U.S. patent application number 17/264277 was filed with the patent office on 2021-12-02 for cdca1-derived peptide and vaccine containing same.
This patent application is currently assigned to ONCOTHERAPY SCIENCE, INC.. The applicant listed for this patent is ONCOTHERAPY SCIENCE, INC.. Invention is credited to Tetsuro HIKICHI, SACHIKO YAMASHITA.
Application Number | 20210371462 17/264277 |
Document ID | / |
Family ID | 1000005664507 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210371462 |
Kind Code |
A1 |
YAMASHITA; SACHIKO ; et
al. |
December 2, 2021 |
CDCA1-DERIVED PEPTIDE AND VACCINE CONTAINING SAME
Abstract
The present invention provides CDCA1-derived epitope peptides
having the ability to induce cytotoxic T cells. The present
invention further provides polynucleotides encoding the peptides,
antigen-presenting cells presenting the peptides, and cytotoxic T
cells targeting the peptides, as well as methods of inducing the
antigen-presenting cells or CTLs. The present invention also
provides compositions and pharmaceutical compositions containing
them as an active ingredient. Further, the present invention
provides methods of treating and/or preventing cancer, and/or
preventing postoperative recurrence thereof, using the peptides,
polynucleotides, antigen-presenting cells, cytotoxic T cells or
pharmaceutical compositions of the present invention. Methods of
inducing an immune response against cancer are also provided.
Inventors: |
YAMASHITA; SACHIKO;
(Kawasaki-shi, JP) ; HIKICHI; Tetsuro;
(Kawasaki-shi, Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ONCOTHERAPY SCIENCE, INC. |
Kawasaki-shi, Kanagawa |
|
JP |
|
|
Assignee: |
ONCOTHERAPY SCIENCE, INC.
Kawasaki-shi, Kanagawa
JP
|
Family ID: |
1000005664507 |
Appl. No.: |
17/264277 |
Filed: |
August 1, 2019 |
PCT Filed: |
August 1, 2019 |
PCT NO: |
PCT/JP2019/030117 |
371 Date: |
January 28, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70539 20130101;
A61P 35/00 20180101; A61K 38/00 20130101; A61K 35/17 20130101; C07K
7/06 20130101; A61K 35/15 20130101 |
International
Class: |
C07K 7/06 20060101
C07K007/06; A61K 35/17 20060101 A61K035/17; A61K 35/15 20060101
A61K035/15; C07K 14/74 20060101 C07K014/74; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 2, 2018 |
JP |
2018-145607 |
Claims
1. An isolated peptide of less than 15 amino acids having cytotoxic
T cell (CTL)-inducing ability, which comprises the amino acid
sequence selected from the group below: (a) the amino acid sequence
selected from the group consisting of SEQ ID NOs: 1, 8, 10, 13, 25,
33 to 35 and 37; and (b) the amino acid sequence in which one, two,
three or several amino acids are substituted, deleted, inserted
and/or added to the amino acid sequence selected from the group
consisting of SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37.
2. The peptide of claim 1, comprising the amino acid sequence in
which one or more substitution(s) selected from (a) to (c) below is
introduced into the amino acid sequence selected from the group
consisting of SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37: (a)
the second amino acid from the N terminus is substituted with an
amino acid selected from the group consisting of threonine and
serine; (b) the third amino acid from the N terminus is substituted
with an amino acid selected from the group consisting of aspartic
acid and glutamic acid; and (c) the C-terminal amino acid is
substituted with tyrosine.
3. The peptide of claim 1, which consists of the amino acid
sequence selected from the group consisting of SEQ ID NOs: 1, 8,
10, 13, 25, 33 to 35 and 37.
4. An isolated polynucleotide, which encodes the peptide of claim
1.
5. A composition comprising a pharmaceutically acceptable carrier
and at least one ingredient selected from the group consisting of
(a) to (e) below: (a) one or more types of peptides of claim 1; (b)
one or more types of polynucleotides encoding the peptide(s) of
claim 1 in an expressible form; (c) an antigen-presenting cell
(APC) that presents on its cell surface a complex of the peptide of
claim 1 and an HLA antigen; (d) an exosome that presents on its
cell surface a complex of the peptide of claim 1 and an HLA
antigen; and (e) a CTL that targets the peptide of claim 1.
6. A composition for inducing a CTL(s), comprising a
pharmaceutically acceptable carrier and at least one ingredient
selected from the group consisting of (a) to (d) below: (a) one or
more types of peptides of claim 1; (b) one or more types of
polynucleotides encoding the peptide(s) of claim 1 in an
expressible form; (c) an antigen-presenting cell (APC) that
presents on its cell surface a complex of the peptide of claim 1
and an HLA antigen; and (d) an exosome that presents on its cell
surface a complex of the peptide of claim 1 and an HLA antigen.
7. The composition of claim 5, which is a pharmaceutical
composition.
8. The composition of claim 7, which is for one or more uses
selected from the group consisting of (i) cancer treatment, (ii)
cancer prevention (prophylaxis) and (iii) prevention (prophylaxis)
of postoperative cancer recurrence.
9. The composition of claim 7, which is for inducing an immune
response against cancer.
10. The composition of claim 8, wherein the cancer is selected from
the group consisting of bladder cancer, breast cancer, cervical
cancer, cholangiocellular cancer, chronic myeloid leukemia (CML),
esophagus cancer, gastric cancer, non-small-cell lung cancer,
lymphoma, osteosarcoma, prostate cancer, kidney cancer, small-cell
lung cancer, head and neck cancer, soft tissue tumor and colon
cancer.
11. The composition of claim 5, which is formulated for
administration to a subject positive for HLA-A01.
12. A method of inducing an APC(s) having CTL-inducing ability,
which comprises a step selected from the group consisting of: (a)
contacting an APC(s) with the peptide of claim 1 in vitro, ex vivo
or in vivo; and (b) introducing a polynucleotide encoding the
peptide of claim 1 into an APC(s).
13. A method of inducing a CTL(s), which comprises a step selected
from the group consisting of: (a) co-culturing a CD8-positive T
cell(s) with an APC(s) that presents on its surface a complex of an
HLA antigen and the peptide of claim 1; (b) co-culturing a
CD8-positive T cell(s) with an exosome(s) that presents on its
surface a complex of an HLA antigen and the peptide of claim 1; and
(c) introducing into a CD8-positive T cell(s) a polynucleotide
encoding each subunit of a T cell receptor (TCR) capable of binding
to the peptide of claim 1 presented by an HLA antigen on a cell
surface.
14. An isolated APC that presents on its surface a complex of an
HLA antigen and the peptide of claim 1.
15. An isolated APC induced by the method of claim 12.
16. An isolated CTL that targets the peptide of claim 1.
17. An isolated CTL induced by the method of claim 13.
18. A method of inducing an immune response against cancer, which
comprises administering to a subject at least one ingredient
selected from the group consisting of (a) to (e) below: (a) one or
more types of peptides of claim 1; (b) one or more types of
polynucleotides encoding the peptide(s) of claim 1 in an
expressible form; (c) an antigen-presenting cell (APC) that
presents on its cell surface a complex of the peptide of claim 1
and an HLA antigen; (d) an exosome that presents on its cell
surface a complex of the peptide of claim 1 and an HLA antigen; and
(e) a CTL that targets the peptide of claim 1.
19. A method of treating and/or preventing cancer, and/or
preventing postoperative recurrence thereof, which comprises
administering to a subject at least one ingredient selected from
the group consisting of (a) to (e) below: (a) one or more types of
peptides of claim 1; (b) one or more types of polynucleotides
encoding the peptide(s) of claim 1 in an expressible form; (c) an
antigen-presenting cell (APC) that presents on its cell surface a
complex of the peptide of claim 1 and an HLA antigen; (d) an
exosome that presents on its cell surface a complex of the peptide
of claim 1 and an HLA antigen; and (e) a CTL that targets the
peptide of claim 1.
20. An antibody that binds to the peptide of claim 1.
21. A method of screening for a peptide having CTL-inducing
ability, which comprises the steps of: (a) generating candidate
sequences consisting of an amino acid sequence in which one, two or
several amino acid residues are substituted, deleted, inserted
and/or added to an original amino acid sequence consisting of the
amino acid sequence selected from among SEQ ID NOs: 1, 8, 10, 13,
25, 33 to 35 and 37; (b) selecting from among the candidate
sequences generated in (a), a candidate sequence that does not have
significant homology (sequence identity) with any known human gene
product other than CDCA1; (c) contacting an APC with a peptide
consisting of the candidate sequence selected in (b); (d)
contacting the APC of (c) with a CD8-positive T cell; and (e)
selecting a peptide having an equal to or higher CTL-inducing
ability than that of a peptide consisting of the original amino
acid sequence.
22. An emulsion comprising one or more types of peptides of claim
1, a water-soluble carrier and an oil adjuvant.
23. A kit comprising a container that houses the composition of
claim 5 and a container that houses an adjuvant.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of biological
science, more specifically to the field of cancer therapy. In
particular, the present invention relates to novel peptides that
are effective as cancer vaccines, methods for either or both of
treating and preventing cancers using the peptide(s), and
pharmaceutical compositions comprising the peptide(s).
BACKGROUND ART
[0002] Cytotoxic T lymphocytes (CTLs) have been known to recognize
epitope peptides derived from the tumor-associated antigens (TAAs)
presented on the major histocompatibility complex (MHC) class I
molecule, which is expressed on tumor cell surface, and then kill
the tumor cells. To date, many TAAs including the melanoma antigen
(MAGE) family have been discovered through immunological approaches
(NPL1: Boon T, Int J Cancer 1993, 54(2):177-80; NPL2: Boon T &
van der Bruggen P, J Exp Med 1996, 183(3):725-9). Immunotherapies
targeting these TAAs are currently undergoing clinical
development.
[0003] In some TAAs, epitope peptides that can be recognized by
CTLs were identified and their application on immunotherapy for
various cancers is anticipated (NPL3: Harris C C, J Natl Cancer
Inst 1996, 88(20): 1442-55; NPL4: Butterfield L H et al., Cancer
Res 1999, 59(13): 3134-42; NPLS: Vissers J L et al., Cancer Res
1999, 59(21): 5554-9; NPL6: van der Burg S H et al., J Immunol
1996, 156(9): 3308-14; NPL7: Tanaka F et al., Cancer Res 1997,
57(20): 4465-8; NPL8: Fujie T et al., Int J Cancer 1999, 80(2):
169-72; NPL9: Kikuchi M et al., Int J Cancer 1999, 81(3): 459-66;
NPL10: Oiso M et al., Int J Cancer 1999, 81(3): 387-94). Until now,
several clinical trials for cancer immunotherapy using these
TAA-derived CTL epitope peptides have been reported. Unfortunately,
however, in many of these clinical trials, the response rate is not
necessarily high (NPLII: Belli F et al., J Clin Oncol 2002, 20(20):
4169-80; NPL12: Coulie P G et al., Immunol Rev 2002, 188: 33-42;
NPL13: Rosenberg SA et al., Nat Med 2004, 10(9): 909-15).
Therefore, there is still demand for identification of novel
epitope peptides that are applicable to cancer immunotherapy.
[0004] CDCA1 (cell division cycle associated 1; also described as
NUF2, NDC80 kinetochore complex component: Nuf2; reference
sequence: GeneBank Accession Number NM_145697 (SEQ ID NO: 44) or
GeneBank Accession Number NM_031423 (SEQ ID NO: 46)) has been
identified as a member of the genes co-expressed with CDC2, cyclin,
topoisomerase II and other cell cycle genes (NPL14: Walker et al.,
Curr Cancer Drug Targets 2001, 1(1): 73-83). CDCA1 has been found
to be related to the centromere of HeLa cells undergoing mitotic
division, and is considered to be a functional homologue of yeast
Nuf2 (NPL15: Wigge P A et al., J Cell Biol 2001, 152(2): 349-60).
