11caagatgtag agaaggggat tga 231220DNAArtificial Sequencean
artificially synthesized primer sequence 12ctctgctcct cctgttcgac
201320DNAArtificial Sequencean artificially synthesized primer
sequence 13acgaccaaat ccgttgactc 201421DNAArtificial Sequencean
artificially synthesized primer sequence 14aagtcccttg ccatcctaaa a
211520DNAArtificial Sequencean artificially synthesized primer
sequence 15atgctatcac ctcccctgtg 20
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