Meanwhile, CDCA1 has been identified as a gene showing elevated
expression in non-small cell lung cancer by gene expression
profiling based on a genome-wide cDNA microarray targeting 27648
genes (NPL16: Hayama et al., Cancer Res 2006, 66(21): 10339-48;
PTL1: WO2007/013480; PTL2: WO2005/089735). The CDCA1 expression is
observed in lung cancer tissues and lung cancer cell lines, but
little expression is observed in 22 normal tissues except the
testis (NPL16; PTL1). Further, as a result of siRNA-mediated
suppression of the CDCA1 expression, suppression of cell
proliferation in CDCA1-expressing lung cancer cell lines is caused
(NPL16; PTLs 1-2). Moreover, elevated expression of CDCA1 is also
observed in various cancers such as cholangiocellular cancer,
bladder cancer and renal cell cancer (NPL17: Harao M et al., Int J
Cancer 2008, 123(11): 2616-25).
[0005] Recently, CDCA1-derived HLA-A02-restricted CTL epitope
peptides (NPL17: Harao et al., Int J Cancer. 2008, 123(11):
2616-25; PTL3: WO2009/025117), HLA-A24-restricted CTL epitope
peptides (PTL4: WO2009/153992), HLA-Al 1-restricted CTL epitope
peptides (PTL5: WO2016/021508), HLA-A33-restricted CTL epitope
peptides (PTL5: WO2016/021508) and HLA-A03-restricted CTL epitope
peptides (PTL5: WO2016/021508) have been identified. Therapeutic
effects by these peptides are expected in cancer patients having
the HLA-A02 type, HLA-A24 type, HLA-Al 1 type, HLA-A33 type or
HLA-A03 type, but for cancer patients having HLA types other than
those, peptides corresponding to respective HLA type are
desired.
CITATION LIST
Patent Literature
[0006] [PTL 1] WO2007/013480
[0007] [PTL 2] WO2005/089735
[0008] [PTL 3] WO2009/025117
[0009] [PTL 4] WO2009/153992
[0010] [PTL 5] WO2016/021508
Non Patent Literature
[0011] [NPL 1] Boon T, Int J Cancer 1993, 54(2): 177-80
[0012] [NPL 2] Boon T & van der Bruggen P, J Exp Med 1996,
183(3): 725-9
[0013] [NPL 3] Harris CC, J Natl Cancer Inst 1996, 88(20):
1442-55
[0014] [NPL 4] Butterfield L H et al., Cancer Res 1999, 59(13):
3134-42
[0015] [NPL 5] Vissers J L et al., Cancer Res 1999, 59(21):
5554-9
[0016] [NPL 6] van der Burg S H et al., J Immunol 1996, 156(9):
3308-14
[0017] [NPL 7] Tanaka F et al., Cancer Res 1997, 57(20): 4465-8
[0018] [NPL 8] Fujie T et al., Int J Cancer 1999, 80(2): 169-72
[0019] [NPL 9] Kikuchi M et al., Int J Cancer 1999, 81(3):
459-66
[0020] [NPL 10] Oiso M et al., Int J Cancer 1999, 81(3): 387-94
[0021] [NPL 11] Belli F et al., J Clin Oncol 2002, 20(20):
4169-80
[0022] [NPL 12] Coulie PG et al., Immunol Rev 2002, 188: 33-42
[0023] [NPL 13] Rosenberg SA et al., Nat Med 2004, 10(9):
909-15
[0024] [NPL 14] Walker et al., Curr Cancer Dnig Targets 2001, 1(1):
73-83
[0025] [NPL 15] Wigge PA et al., J Cell Biol 2001, 152(2):
349-60
[0026] [NPL 16] Hayama et al., Cancer Res 2006, 66(21):
10339-48
[0027] [NPL 17] Harao et al., Int J Cancer 2008, 123(11):
2616-25
SUMMARY OF THE INVENTION
[0028] The present invention relates to peptides that can induce
cytotoxic T cells (CTLs) specifically responding to
CDCA1-expressing cells. When these peptides are presented on
antigen-presenting cells (APCs) by the human leukocyte antigen
(HLA) and recognized by CD8-positive T cells, CTLs that show a
peptide-specific cytotoxic activities are induced. CDCA1-derived
peptides that have been identified so far to have CTL-inducing
ability (CTL inducibility) are HLA-A02-restricted,
HLA-A24-restricted, HLA-A11-restricted, HLA-A33-restricted, or
HLA-A03-restricted peptides, and when antigen-presenting cells do
not express these HLAs, the peptides cannot induce CTLs. Therefore,
conventional peptides are not suitable for performing immunotherapy
on cancer patients (subjects) that do not have these HLAs. HLA-A01
is an HLA allele highly frequently observed in Caucasians (Cao K et
al., Hum Immunol 2001, 62(9): 1009-30). It is desirable to
administer HLA-A01-restricted peptides to HLA-A01-positive cancer
patients. Hence, the present invention relates to CDCA1l-derived
peptides with CTL-inducing ability that are restrictive to HLA-A01.
Based on results disclosed herein, the peptides of the present
invention have been proven to be epitope peptides that can induce a
potent and specific immune response against cancer cells expressing
CDCA1 and HLA A01.
[0029] Therefore, one of the objectives of the present invention is
to provide CDCA1-derived peptides that can induce CTLs in an
HLA-A01-restrictive manner. These peptides can be used to induce
CTLs in vitro, ex vivo or in vivo, or can be used to administer to
subjects for the purpose of inducing an immune response against
CDCA1-expressing cancer cells. Preferable peptides are peptides
comprising the amino acid sequence selected from among SEQ ID NOs:
1, 8, 10, 13, 25, 33 to 35 and 37; more preferable peptides are
nonapeptides or decapeptides; and even more preferable peptides are
peptides consisting of the amino acid sequence selected from among
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37.
[0030] The peptides of the present invention encompass peptides in
which one, two, or more amino acid(s) is/are substituted, deleted,
inserted and/or added, as long as the resultant modified peptides
retain the CTL-inducing ability of the original peptide.
[0031] The present invention further provides isolated
polynucleotides encoding any one of the peptides of the present
invention. Similar to the peptides of the present invention, these
polynucleotides can be used for inducing APCs with CTL-inducing
ability, and can be administered to subjects for inducing an immune
response against CDCA1-expressing cancer cells.
[0032] The present invention also provides compositions comprising
one or more types of peptides of the present invention, one or more
types of polynucleotides encoding one or more types of peptides of
the present invention, APCs of the present invention, exosomes
presenting peptides of the present invention, and/or CTLs of the
present invention. The compositions of the present invention are
preferably pharmaceutical compositions. The pharmaceutical
compositions of the present invention can be used for treating
and/or preventing cancer, as well as preventing postoperative
recunence thereof. They can also be used for inducing an immune
response against cancer. When administered to a subject, a peptide
of the present invention is presented on the surface of an APC, and
as a result CTLs targeting the peptide are induced. Therefore,
another objective of the present invention is to provide
compositions for inducing CTLs, wherein the compositions comprise
one or more types of peptides of the present invention, one or more
types of polynucleotides encoding one or more types of peptides of
the present invention, APCs of the present invention, and/or
exosomes presenting peptides of the present invention.
[0033] A further objective of the present invention is to provide
methods of inducing APCs having CTL-inducing ability, wherein the
methods comprise a step of contacting one or more types of peptides
of the present invention with an APC, or a step of introducing a
polynucleotide encoding any one peptide of the present invention
into an APC.
[0034] The present invention further provides a method of inducing
CTLs, comprising a step of co-culturing a CD8-positive T cell with
an APC that presents on its surface a complex of an HLA antigen and
a peptide of the present invention, a step of co-culturing a
CDR-positive T cell with an exosome that presents on its surface a
complex of an HLA antigen and a peptide of the present invention,
or a step of introducing into a CD8-positive T cell a vector
comprising a polynucleotide encoding each subunit of a T cell
receptor (TCR) capable of binding to a peptide of the present
invention presented by an HLA antigen on a cell surface.
[0035] A further objective of the present invention is to provide
isolated APCs that present on their surface a complex of an HLA
antigen and a peptide of the present invention. The present
invention further provides isolated CTLs targeting a peptide of the
present invention. These APCs and CTLs can be used in immunotherapy
for CDCA1-expressing cancers.
[0036] Another objective of the present invention is to provide
methods of inducing an immune response against cancer in a subject,
wherein the methods comprise a step of administering to the subject
a composition(s) comprising a peptide(s) of the present invention
or a polynucleotide(s) encoding the peptide(s), an APC(s) of the
present invention, an exosome(s) presenting a peptide(s) of the
present invention, and/or a CTL(s) of the present invention.
Another objective of the present invention is to provide methods of
treating and/or preventing cancer, as well as preventing
postoperative recurrence thereof in a subject, wherein the methods
comprise a step of administering to the subject a peptide(s) of the
present invention, a polynucleotide(s) encoding the peptide(s), an
APC(s) of the present invention, an exosome(s) presenting a
peptide(s) of the present invention, and/or a CTL(s) of the present
invention.
[0037] In addition to the above, other objects and features of the
present invention will become more fully apparent when the
following detailed description is read in conjunction with the
accompanying figures and examples. However, it is to be understood
that both the foregoing summary of the present invention and the
following detailed description are of exemplified embodiments, and
not restrictive of the present invention or other alternate
embodiments of the present invention. In particular, while the
present invention is described herein with reference to a number of
specific embodiments, it will be appreciated that the description
is illustrative of the present invention and is not constructed as
limiting of the present invention. Various modifications and
applications may occur to those who are skilled in the art, without
departing from the spirit and the scope of the present invention,
as described by the appended claims. Likewise, other objects,
features, benefits and advantages of the present invention will be
apparent from this summary and certain embodiments described below,
and will be readily apparent to those skilled in the art. Such
objects, features, benefits and advantages will be apparent from
the above in conjunction with the accompanying examples, data,
figures and all reasonable inferences to be drawn therefrom, alone
or with consideration of the references incorporated herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 is comprised of photos (a) to (j) showing the results
of the IFN-gamma enzyme-linked immunospot (ELISPOT) assay performed
using cells induced using peptides derived from CDCA1. In the
figure, "+" shows IFN-gamma production against target cells pulsed
with a peptide of interest and "-" shows IFN-gamma production
against target cells that have not been pulsed with any peptides
(negative target). In comparison with the negative control,
peptide-specific IFN-gamma production was observed in:
[0039] Well #1 with CDCA1-A01-8-138 (SEQ ID NO: 1) (a),
[0040] Well #7 with CDCA1-A01-9-290 (SEQ ID NO: 25) (b),
[0041] Well #5 with CDCA1-A01-9-130 (SEQ ID NO: 33) (c),
[0042] Well #7 with CDCA1-A01-9-246 (SEQ ID NO: 34) (d),
[0043] Well #1 with CDCA1-A01-9-268 (SEQ ID NO: 35) (e),
[0044] Well #3 with CDCA1-A01-9-288 (SEQ ID NO: 37) (f),
[0045] Well #2 with CDCA1-A01-10-136 (SEQ ID NO: 8) (g),
[0046] Well #6 with CDCA1-A01-10-56 (SEQ ID NO: 10) (h), and
[0047] Well #6 with CDCA1-A01-10-48 (SEQ ID NO: 13) (i).
[0048] CTL clones were established from the boxed cells in the
photos that showed a reaction. In contrast, CDCA1-A01-10-66 (SEQ ID
NO: 9) (j) is shown as an example of typical negative data where
peptide-specific IFN-gamma production was not observed.
[0049] FIG. 2 is comprised of a series of line graphs (a) to (c)
showing IFN-gamma production of CTL clones established by limiting
dilution method following induction by CDCA1-A01-10-136 (SEQ ID NO:
8), CDCA1-A01-10-56 (SEQ ID NO: 10), or CDCA1-A01-10-48 (SEQ ID NO:
13). These results show peptide-specific IFN-gamma production of
the CTL clones. In the figure, "+" shows IFN-gamma production of
the CTL clones against target cells pulsed with a peptide of
interest and "-" shows IFN-gamma production of the CTL clones
against target cells that have not been pulsed with any peptides.
The R/S ratio indicates the ratio of the cell number of the CTL
clones (Responder cells) and the cell number of target cells that
stimulate these cells (Stimulator cells).
[0050] FIG. 3 is a line graph showing IFN-gamma production of a CTL
clone against target cells expressing both CDCA1 and HLA-A*01:01.
COS7 cells expressing either HLA-A*01:01 or the full-length CDCA1
gene served as a negative control. The CTL clone established
following induction by CDCA1-A01-10-136 (SEQ ID NO: 8) showed
IFN-gamma production in COS7 cells into which both CDCA1 and
HLA-A*01:01 genes were introduced (black diamond). Meanwhile, it
did not show significant IFN-gamma production in COS7 cells into
which either HLA-A*01:01 (triangle) or CDCA1 (white circle) was
introduced.
MODE FOR CARRYING OUT THE INVENTION
[0051] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods,
devices, and materials are now described. However, before the
present materials and methods are described, it is to be understood
that the present invention is not limited to the particular sizes,
shapes, dimensions, materials, methodologies, protocols, etc.
described herein, as these may vary in accordance with routine
experimentation and optimization. It is also to be understood that
the terminology used in the description is for the purpose of
describing the particular versions or embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
I. Definitions
[0052] The words "a", "an", and "the" as used herein mean "at least
one" unless otherwise specifically indicated.
[0053] The terms "isolated" and "purified" used in relation with a
substance (for example, peptide, antibody, polynucleotide or such)
indicate that the substance does not substantially contain at least
one substance that may else be included in a natural source. Thus,
an isolated or purified peptide refers to a peptide that does not
substantially contain another cellular material, for example,
carbohydrate, lipid and other contaminating proteins from the cell
or tissue source from which the peptide is derived. When the
peptide is chemically synthesized, an isolated or purified peptide
refers to a peptide that does not substantially contain a precursor
substance or another chemical substance. The phrase "does not
substantially contain a cellular material" includes peptide
preparations in which the peptide is separated from cellular
components of the cells from which it is isolated or recombinantly
produced. Thus, a peptide that does not substantially contain a
cellular material encompasses peptide preparations that contain
less than about 30%, 20%, 10%, or 5%, 3%, 2% or 1% (dry weight
basis) of other cellular materials. When the peptide is
recombinantly produced, an isolated or purified peptide does not
substantially contain culture medium, which encompasses peptide
preparations that contain culture medium less than about 20%, 10%,
or 5%, 3%, 2% or 1% (dry weight basis) of the volume of the peptide
preparation. When the peptide is generated by chemical synthesis,
an isolated or purified peptide does not substantially contain a
precursor substance or other chemical substances, which encompasses
peptide preparations that contain a precursor substance or other
chemical substances less than about 30%, 20%, 10%, 5%, 3%, 2% or 1%
(dry weight basis) of the volume of the peptide preparation. That a
particular peptide preparation is an isolated or purified peptide
can be confirmed, for example, by the appearance of a single band
following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis and Coomassie Brilliant Blue staining or such of
the gel. In a preferred embodiment, the peptides and
polynucleotides of the present invention are isolated or
purified.
[0054] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein, and refer to polymers of amino acid
residues. These terms are applied to also non-naturally occurring
amino acid polymers comprising one or more non-naturally occurring
amino acid residues, in addition to naturally occurring amino acid
polymers. Non-naturally occurring amino acids include amino acid
analogs, amino acid mimetics, and such.
[0055] The term "amino acid" as used herein refers to naturally
occurring amino acids, as well as amino acid analogs and amino acid
mimetics that functions similarly to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those modified after translation in cells
(e.g., hydroxyproline, gamma-carboxygiutamate, and O-phosphoserine,
etc.). The phrase "amino acid analog" refers to compounds that have
the same basic chemical structure (an alpha carbon bound to a
hydrogen, a carboxy group, an amino group, and an R group) as a
naturally occurring amino acid but have a modified R group or
modified backbones (e.g., homoserine, norleucine, methionine
sulfoxide, methionine methyl sulfonium, and such). The phrase
"amino acid mimetic" refers to chemical compounds that have
different structures but similar functions to general amino acids.
Amino acids can be either L-amino acids or D-amino acids, and the
peptides of the present invention are preferably L-amino acid
polymers.
[0056] The terms "polynucleotide", "oligonucleotide" and "nucleic
acid" are used interchangeably herein, and refer to a polymer of
nucleotides.
[0057] The term "composition" used in the present specification is
intended to encompass products that include specified ingredients
in specified amounts, and any products generated directly or
indirectly from combination of specified ingredients in the
specified amounts. When the composition is a pharmaceutical
composition, the term "composition" is intended to encompass
products including active ingredient(s) and inert ingredient(s), as
well as any products generated directly or indirectly from
combination, complexation or aggregation of any two or more
ingredients, from dissociation of one or more ingredients, or from
other types of reactions or interactions of one or more
ingredients. Thus, the pharmaceutical compositions of the present
invention encompass any compositions made by admixing compounds or
cells of the present invention with a pharmaceutically or
physiologically acceptable carrier. Without being limited thereto,
the terms "pharmaceutically acceptable carrier" or "physiologically
acceptable carrier" used in the present specification include
liquid or solid bulking agents, diluents, excipients, solvents, and
encapsulation materials; and mean pharmaceutically or
physiologically acceptable materials, compositions, substances or
media.
[0058] Unless otherwise specified, the term "cancer" refers to a
cancer that overexpresses the CDCA1 gene; and examples thereof
include bladder cancer, breast cancer, cervical cancer,
cholangiocellular cancer, chronic myeloid leukemia (CML), esophagus
cancer, gastric cancer, non-small-cell lung cancer, lymphoma,
osteosarcoma, prostate cancer, kidney cancer, small-cell lung
cancer, head and neck cancer, soft tissue tumor, colon cancer and
such, without being limited thereto. In an exemplary embodiment,
the "cancer" is a cancer that expresses CDCA1 and HLA-A01.
[0059] Unless otherwise specified, the terms "cytotoxic T
lymphocyte" and "cytotoxic T cell" and "CTL" are used
interchangeably herein. Unless otherwise specifically indicated,
they refer to a sub-group of T lymphocytes that can recognize
non-self cells (for example, tumor/cancer cells, virus-infected
cells) and induce the death of such cells.
[0060] Unless otherwise specified, the term "HLA-A01 (HLA-A1)"
refers to the HLA-A01 type which includes subtypes such as
HLA-A*01:01, HLA-A*01:02, HLA-A*01:03, and HLA-A*01:04.
[0061] In the context of a subject or patient, the phrase "HLA
antigen of a subject (or patient) is HLA-A01" used herein refers to
that a subject or patient has the HLA-A01 antigen gene homozygously
or heterozygously as the MHC (Major Histocompatibility Complex)
Class I molecule, and that the HLA-A01 antigen is expressed in the
cells of the subject or patient as the HLA antigen.
[0062] As long as the methods and compositions of the present
invention are useful in the context of cancer "treatment", the
treatment is considered "efficacious" when it achieves clinical
advantages, for example, reduction in the size, spreading or
metastatic ability of cancer, retardation of cancer progression,
alleviation of clinical symptoms of cancer, prolongation of
survival period, suppression of postoperative recurrence in a
subject. When the treatment is applied prophylactically,
"efficacious" means that the treatment retards or prevents cancer
formation, or prevents or alleviates clinical symptoms of cancer.
Effectiveness is determined in relation to any publicly known
method for diagnosing or treating a specific tumor type.
[0063] As long as the methods and compositions of the present
invention are useful in the context of cancer "prevention
(prophylaxis)", the term "prevention (prophylaxis)" herein includes
any work that eases the load of disease-associated mortality or
morbidity. Prevention (Prophylaxis) can be carried out at the
"primary, secondary and tertiary prevention (prophylaxis) levels".
Whereas the primary prevention (prophylaxis) avoids the development
of a disease, prevention (prophylaxis) at the secondary and
tertiary levels encompasses prevention (prophylaxis) of disease
progression and appearance of symptoms, as well as workings
intended to reduce adverse effects of the existing disease by
restoring functions and reducing disease-associated complications.
Alternately, prevention (prophylaxis) can include alleviation of
severity of a specific disorder, for example, extensive preventive
therapy intended to reduce tumor growth and metastasis.
[0064] In the context of the present invention, the treatment
and/or prevention (prophylaxis) of cancer and/or prevention
(prophylaxis) of postoperative recurrence thereof include either of
the events such as inhibition of cancer cell proliferation, tumor
involution or regression, induction of remission and suppression of
cancer development, tumor regression, as well as reduction or
inhibition of metastasis, suppression of postoperative recurrence
of cancer, and prolongation of survival period. Effective treatment
and/or prevention (prophylaxis) of cancer reduce mortality, improve
prognosis of an individual with cancer, reduce the blood levels of
tumor markers, and alleviate detectable symptoms associated with
cancer. For example, alleviation or improvement of symptoms
constitutes effective treatment and/or prevention (prophylaxis),
and includes a condition in which the symptoms are alleviated or
stable by 10%, 20%, 30% or more.
[0065] In the context of the present invention, the term "antibody"
refers to immunoglobulins and fragments thereof that are
specifically reactive to a designated protein or peptide thereof.
An antibody can include human antibodies, primatized antibodies,
chimeric antibodies, bispecific antibodies, humanized antibodies,
antibodies fused to other proteins or radiolabels, and antibody
fragments. Furthermore, an "antibody" herein is used in the
broadest sense and specifically covers intact monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (e.g.,
bispecific antibodies) formed from two or more intact antibodies,
and antibody fragments so long as they exhibit the desired
biological activity. An "antibody" may be antibodies of all classes
(e.g., IgA, IgD, IgE, IgG and IgM).
[0066] Unless otherwise specified, the technical terms and
scientific terms used herein all have the same meanings as terms
commonly understood by one of ordinary skill in the art to which
the present invention belongs.
II. Peptides
[0067] HLA-A01 is an HLA allele commonly seen in Caucasians (Cao et
al., Hum Immunol 2001, 62(9): 1009-30). Thus, an effective method
of treating CDCA1-expressing cancers for a great population of
Caucasians can be provided by providing CDCA1-derived CTL-inducing
peptides restricted to HLA-A01. Thus, the present invention
provides CDCA1-derived peptides that are capable of inducing CTLs
in an HLA-A01-restrictive manner.
[0068] The peptides of the present invention are CDCA1-derived
peptides that are capable of inducing CTLs in an
HLA-A01-restrictive manner Peptides capable of inducing CTLs in an
HLA-A01-restrictive manner include peptides having the amino acid
sequence selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37.
[0069] CTLs having a cytotoxic activity specific to these peptides
can be established by in vitro stimulation of T cells by dendritic
cells pulsed with these peptides. The established CTLs show a
specific cytotoxic activity against target cells pulsed with each
of the peptides.
[0070] The CDCA1 gene is overexpressed in cancer cells such as
cancer cells in, for example, bladder cancer, breast cancer,
cervical cancer, cholangiocellular cancer, chronic myeloid leukemia
(CML), esophagus cancer, gastric cancer, non-small-cell lung
cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer,
small-cell lung cancer, head and neck cancer, soft tissue tumor,
colon cancer and such, but is not expressed in most normal organs.
It is thus an excellent target for immunotherapy. Therefore, the
peptides of the present invention can be suitably used for cancer
immunotherapy. A preferred peptide is a nonapeptide (a peptide
consisting of 9 amino acid residues) or a decapeptide (a peptide
consisting of 10 amino acid residues), and it is more preferably a
peptide consisting of the amino acid sequence selected from among
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37. For example, a
peptide having the amino acid sequence of SEQ ID NO: 8 is suitable
for induction of CTLs that show a specific cytotoxic activity
against cells expressing HLA-A01 and CDCA1, and can be suitably
used for cancer immunotherapy in HLA-A01-positive patients. In a
more preferred embodiment, the peptide of the present invention is
a peptide consisting of the amino acid sequence of SEQ ID NO:
8.
[0071] For the peptides of the present invention, an additional
amino acid residue(s) can be made to adjoin the amino acid sequence
of the peptide of the present invention, as long as the resultant
peptides retain the CTL-inducing ability of the original peptide.
The additional amino acid residue(s) may be composed of any types
of amino acid(s), as long as they do not impair the CTL-inducing
ability of the original peptide. Therefore, the peptides of the
present invention encompass peptides having CTL-inducing ability,
comprising the amino acid sequence selected from among SEQ ID NOs:
1, 8, 10, 13, 25, 33 to 35 and 37. Such peptides are, for example,
less than about 40 amino acids, in many cases less than about 20
amino acids, and usually less than about 15 amino acids. Therefore,
if the original peptide is a nonapeptide, the peptide of the
present invention encompasses peptides that are 10 amino-acid long
or 11-40 amino-acid long, which are produced by adjoining
additional amino acid(s) to the peptide. Furthermore, if the
original peptide is a decapeptide, the peptide of the present
invention encompasses peptides that are 11-40 amino-acid long. Such
a peptide can be, for example, a peptide that is 11-20 amino-acid
long or a peptide that is 11-15 amino-acid long. A preferred
example of an additional amino acid residue(s) is an amino acid
residue(s) adjacent to the amino acid sequence of the peptide of
the present invention in the full-length amino acid sequence of
CDCA1 (for example, SEQ ID NO: 64). Therefore, the peptides of the
present invention encompass peptides comprising the amino acid
sequence selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37, and wherein the peptides are peptide fragments of CDCA1 and
have CTL-inducing ability.
[0072] In general, modifications of one, two or more amino acids in
a certain peptide do not affect the functions of the peptide, or in
some cases even enhance the desired functions of the original
peptide. In fact, modified peptides (i.e., peptides composed of the
amino acid sequence in which one, two or several amino acid
residues are modified (i.e., substituted, deleted, inserted, and/or
added) compared to the original reference sequence) are known to
retain the biological activity of the original peptide (Mark et
al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and Smith,
Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et al.,
Proc Nati Acad Sci USA 1982, 79: 6409-13). Thus, in one embodiment,
the peptides of the present invention can be peptides comprising
the amino acid sequence in which one, two or several amino acids
are substituted, deleted, inserted and/or added to the amino acid
sequence selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37 and having CTL-inducing ability.
[0073] On skilled in the art can recognize that individual
substitutions to an amino acid sequence that alter a single amino
acid or a small percentage of amino acids tend to result in the
conservation of the properties of the original amino acid side
chain(s). Accordingly, those are frequently referred to as
"conservative substitutions" or "conservative modifications"; and
modification of a protein by "conservative substitution" or
"conservative modification" may result in a modified protein that
has similar functions as the original protein. Tables of
conservative substitutions presenting functionally similar amino
acids are well known in the art. Examples of amino acid side chain
characteristics that functionally resemble include, for example,
hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic
amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side chains
having the following functional groups or characteristics in
common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl
group containing side-chain (S, T, Y); a sulfur atom containing
side-chain (C, M); a carboxylic acid and amide containing
side-chain (D, N, E, Q); a base containing side-chain (R, K, H);
and an aromatic containing side-chain (H, F, Y, W). In addition,
the following eight groups each contain amino acids that are
accepted in the art as conservative substitutions for one another:
[0074] 1) Alanine (A), Glycine (G); [0075] 2) Aspartic acid (D),
Glutamic acid (E); [0076] 3) Asparagine (N), Glutamine (Q); [0077]
4) Arginine (R), Lysine (K); [0078] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); [0079] 6) Phenylalanine (F), Tyrosine
(Y), Tryptophan (W); [0080] 7) Serine (5), Threonine (T); and
[0081] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton,
Proteins 1984).
[0082] Such conservatively modified peptides are also encompassed
in peptides of the present invention. However, peptides of the
present invention are not restricted thereto and can include
non-conservative modifications, so long as the modified peptide
retains the CTL-inducing ability of the original peptide.
Furthermore, modified peptides do not exclude CTL inducible
peptides derived from polymorphic variants, interspecies
homologues, and alleles of CDCA1.
[0083] So long as a peptide retains the CTL-inducing ability of an
original peptide, one can modify (i.e., substitute, delete, insert
and/or add) a small number (for example, 1, 2 or several) or a
small percentage of amino acids. Herein, the term "several" means 5
or fewer amino acids, for example, 4 or 3 or fewer. The percentage
of amino acids to be modified is preferably 20% or less, more
preferably 15% or less, even more preferably 10% or less or 1 to
5%.
[0084] When used in the context of immunotherapy, peptides of the
present invention are presented on the surface of a cell or
exosome, preferably as a complex with an HLA antigen. Therefore, it
is preferable that the peptides of the present invention possess
high binding affinity to the HLA antigen. To that end, the peptides
can be modified by substitution, deletion, insertion, and/or
addition of the amino acid residues to yield a modified peptide
having improved binding affinity. Since the regularity of the
sequences of peptides displayed by binding to HLA antigens is
already known (Falk et al., Immunogenetics 1994, 40 232-41; Chujoh,
et al., Tissue Antigens 1998, 52: 501-9; Takiguchi et al., Tissue
Antigens 2000, 55: 296-302.), modifications based on such
regularity can be introduced into the peptides of the present
invention.
[0085] For example, in peptides having binding affinity for HLA
Class I, the second amino acid from the N terminus and the
C-terminal amino acid are generally anchor residues involved in the
binding to HLA Class I (Rainmensee HG, et al., Immunogenetics.
1995, 41(4): 178-228.). For example, for HLA-A01, aspartic acid and
glutamic acid for the third amino acid from the N terminus, and
tyrosine for the C-terminal amino acid are known as anchor residues
with high binding affinity for HLA-A01. Further, in HLA-A01, there
is auxiliary anchor residues at position 2 from the N terminus; and
it is known that threonine and serine are preferred as the second
amino acid from the N terminus (Kubo, R. T Journal of Immunology
1994, 152: 3913-24; Gambacorti-Passerini, C. Clinical Cancer
Research 1997, 3: 675-83; Falk, K. Immunogenetics 1994, 40:
238-41). Thus, to enhance the HLA-A01-binding affinity, there is a
possibility that it is desirable to substitute the third amino acid
from the N terminus with aspartic acid or glutamic acid, and/or to
substitute the C-terminal amino acid with tyrosine. Further, there
is a possibility that it is also desirable to substitute the second
amino acid from the N terminus with threonine or serine. Thus,
peptides with CTL-inducing ability, comprising an amino acid
sequence in which, in the amino acid sequence selected from among
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37, the second amino
acid from the N terminus is substituted with threonine or serine;
the third amino acid from the N terminus is substituted with
aspartic acid or glutamic acid; and/or the C-terminal amino acid is
substituted with tyrosine are encompassed by the peptides of the
present invention. In a preferred embodiment, the peptide of the
present invention can be a peptide having CTL-inducing ability that
consists of an amino acid sequence in which, in the amino acid
sequence selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37, the second amino acid from the N terminus is substituted
with threonine or serine; the third amino acid from the N terminus
is substituted with aspartic acid or glutamic acid; and/or the
C-terminal amino acid is substituted with tyrosine. That is, the
peptides of the present invention encompass peptides having
CTL-inducing ability, which comprise an amino acid sequence with
one or more substitutions selected from (a) to (c) below in the
amino acid sequence selected from among SEQ ID NOs: 1, 8, 10, 13,
25, 33 to 35 and 37: [0086] (a) the second amino acid from the N
terminus is substituted with threonine or serine; [0087] (b) the
third amino acid from the N terminus is substituted with aspartic
acid or glutamic acid; and [0088] (c) the C-terminal amino acid is
substituted with tyrosine.
[0089] In a preferred embodiment, the peptide of the present
invention may be a peptide having CTL-inducing ability that
consists of an amino acid sequence in which one or more
substitutions selected from (a) to (c) above are introduced into
the amino acid sequence selected from among SEQ ID NOs: 1, 8, 10,
13, 25, 33 to 35 and 37. In the present invention, the preferred
number of substitutions is 1, 2 or 3 substitutions selected from
(a) to (c) above.
[0090] Furthermore, the peptide of the present invention may be a
peptide having CTL-inducing ability, which comprises an amino acid
sequence in which, in the amino acid sequence selected from among
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37, the third amino acid
from the N terminus is substituted with aspartic acid or glutamic
acid, and/or the C-terminal amino acid is substituted with
tyrosine. Preferably, the peptide of the present invention may be a
peptide having CTL-inducing ability, which consists of an amino
acid sequence in which, in the amino acid sequence selected from
among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37, the third
amino acid from the N terminus is substituted with aspartic acid or
glutamic acid, and/or the C-terminal amino acid is substituted with
tyrosine. That is, the peptide of the present invention may be a
peptide having CTL-inducing ability, which comprises an amino acid
sequence in which one or more substitutions selected from (a) and
(b) below are introduced into the amino acid sequence selected from
among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37: [0091] (a) the
third amino acid from the N terminus is substituted with aspartic
acid or glutamic acid; and [0092] (b) the C-terminal amino acid is
substituted with tyrosine.
[0093] In a preferred embodiment, the peptide of the present
invention may be a peptide having CTL-inducing ability, which
consists of an amino acid sequence in which one or more
substitutions selected from (a) to (b) above are introduced into
the amino acid sequence selected from among SEQ ID NOs: 1, 8, 10,
13, 25, 33 to 35 and 37.
[0094] Substitution(s) may be introduced into amino acid(s) not
only at the anchor site(s), but also at a position(s) of potential
T cell receptor (TCR) recognition site(s) of the peptides. Several
research studies have demonstrated that a peptide that has amino
acid substitutions, such as CAP1, p53.sub.(264-272),
Her-2/neu.sub.(369-377) or gp100.sub.(209-217), may have equal to
or better activity than that of the original peptide (Zaremba et
al. Cancer Res. 1997, 57, 4570-7; T. K. Hoffmann et al. J Immunol.
2002, 168(3): 1338-47; S. O. Dionne et al. Cancer Immunol
immunother. 2003, 52: 199-206; and S. 0. Dionne et al. Cancer
Immunology, Immunotherapy 2004, 53, 307-14).
[0095] The present invention also contemplates that one, two or
several amino acids can be added to the N terminus and/or C
terminus of the peptides of the present invention (for example,
peptides consisting of the amino acid sequence selected from among
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37). Such modified
peptides that retain CTL-inducing ability are also included in the
present invention. For example, when a peptide in which one, two or
several amino acids are added to the N terminus and/or C terminus
of a peptide consisting of the amino acid sequence of SEQ ID NO: 8
is contacted with an APC(s), it is incorporated into the APC(s) and
processed to become a peptide consisting of the amino acid sequence
of SEQ ID NO: 8. It can then induce CTLs through presentation on
the cell surface of an APC via the antigen presentation pathway.
More specifically, peptides of the present invention can be
peptides in which one, two or several amino acids are added to
either or both of the N terminus and C terminus.
[0096] In yet another embodiment of the present invention, peptides
consisting of an amino acid sequence which comprises one, two, or
several amino acid substitutions in the amino acid sequence
referenced by each SEQ ID NO and which further comprises one, two,
or several amino acids added to either or both of the N-terminus
and the C-terminus of the substitution-bearing amino acid sequence
are provided.
[0097] When a peptide of the present invention comprises an amino
acid substitution, a desired position for the substitution can be,
for example, one, two, or three selected from position 2 from the
N-terminus, position 3 from the N-terminus, and the C-terminus in
the amino acid sequence referenced by SEQ ID NO: 1, 8, 10, 13, 25,
33 to 35 or 37 which is comprised in said peptide of the present
invention.
[0098] However, when the amino acid sequence of a peptide is
identical to a portion of the amino acid sequence of an endogenous
or exogenous protein having a different function, side effects such
as autoimmune disorders and/or allergic symptoms against specific
substances may be induced. Therefore, it is preferable to perform
homology searches using available databases to avoid situations in
which the amino acid sequence of the peptide matches the amino acid
sequence of another protein. When it becomes clear from the
homology searches that no peptide exists with as few as 1 or 2
amino acid differences as compared to the objective peptide, the
objective peptide can be modified in order to increase its binding
affinity with HLA antigens, and/or increase its CTL-inducing
ability without danger of such side effects.
[0099] Peptides in which one, two or several amino acids of a
peptide of the present invention are modified are predicted to be
able to retain CTL-inducing ability of the original peptide;
however, it is preferable to verify the CTL-inducing ability of the
modified peptides. Herein, the "peptide having CTL-inducing ability
(CTL inducibility)" refers to a peptide that induces CTLs through
APCs stimulated with the peptide. "CTL induction" includes
induction of differentiation into CTLs, induction of CTL
activation, induction of CTL proliferation, induction of CTL's
cytotoxic activity, induction of CTL-mediated dissolution of target
cells, and induction of increase of IFN-gamma production of
CTLs.
[0100] The CTL-inducing ability can be confirmed by inducing and
stimulating APCs that retain an HLA antigen (for example, B
lymphocytes, macrophages, and dendritic cells) with a peptide, and
mixing them with CD8-positive T cells; and then measuring IFN-gamma
released by CTLs against the target cells. For the APCs, human
peripheral blood mononuclear leukocyte-derived dendritic cells can
be preferably used. As a reaction system, transgenic animals
generated to express an HLA antigen can be used. Alternatively, for
example, the target cells may be radio-labelled with .sup.51Cr or
such, and the cytotoxic activity of the peptide-induced CTLs may be
calculated from the radioactivity emitted from the target cells.
Alternatively, in the presence of peptide-stimulated APCs, it is
possible to evaluate the CTL-inducing ability by measuring the
IFN-gamma produced and released by CTLs, and visualizing the
inhibition zone on the media using anti-IFN-gamma monoclonal
antibodies.
[0101] In addition to the modifications above, the peptides of the
present invention can be linked to other peptides as long as the
resultant linked peptide retains the CTL-inducing ability. An
example of an appropriate peptide to be linked with the peptides of
the present invention includes a TAA-derived CTL-inducing peptide.
Further, the peptides of the present invention can also be linked
with each other. Suitable linkers for use in linking peptides are
known in the art, and for example, linkers such as AAY (P. M.
Daftarian et al., J Trans Med. 2007, 5: 26), AAA, NKRK (SEQ ID NO:
48) (R. P. M. Sutmuller et al., J Immunol. 2000, 165: 7308-15), or
K (S. Ota et al., Can Res. 2002, 62, 1471-6; K. S. Kawamura et al.,
J Immunol. 2002, 168: 5709-15) can be used. Peptides can be linked
in various arrangements (for example, catenulate, repeated, etc.),
and one can also link three or more peptides.
[0102] The peptides of the present invention can also be linked to
other substances as long as the resultant linked peptide retains
the CTL-inducing ability. Examples of an appropriate substance to
be linked with a peptide of the present invention include, for
example, a peptide, a lipid, a sugar or sugar chain, an acetyl
group, and a naturally-occurring or synthetic polymer. The peptides
of the present invention can be modified by glycosylation,
side-chain oxidation, phosphorylation or such, as long as their
CTL-inducing ability is not impaired. One can also perform such
types of modifications to confer additional functions (for example,
targeting function and delivery function) or to stabilize the
peptide.
[0103] For example, to increase the in vivo stability of a peptide,
it is known in the art to introduce D-amino acids, amino acid
mimetics or non-naturally occurring amino acids, and this concept
may also be applied to peptides of the present invention. Peptide
stability can be assayed by several methods. For example, stability
can be tested by using a peptidase as well as various biological
media such as human plasma and serum (see, e.g., Verhoef et al.,
Eur J Drug Metab Phannacokin 1986, 11: 291-302).
[0104] Further, as stated above, among the modified peptides in
which one, two, or several amino acid residues have been
substituted, deleted, inserted and/or added, those having equal to
or higher activity as compared to original peptides can be screened
for or selected. Thus, the present invention also provides methods
of screening for or selecting modified peptides that have equal to
or higher activity than that of the original peptide. Specifically,
the present invention provides a method of screening for a peptide
having CTL-inducing ability, wherein the method comprises the steps
of: [0105] (a) generating candidate sequences consisting of an
amino acid sequence in which one, two, or several amino acid
residues are substituted, deleted, inserted and/or added to the
original amino acid sequence consisting of the amino acid sequence
selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37;
[0106] (b) selecting from among the candidate sequences generated
in (a), a candidate sequence that does not have a significant
homology (sequence identity) with any known human gene product
other than CDCA1; [0107] (c) contacting a peptide consisting of the
candidate sequence selected in (b) with APCs; [0108] (d) contacting
the APCs of (c) with CD8-positive T cells; and [0109] (e) selecting
a peptide that has an equal to or higher CTL-inducing ability than
that of a peptide consisting of the original amino acid
sequence.
[0110] In the present invention, all the peptides consisting of SEQ
ID NO: 1, 8, 10, 13, 25, 33 to 35 or 37 have an HLA-A01-restrictive
CTL-inducing activity. Therefore, in order to select ones with the
CTL-inducing ability from their amino-acid-sequence-modified
variants, the APCs in step (c) above are desirably cells having
HLA-A01.
[0111] Herein, the peptide of the present invention is also
described as a "CDCA1 peptide(s)" or a "CDCA1 polypeptide(s)".
III. Preparation of Peptides of the Present Invention
[0112] Well known techniques can be used to prepare peptides of the
present invention. For example, recombinant DNA technology or
chemical synthesis can be used to prepare peptides of the present
invention. Peptides of the present invention can be synthesized
individually, or as longer polypeptides including two or more
peptides. Peptides of the present invention can be isolated from
host cells or synthesis reaction products after they are produced
in the host cells using recombinant DNA technology or after they
are chemically synthesized. That is, peptides of the present
invention can be purified or isolated so as not to substantially
contain other host-cell proteins and fragments thereof, or any
other chemical substances.
[0113] The peptides of the present invention may contain
modifications, such as glycosylation, side chain oxidation, or
phosphorylation provided such modifications do not destroy the
biological activity of the original peptide. Other illustrative
modifications include incorporation of D-amino acids or other amino
acid mimetics that may be used, for example, to increase the serum
half life of the peptides.
[0114] A peptide of the present invention can be obtained through
chemical synthesis based on the selected amino acid sequence.
Examples of conventional peptide synthesis methods that can be
adapted to the synthesis include the methods described in the
documents below: [0115] (i) Peptide Synthesis, Interscience, New
York, 1966; [0116] (ii) The Proteins, Vol. 2, Academic Press, New
York, 1976; [0117] (iii) "Peptide Synthesis" (in Japanese), Maruzen
Co., 1975; [0118] (iv) "Basics and Experiment of Peptide Synthesis"
(in Japanese), Maruzen Co., 1985; [0119] (v) "Development of
Pharmaceuticals" (in Japanese), Continued Vol. 14 (peptide
synthesis), Hirokawa, 1991; [0120] (vi) WO99/67288; and [0121]
(vii) Barany G. & Merrifield R. B., Peptides Vol. 2, Solid
Phase Peptide Synthesis, Academic Press, New York, 1980,
100-118.
[0122] Alternatively, the peptides of the present invention can be
obtained by adapting any known genetic engineering methods for
producing peptides (e.g., Morrison J, J Bacteriology 1977, 132:
349-51; Clark-Curtiss & Curtiss, Wu et al., Methods in
Enzymology 1983, 101: 347-62). For example, first, a suitable
vector harboring a polynucleotide encoding the peptide of the
present invention in an expressible form (e.g., downstream of a
regulatory sequence corresponding to a promoter sequence) is
prepared and transformed into a suitable host cell. The host cell
is then cultured to produce the peptide of the present invention.
The peptide of the present invention can also be produced in vitro
using an in vitro translation system.
IV. Polynucleotides
[0123] The present invention also provides a polynucleotide which
encodes any of the peptides of the present invention. These include
polynucleotides derived from the naturally occurring CDCA1 gene
(e.g., GenBank Accession No. NM_145697 (SEQ ID NO: 44) or GenBank
Accession No. NM_031423 (SEQ ID NO: 46)) as well as those having a
conservatively modified nucleotide sequence thereof. Herein, the
phrase "conservatively modified nucleotide sequence" refers to
sequences which encode identical or essentially identical amino
acid sequences. Due to the degeneracy of the genetic code, a large
number of functionally identical nucleic acids encode any given
protein. For instance, the codons GCA, GCC, GCG, and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described above without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations",
which are one species of conservatively modified variations. Every
nucleic acid sequence herein which encodes a peptide also describes
every possible silent variation of the nucleic acid. One of
ordinary skill will recognize that each codon in a nucleic acid
(except AUG, which is ordinarily the only codon for methionine, and
TGG, which is ordinarily the only codon for tryptophan) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid that encodes a peptide is
implicitly described in each disclosed sequence.
[0124] The polynucleotide of the present invention can be composed
of DNA, RNA, and derivatives thereof A DNA is suitably composed of
bases such as A, T, C, and G, and T is replaced by U in an RNA.
[0125] The polynucleotide of the present invention can encode
multiple peptides of the present invention with or without
intervening amino acid sequences in between. For example, the
intervening amino acid sequence can provide a cleavage site (e.g.,
enzyme recognition sequence) of the polynucleotide or the
translated peptides. Furthermore, the polynucleotide can include
any additional sequences to the coding sequence encoding the
peptide of the present invention. For example, the polynucleotide
can be a recombinant polynucleotide that includes regulatory
sequences required for the expression of the peptide or can be an
expression vector (e.g., plasmid) with marker genes and such. In
general, such recombinant polynucleotides can be prepared by the
manipulation of polynucleotides through conventional recombinant
techniques using, for example, polymerases and endonucleases.
[0126] Both recombinant and chemical synthesis techniques can be
used to produce the polynucleotides of the present invention. For
example, a polynucleotide can be produced by insertion into an
appropriate vector, which can be expressed when transfected into a
competent cell. Alternatively, a polynucleotide can be amplified
using PCR techniques or expression in suitable hosts (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1989). Alternatively, a
polynucleotide can be synthesized using the solid phase techniques,
as described in Beaucage SL & Iyer RP, Tetrahedron 1992, 48:
2223-311; Matthes et al., EMBO J 1984, 3: 801-5.
V. Exosomes
[0127] The present invention further provides intracellular
vesicles, referred to as exosomes, that present complexes formed
between the peptides of the present invention and HLA antigens on
their surface. Exosomes can be prepared, for example, using the
methods detailed in JPH11-510507 and WO99/03499, and can be
prepared using APCs obtained from patients who are subject to
treatment and/or prevention (prophylaxis). The exosomes of the
present invention can be inoculated as vaccines, in a fashion
similar to the peptides of the present invention.
[0128] The type of the HLA antigens included in the above-described
complexes must match that of the subject in need of treatment
and/or prevention (prophylaxis). For example, HLA-A01 (for example,
HLA-A*01:01) is an HLA allele highly frequently observed in
Caucasians, and this HLA antigen type is considered to be suitable
for treatment in Caucasian patients. Typically in clinical
practice, it is possible to select an appropriate peptide that has
a high level of binding affinity for a specific HLA antigen or that
has CTL-inducing ability by antigen presentation mediated by a
specific HLA antigen, by studying in advance the HLA antigen type
of the patient in need of treatment.
[0129] The exosomes of the present invention present on their
surface a complex of a peptide of the present invention and
HLA-A01. When the HLA that forms a complex with a peptide of the
present invention is HLA-A01, the peptide of the present invention
is preferably a peptide having the amino acid sequence selected
from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37 or a
modified peptide thereof, and more preferably a peptide consisting
of the amino acid sequence selected from among SEQ ID NOs: 1, 8,
10, 13, 25, 33 to 35 and 37 or a modified peptide thereof.
VI. Antigen-Presenting Cells (APCs)
[0130] The present invention further provides APCs that present on
their surface complexes formed between HLA antigens and peptides of
the present invention. Alternatively, the present invention
provides APCs having on their cell surface complexes formed between
HLA antigens and peptides of the present invention. The APCs of the
present invention can be isolated APCs. When used in the context of
cells (APCs, CTLs, etc.), the teliu "isolated" means that the cells
are separated from another type of cells. The APCs of the present
invention may be APCs induced from APCs derived from the patient to
be subjected to treatment and/or prevention (prophylaxis), and can
be administered as a vaccine by themselves or in combination with
other drugs including a peptide(s), an exosome(s) or a CTL(s) of
the present invention.
[0131] The APCs of the present invention are not limited to
specific types of cells, and include cells known to present
proteinaceous antigens on their cell surface so as to be recognized
by lymphocytes, for example, dendritic cells (DCs), Langerhans
cells, macrophages, B cells, and activated T cells. Since DC is a
representative APC that has the strongest CTL-inducing activity
among APCs, DCs can be preferably used as the APCs of the present
invention.
[0132] For example, APCs of the present invention can be obtained
by inducing DCs from peripheral blood monocytes and then
stimulating them in vitro, ex vivo or in vivo with the peptides of
the present invention. When the peptide of the present invention is
administered to a subject, APCs presenting the peptide of the
present invention are induced in the body of the subject.
Therefore, after the peptides of the present invention are
administered to a subject, the APCs of the present invention can be
obtained by collecting APCs from the subject. Alternatively, the
APCs of the present invention can be obtained by contacting APCs
collected from a subject with a peptide of the present
invention.
[0133] In order to induce an immune response against
CDCA1-expressing cancer cells in a subject, the APCs of the present
invention can be administered to the subject by themselves or in
combination with other drugs including peptide(s), exosome(s) or
CTL(s) of the present invention. For example, the ex vivo
administration can comprise the following steps of: [0134] (a)
collecting APCs from a first subject; [0135] (b) contacting the
APCs of step (a) with a peptide; and [0136] (c) administering the
APCs of step (b) to a second subject.
[0137] The first subject and the second subject may be the same
individual, or may be different individuals. When the first subject
and the second subject are different individuals, it is preferable
that the HLAs of the first subject and the second subject are of
the same type. The APC obtained in step (b) above can be a vaccine
for cancer treatment and/or prevention (prophylaxis).
[0138] The APCs of the present invention obtained by a method such
as described above have CTL-inducing ability. The term
"CTL-inducing ability (CTL inducibility)" used in the context of an
APC(s) refers to the ability of the APC to be able to induce a
CTL(s) when placed in contact with a CD8-positive T cell(s). The
CTL(s) induced by the APC of the present invention is a CTL(s)
specific to CDCA1 and demonstrates a specific cytotoxic activity
against CDCA1-expressing cells.
[0139] In addition to the above-described methods, the APCs of the
present invention can be prepared by introducing a polynucleotide
encoding a peptide of the present invention into APCs in vitro. The
polynucleotide to be introduced can be in the form of DNA or RNA.
The method of introduction is not particularly limited, and
examples thereof include various methods conventionally performed
in the art such as lipofection, electroporation and the calcium
phosphate method. More specifically, methods described in Cancer
Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996,
184: 465-72; and JP2000-509281 can be used. By introducing a
polynucleotide encoding a peptide of the present invention into an
APC, the polynucleotide is transcribed and translated in the cell,
and then the produced peptide is processed by MHC Class I and
proceeds through a presentation pathway to present the peptide of
the present invention on the cell surface of the APC.
[0140] In a preferred embodiment, the APC of the present invention
is an APC that presents on its cell surface a complex formed
between HLA-A01 (more preferably HLA-A*01:01) and a peptide of the
present invention. When the HLA that forms a complex with a peptide
of the present invention is HLA-A01, the peptide of the present
invention is preferably a peptide having the amino acid sequence
selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37
or a modified peptide thereof, and more preferably a peptide
consisting of the amino sequence selected from among SEQ ID NOs: 1,
8, 10, 13, 25, 33 to 35 and 37.
[0141] The APC(s) of the present invention is preferably an APC(s)
induced by a method comprising a step described (a) or (b) below:
[0142] (a) contacting an APC(s) expressing HLA-A01 (more preferably
HLA-A*01:01) with a peptide of the present invention; or [0143] (b)
introducing a polynucleotide encoding a peptide of the present
invention into an APC(s) expressing HLA-A01 (more preferably
HLA-A*01:01).
[0144] The peptide of the present invention to be contacted with
the HLA-A01-expressing APC(s) is preferably a peptide having the
amino acid sequence selected from among SEQ ID NOs: 1, 8, 10, 13,
25, 33 to 35 and 37 or a modified peptide thereof, and more
preferably a peptide consisting of the amino acid sequence selected
from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37.
[0145] The present invention provides use of a peptide of the
present invention for the manufacture of a pharmaceutical
composition that induces an APC(s) having CTL-inducing ability. In
addition, the present invention provides a method or process of
manufacturing a pharmaceutical composition that induces an APC(s)
having CTL-inducing ability. Further, the present invention
provides a peptide of the present invention for inducing an APC(s)
having CTL-inducing ability.
VII. Cytotoxic T Lymphocytes (CTLs)
[0146] The CTL induced by a peptide of the present invention can be
used as a vaccine in a similar manner to the peptide of the present
invention since it enhances an immune response targeting CDCA
I-expressing cancer cell in vivo. Thus, the present invention
provides CTLs that are induced or activated by a peptide of the
present invention. The CTLs of the present invention are CTLs that
target a peptide of the present invention, and are capable of
binding to a complex of a peptide of the present invention and an
HLA antigen. Binding of a CTL to the complex is mediated via a T
cell receptor (TCR) present on the cell surface of the CTL. The
CTLs of the present invention can be isolated CTLs.
[0147] The CTLs of the present invention can be obtained by (1)
administering a peptide of the present invention to a subject, (2)
stimulating APCs and CD8-positive T cells, or peripheral blood
mononuclear cells (PBMCs) derived from a subject with a peptide of
the present invention in vitro, (3) contacting in vitro
CD8-positive T cells or PBMCs with APCs or exosomes that present on
their surface a complex of an HLA antigen and a peptide of the
present invention, or (4) introducing into CD8-positive T cells a
vector comprising a polynucleotide encoding each subunit of a T
cell receptor (TCR) capable of binding to a peptide of the present
invention presented on cell surface via an HLA antigen. The
exosomes and APCs used in the method of (2) or (3) above can be
prepared by methods described in the "V. Exosomes" and "VI.
Antigen-presenting cells (APCs)" sections, respectively, and the
details of the method of (4) above will be described in the "VIII.
T cell receptors (TCRs)" section.
[0148] The CTLs of the present invention can be administered by
themselves to patients who are subject to treatment and/or
prevention (prophylaxis), or in combination with other drugs
including peptide(s), APC(s) or exosome(s) of the present invention
for the purpose of regulating effects. Further, the CTLs of the
present invention can be CTLs induced from CD8-positive T cells
derived from the patients who are subject to administration of the
CTLs. The CTLs of the present invention act specifically on target
cells that present the peptides of the present invention, for
example, the same peptides used to induce the CTLs of the present
invention. The target cells may be cells that endogenously express
CDCA1, such as cancer cells, or cells transfected with the CDCA1
gene. Cells that present a peptide of the present invention on
their cell surface due to stimulation by the peptide can become a
target of attack by the CTLs of the present invention. The cells
targeted by the CTLs of the present invention are preferably cells
that are positive for HLA-A01 (more preferably HLA-A*01:01).
[0149] In a preferred embodiment, the CTLs of the present invention
target specifically cells that express both HLA-A01 (more
preferably HLA-A*01:01) and CDCA1. Herein, that the CTL "targets"
cells refers to CTL recognition of cells that present on their cell
surface a complex of HLA and a peptide of the present invention and
demonstration of a cytotoxic activity against the cells. Further,
"specifically target" refers to that the CTLs demonstrate a
cytotoxic activity against those cells, but do not show a damaging
activity to other cells. The expression "recognize cells" used in
the context of CTLs refers to binding to a complex of HLA and a
peptide of the present invention presented on cell surface via its
TCR, and demonstrating a specific cytotoxic activity against the
cell. Therefore, the CTLs of the present invention are preferably
CTLs that can bind via TCR to a complex formed between HLA-A01
(more preferably HLA-A*01:01) and a peptide of the present
invention presented on cell surface.
[0150] Furthermore, the CTLs of the present invention are
preferably CTLs induced by a method comprising a step described in
(a) or (b) below: [0151] (a) contacting in vitro CD8-positive T
cells with APCs or exosomes that present on their surface a complex
of HLA-A01 (more preferably HLA-A*01:01) and a peptide of the
present invention; or [0152] (b) introducing into CD8-positive T
cells a polynucleotide encoding each subunit of a TCR capable of
binding to a peptide of the present invention presented on cell
surface by HLA-A01 (more preferably HLA-A*01:01).
VIII. T Cell Receptors (TCRs)
[0153] The present invention also provides compositions comprising
a polynucleotide encoding each subunit of a TCR capable of binding
to a peptide of the present invention presented on cell surface by
an HLA antigen, and methods of using the same. The polynucleotide
confers CD8-positive T cells with specificity against
CDCA1-expressing cancer cells through expression of a TCR on cell
surface capable of binding to a peptide of the present invention
presented on cell surface by an HLA antigen. Polynucleotides
encoding an alpha chain(s) and a beta chain(s) can be identified as
the TCR subunit of the CTL induced by a peptide of the present
invention by using known methods in the art (WO2007/032255 and
Morgan et al., J Immunol, 2003, 171, 3288). For example, PCR
methods are preferred for TCR analysis. PCR primers for analysis
may be, for example, 5'-R Primer (5'-gtctaccaggcattcgcttcat-3')
(SEQ ID NO: 40) as a 5' side primer; and 3-TRa-C Primer
(5'-tcagctggaccacagccgcagcgt-3') (SEQ ID NO: 41) specific to
TCR-alpha-chain C-region, 3-TRb-C1 Primer
(5'-tcagaaatectttctcttgac-3') (SEQ ID NO: 42) specific to
TCR-beta-chain C1-region, or 3-TRb-C2 Primer
(5'-ctagcctctggaatcctttctctt-3') (SEQ ID NO: 43) specific to
TCR-beta-chain C2-region as 3' side primers, but are not limited
thereto. The TCRs formed by introducing the identified
polynucleotides into CD8-positive T cells can bind with high
binding affinity to the target cells that present a peptide of the
present invention, and mediates efficient killing of the target
cells presenting a peptide of the present invention in vivo and in
vitro.
[0154] A polynucleotide encoding each TCR subunit can be
incorporated into an appropriate vector, for example, retrovirus
vector. These vectors are well known in the art. The polynucleotide
or a vector comprising thereof in an expressible form can be
introduced into a CD8-positive T cell, for example, a CD8-positive
T cell derived from a patient. The present invention provides
off-the-shelf compositions that allow rapid and easy production of
modified T cells that have superior cancer cell-killing properties
by rapid modification of the patient's own T cells (or T cells
derived from another subject).
[0155] Herein, a specific TCR is a TCR that can confer a specific
cytotoxic activity against target cells by specifically recognizing
a complex of a peptide of the present invention and an HLA antigen
presented on the surface of the target cell when the TCR is present
on the surface of a CD8-positive T cell. Specific recognition of
the above-described complex can be confirmed by any known method,
and preferable examples thereof include HLA multimer staining
analysis using HLA molecules and peptides of the present invention
and ELISPOT assay methods. Specific TCR-mediated recognition of
target cell by T cell introduced with the above-described
polynucleotide and signal transduction in the cell can be confirmed
by carrying out an ELISPOT assay. When the above-described TCR is
present on the surface of a CD8-positive T cell, whether the TCR
can confer a target cell-specific cytotoxic activity against the
CD8-positive T cell can also be confirmed by known methods.
Preferable methods include, for example, measuring the cytotoxic
activity against target cells by a chrome release assay method or
such.
[0156] The present invention provides, in the context of HLA-A01,
CTLs prepared by transforming CD8-positive T cells with a
polynucleotide encoding each subunit of TCR that binds to, for
example, a peptide having the amino acid sequence selected from
among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37.
[0157] The transformed CTLs are capable of homing in vivo and may
be propagated by a well-known in vitro culturing method (for
example, Kawakami et al., J Immunol., 1989, 142, 3452-61). The CTLs
of the present invention can be used to form an immunogenic
composition useful for disease treatment or prevention
(prophylaxis) in a patient in need of treatment or prevention
(prophylaxis) (the contents are incorporated herein for reference
WO2006/031221).
IX. Pharmaceutical Compositions
[0158] The present invention further provides compositions or
pharmaceutical compositions, comprising at least one active
ingredient selected from below: [0159] (a) a peptide of the present
invention; [0160] (b) a polynucleotide encoding a peptide of the
present invention in an expressible form; [0161] (c) an APC of the
present invention; [0162] (d) an exosome of the present invention;
and [0163] (e) a CTL of the present invention.
[0164] The pharmaceutical compositions of the present invention can
comprise as needed a carrier(s), an excipient(s) or such commonly
used in pharmaceuticals without particular limitations, in addition
to the active ingredient(s) described above. An example of a
carrier that can be used in a pharmaceutical composition of the
present invention includes sterilized water, physiological saline,
phosphate buffer, culture fluid and such. Therefore, the present
invention also provides pharmaceutical compositions, comprising at
least one active ingredient selected from (a) to (e) below and a
pharmaceutically acceptable carrier: [0165] (a) a peptide of the
present invention; [0166] (b) a polynucleotide encoding a peptide
of the present invention in an expressible form; [0167] (c) an APC
of the present invention; [0168] (d) an exosome of the present
invention; and [0169] (e) a CTL of the present invention.
[0170] Further, the pharmaceutical compositions of the present
invention can comprise, as needed, stabilizers, suspensions,
preservatives, surfactants, solubilizing agents, pH adjusters,
aggregation inhibitors and such.
[0171] The CDCA1 expression significantly up-regulates in cancer
cells compared with normal tissues. Thus, a peptide of the present
invention or a polynucleotide encoding the peptide can be used to
treat and/or prevent cancer, and/or prevent postoperative
recurrence thereof. Therefore, the present invention provides
pharmaceutical compositions for treating and/or preventing cancer,
and/or preventing postoperative recurrence thereof, comprising one
or more types of peptides or polynucleotides of the present
invention as an active ingredient. Alternatively, the peptides of
the present invention can be made to be presented on the surface of
exosomes or APCs for use as pharmaceutical compositions. In
addition, CTLs of the present invention targeting any one of the
peptides of the present invention can also be used as an active
ingredient of the pharmaceutical compositions of the present
invention. The pharmaceutical compositions of the present invention
may comprise a therapeutically effective amount or a
pharmaceutically effective amount of the above-described active
ingredient.
[0172] The pharmaceutical compositions of the present invention may
also be used as a vaccine. In the context of the present invention,
the term "vaccine" (also called "immunogenic composition") refers
to a composition that has a function of inducing an immune response
that leads to antitumor action when inoculated into an animal.
Thus, a pharmaceutical composition of the present invention can be
used to induce an immune response that leads to antitumor action.
The immune response induced by a peptide, a polynucleotide, an APC,
a CTL and a pharmaceutical composition of the present invention is
not particularly limited as long as it is an immune response that
leads to antitumor action, and examples include induction of cancer
cell-specific CTLs and induction of cancer cell-specific cytotoxic
activity.
[0173] The pharmaceutical compositions of the present invention can
be used to treat and/or prevent cancer, and/or prevent
postoperative recurrence thereof in human subjects or patients. The
pharmaceutical compositions of the present invention can be used
preferably to a subject positive for HLA-A01. Further, the
pharmaceutical compositions of the present invention can be used
preferably to treat and/or prevent cancers expressing HLA A01 and
CDCA1, and/or prevent postoperative recurrence thereof.
[0174] In another embodiment, the present invention provides use of
an active ingredient selected from below in the manufacture of a
pharmaceutical composition for treating or preventing cancer
expressing HLA-A01 and CDCA1: [0175] (a) a peptide of the present
invention; [0176] (b) a polynucleotide encoding a peptide of the
present invention in an expressible form; [0177] (c) an APC that
presents a peptide of the present invention on its surface; [0178]
(d) an exosome that presents a peptide of the present invention on
its surface; and [0179] (e) a CTL of the present invention.
[0180] Alternatively, the present invention further provides an
active ingredient selected from below for use in treating or
preventing cancer expressing HLA-A01 and CDCA1: [0181] (a) a
peptide of the present invention; [0182] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0183] (c) an APC that presents a peptide of the present invention
on its surface; [0184] (d) an exosome that presents a peptide of
the present invention on its surface; and [0185] (e) a CTL of the
present invention.
[0186] Alternatively, the present invention further provides a
method or process for manufacturing a pharmaceutical composition
for treating or preventing cancer expressing HLA-A01 and CDCA1,
wherein the method or process comprises a step of formulating at
least one active ingredient selected from below with a
pharmaceutically or physiologically acceptable carrier: [0187] (a)
a peptide of the present invention; [0188] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0189] (c) an APC that presents a peptide of the present invention
on its surface; [0190] (d) an exosome that presents a peptide of
the present invention on its surface; and [0191] (e) a CTL of the
present invention.
[0192] In another embodiment, the present invention further
provides a method or process for manufacturing a pharmaceutical
composition for treating or preventing cancer expressing HLA-A01
and CDCA1, wherein the method or process comprises a step of mixing
an active ingredient selected from below with a pharmaceutically or
physiologically acceptable carrier: [0193] (a) a peptide of the
present invention; [0194] (b) a polynucleotide encoding a peptide
of the present invention in an expressible form; [0195] (c) an APC
that presents a peptide of the present invention on its surface;
[0196] (d) an exosome that presents a peptide of the present
invention on its surface; and [0197] (e) a CTL of the present
invention.
[0198] In another embodiment, the present invention further
provides a method for treating or preventing cancer expressing
HLA-A01 and CDCA1, which comprises a step of administering to a
subject at least one active ingredient selected from below: [0199]
(a) a peptide of the present invention; [0200] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0201] (c) an APC that presents a peptide of the present invention
on its surface; [0202] (d) an exosome that presents a peptide of
the present invention on its surface; and [0203] (e) a CTL of the
present invention.
[0204] In the present invention, peptides having the amino acid
sequence selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37 are identified as HLA-A01-restricted epitope peptides that
can induce a potent and specific immune response. Therefore,
pharmaceutical compositions of the present invention comprising at
least one peptide having the amino acid sequence selected from
among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37 are suitable
particularly for administration to a subject having HLA-A01 (for
example, HLA-A*01:01) as an HLA antigen. The same applies to
pharmaceutical compositions comprising a polynucleotide encoding
any of these peptides (i.e., polynucleotides of the present
invention), an APC or exosome that presents these peptides (i.e.,
APCs or exosomes of the present invention), or a CTL targeting
these peptides (i.e., CTLs of the present invention). That is,
pharmaceutical compositions comprising an active ingredient in
association with a peptide having the amino acid sequence selected
from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37 are
suitable for administration to subjects having HLA-A01 (i.e.,
HLA-A01-positive subjects). In a more preferred embodiment, the
pharmaceutical composition of the present invention is a
pharmaceutical composition that comprises a peptide having the
amino acid sequence of SEQ ID NO: 8.
[0205] Cancers to be treated and/or prevented by pharmaceutical
compositions of the present invention are not particularly limited
as long as they are cancers that express CDCA1, and include various
cancers, bladder cancer, breast cancer, cervical cancer,
cholangiocellular cancer, chronic myeloid leukemia (CML), esophagus
cancer, gastric cancer, non-small-cell lung cancer, lymphoma,
osteosarcoma, prostate cancer, kidney cancer, small-cell lung
cancer, head and neck cancer, soft tissue tumor, colon cancer and
such.
[0206] In addition to the active ingredients described above, the
pharmaceutical compositions of the present invention can comprise
the other peptides that have the ability to induce CTLs against
cancer cells (for example, the other TAA-derived CTL-inducing
peptides), the other polynucleotides encoding the other peptides,
the other cells that present the other peptides, or such.
[0207] The pharmaceutical compositions of the present invention may
also optionally comprise the other therapeutic substances as an
active ingredient, as long as they do not inhibit the anti-tumor
effects of the above-described active ingredients such as peptides
of the present invention. For example, the pharmaceutical
compositions of the present invention may optionally comprise
anti-inflammatory compositions, analgesics, chemotherapeutics and
the like. In addition to including the other therapeutic substances
to a pharmaceutical composition of the present invention itself,
one can also administer the pharmaceutical composition of the
present invention sequentially or concurrently with one or more
other pharmaceutical compositions. The dose of the pharmaceutical
composition of the present invention and the other pharmaceutical
compositions depend on, for example, the type of pharmaceutical
composition used and the disease being treated, as well as the
scheduling and routes of administration.
[0208] It should be understood that in consideration of the
formulation type, the pharmaceutical composition of the present
invention may include other components conventional in the art, in
addition to the ingredients specifically mentioned herein.
[0209] The present invention also provides articles of manufacture
or kits that comprise a pharmaceutical composition of the present
invention. The articles of manufacture or kits of the present
invention can include a container that houses the pharmaceutical
composition of the present invention. An example of an appropriate
container includes a bottle, a vial or a test tube, but is not
limited thereto. The container may be formed of various materials
such as glass or plastic. A label may be attached to the container,
and the disease or disease state to which the pharmaceutical
composition of the present invention should be used may be
described in the label. The label may also indicate directions for
administration and such.
[0210] The articles of manufacture or kits of the present invention
may further comprise a second container that houses
pharmaceutically acceptable diluents optionally, in addition to the
container that houses the pharmaceutical composition of the present
invention. The articles of manufacture or kits of the present
invention may further comprise the other materials desirable from a
commercial standpoint and the user's perspective, such as the other
buffers, diluents, filters, injection needles, syringes, package
inserts with instructions for use.
[0211] As needed, the pharmaceutical composition of the present
invention can be provided in a pack or dispenser device that can
contain one or more units of dosage forms containing active
ingredients. The pack can include, for example, a metallic foil or
a plastic foil such as a blister pack. Instructions for
administration can be attached to the pack or dispenser device.
(1) Pharmaceutical Compositions Comprising Peptide(s) as an Active
Ingredient
[0212] The pharmaceutical composition comprising a peptide of the
present invention can be formulated by conventional formulation
methods as needed. The pharmaceutical compositions of the present
invention may comprise as needed in addition to the peptide of the
present invention, carriers, excipients and such commonly used in
pharmaceuticals without particular limitations. Examples of
carriers that can be used in pharmaceutical compositions of the
present invention include sterilized water (for example, water for
injection), physiological saline, phosphate buffer, phosphate
buffered saline, Tris buffered saline, 0.3% glycine, culture fluid,
and such. Further, the pharmaceutical compositions of the present
invention may comprise as needed stabilizers, suspensions,
preservatives, surfactants, solubilizing agents, pH adjusters,
aggregation inhibitors, and such. The pharmaceutical compositions
of the present invention can induce specific immunity against
CDCA1-expressing cancer cells, and thus can be applied for the
purpose of cancer treatment or prevention (prophylaxis).
[0213] For example, the pharmaceutical compositions of the present
invention can be prepared by dissolving in pharmaceutically or
physiologically acceptable water-soluble carriers such as
sterilized water (for example, water for injection), physiological
saline, phosphate buffer, phosphate buffered saline, and Tris
buffered saline and adding, as needed, stabilizers, suspensions,
preservatives, surfactants, solubilizing agents, pH adjusters,
aggregation inhibitors and such, and then sterilizing the peptide
solution. The method of sterilizing a peptide solution is not
particularly limited, and is preferably carried out by filtration
sterilization. Filtration sterilization can be performed using, for
example, a filtration sterilization filter of 0.22 micro-m or less
in pore diameter. The filtration-sterilized peptide solution can be
administered to a subject, for example, as an injection, without
being limited thereto. The pharmaceutical compositions of the
present invention may be prepared as a freeze-dried formulation by
freeze-drying the above-described peptide solution. The
freeze-dried formulation can be prepared by filling the peptide
solution prepared as described above into an appropriate container
such as an ampule, a vial or a plastic container, followed by
freeze drying and encapsulation into the container with a
wash-sterilized rubber plug or such after pressure recovery. The
freeze-dried formulation can be administered to a subject after it
is re-dissolved in pharmaceutically or physiologically acceptable
water-soluble carriers such as sterilized water (for example, water
for injection), physiological saline, phosphate buffer, phosphate
buffered saline, Tris buffered saline and such before
administration. Preferred examples of pharmaceutical compositions
of the present invention include injections of such a
filtration-sterilized peptide solution, and freeze-dried
formulations that result from freeze-drying the peptide solution.
The present invention further encompasses kits comprising such a
freeze-dried formulation and re-dissolving solution. The present
invention also encompasses kits comprising a container that houses
the freeze-dried formulation, which is a pharmaceutical composition
of the present invention, and a container that stores a
re-dissolving solution thereof.
[0214] The pharmaceutical compositions of the present invention can
comprise a combination of two or more types of the peptides of the
present invention. The combination of peptides can take a cocktail
form of mixed peptides, or can be conjugated with each other using
standard techniques. For example, peptides can be chemically linked
or expressed as single fusion polypeptide sequences. By
administering a peptide of the present invention, the peptide is
presented on APCs by an HLA antigen at a high density, and then
subsequently CTLs that react specifically to a complex formed
between the presented peptide and the HLA antigen are induced.
Alternatively, APCs (for example, DCs) are removed from a subject,
and subsequently stimulated with peptides of the present invention
to obtain APCs that present any of the peptides of the present
invention on their cell surface. These APCs are re-administered to
a subject to induce CTLs in the subject, and as a result, the
aggressiveness towards CDCA1-expressing cancer cells can be
increased.
[0215] The pharmaceutical compositions of the present invention may
also comprise an adjuvant known for effectively establishing
cellular immunity. An adjuvant refers to a compound that enhances
the immune response against an antigen that has immunological
activity when administered together (or successively) with the
antigen. Known adjuvants described in literatures, for example,
Clin Microbiol Rev 1994, 7: 277-89, can be used. Examples of a
suitable adjuvant include aluminum salts (aluminum phosphate,
aluminum hydroxide, aluminum oxyhydroxide and such), alum, cholera
toxin, Salmonella toxin,
[0216] Incomplete Freund's adjuvant (IFA), Complete Freund's
adjuvant (CFA), ISCOMatrix, GM-CSF and other immunostimulatory
cytokines, oligodeoxynucleotide containing the CpG motif (CpG7909
and such), oil-in-water emulsions, Saponin or its derivatives (QS21
and such), lipopolysaccharide such as Lipid A or its derivatives
(MPL, RC529, GLA, E6020 and such), lipopeptides, lactoferrin,
flagellin, double-stranded RNA or its derivatives (poli IC and
such), bacterial DNA, imidazoquinolines (Imiquimod, R848 and such),
C-type lectin ligand (trehalose-6,6'-dibehenate (TDB) and such),
CD1d ligand (alpha-galactosylceramide and such), squalene emulsions
(MF59, AS03, AF03 and such), PLGA, and such, without being limited
thereto. These adjuvants can usually be mixed with an antigen
having immunological activity (i.e., the peptide of the present
invention) in an amount effective for enhancing or strengthening
the antigen's immunogenicity. The adjuvant may be contained in
another container separate from the pharmaceutical composition
comprising a peptide of the present invention in the kits
comprising the pharmaceutical composition of the present invention.
In this case, the adjuvant and the pharmaceutical composition may
be administered to a subject in succession, or mixed together
immediately before administration to a subject. Such kits
comprising a pharmaceutical composition comprising a peptide of the
present invention and an adjuvant are also provided by the present
invention. When the pharmaceutical composition of the present
invention is a freeze-dried formulation, the kit can further
comprise a re-dissolving solution. Further, the present invention
provides kits comprising a container that houses a pharmaceutical
composition of the present invention and a container that stores an
adjuvant. The kit can further comprise as needed a container that
stores the re-dissolving solution.
[0217] When an oil adjuvant is used as an adjuvant, the
pharmaceutical composition of the present invention may be prepared
as an emulsion. Emulsions can be prepared, for example, by mixing
and stirring the peptide solution prepared as described above and
an oil adjuvant. The peptide solution may be one that has been
re-dissolved after freeze-drying. The emulsion may be either of the
W/O-type emulsion and O/W-type emulsion, and the W/O-type emulsion
is preferred for obtaining a high immune response-enhancing effect.
IFA can be preferably used as an oil adjuvant, without being
limited thereto. Preparation of an emulsion can be carried out
immediately before administration to a subject, and in this case,
the pharmaceutical composition of the present invention may be
provided as a kit comprising the peptide solution of the present
invention and an oil adjuvant. When the pharmaceutical composition
of the present invention is a freeze-dried formulation, the kit can
further comprise a re-dissolving solution.
[0218] Further, the pharmaceutical composition of the present
invention may be a liposome formulation within which a peptide of
the present invention is encapsulated, a granular formulation in
which a peptide is bound to beads with several micrometers in
diameter, or a formulation in which a lipid is bound to a
peptide.
[0219] In another embodiment of the present invention, the peptide
of the present invention may also be administered in the form of a
pharmaceutically acceptable salt. Preferred examples of salts
include salts with alkali metals (lithium, potassium, sodium and
such), salts with alkaline-earth metals (calcium, magnesium and
such), salts with other metals (copper, iron, zinc, manganese and
such), salts with organic bases, salts with amines, salts with
organic acids (acetic acid, formic acid, propionic acid, fumaric
acid, maleic acid, succinic acid, tartaric acid, citric acid, malic
acid, oxalic acid, benzoic acid, methanesulfonic acid and such),
and salts with inorganic acids (hydrochloric acid, phosphoric acid,
hydrobromic acid, sulfuric acid, nitric acid and such). Therefore,
pharmaceutical compositions comprising a pharmaceutically
acceptable salt of a peptide of the present invention are also
encompassed by the present invention. Further, the "peptide of the
present invention" also encompasses, in addition to the free
peptide, pharmaceutically acceptable salts thereof.
[0220] In some embodiments, the pharmaceutical compositions of the
present invention may further include a component which primes
CTLs. Lipids have been identified as substances capable of priming
CTLs in vivo against viral antigens. For example, palmitic acid
residues can be attached to the epsilon- and alpha-amino groups of
a lysine residue and then linked to a peptide of the present
invention. The lipidated peptide can then be administered either
directly in a micelle or particle, incorporated into a liposome, or
emulsified in an adjuvant. As another example of lipid priming of
CTL responses, E. coil lipoproteins, such as
tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P3CSS) can be used
to prime CTLs when covalently attached to an appropriate peptide
(see, e.g., Deres et al., Nature 1989, 342: 561-4).
[0221] Examples of methods for administering the peptides or
pharmaceutical compositions of the present invention include oral,
epidermal, subcutaneous, intramuscular, intraosseous, peritoneal,
and intravenous injections, as well as systemic administration or
local administration to the vicinity of the targeted sites, but are
not limited thereto. A preferred administration method includes
subcutaneous injection to the vicinity of lymph nodes such as the
armpit or groin. The administration can be performed by single
administration or boosted by multiple administrations. The peptides
of the present invention can be administered to a subject in a
therapeutically or pharmaceutically effective amount for treating
cancer or in a therapeutically or pharmaceutically effective amount
for inducing immunity (more specifically CTLs) against
CDCA1-expressing cancer cells. The dose of the peptides of the
present invention can be appropriately adjusted according to the
disease to be treated, the patient's age and weight, the method of
administration and such. For each of the peptides of the present
invention, the dose is usually 0.001 mg-1000 mg, for example, 0.01
mg-100 mg, for example, 0.1 mg-30 mg, for example, 0.1 mg-10 mg,
for example, 0.5 mg-5 mg. The dosing interval can be once every
several days to several months, and for example, the dosing can be
done in a once-per-week interval. A skilled artisan can
appropriately select a suitable dosage.
[0222] In a preferred embodiment, the pharmaceutical compositions
of the present invention comprise a therapeutically effective
amount of a peptide of the present invention and a pharmaceutically
or physiologically acceptable carrier. In another embodiment, the
pharmaceutical compositions of the present invention comprise a
therapeutically effective amount of a peptide of the present
invention, a pharmaceutically or physiologically acceptable cancer,
and an adjuvant. The pharmaceutical compositions of the present
invention can comprise 0.001 mg-1000 mg, preferably 0.01 mg-100 mg,
more preferably 0.1 mg-30 mg, even more preferably 0.1 mg-10 mg,
for example, 0.5 mg-5 mg of a peptide of the present invention.
[0223] When a pharmaceutical composition of the present invention
is an injection, it can comprise a peptide of the present invention
at a concentration of 0.001 mg/ml-1000 mg/ml, preferably 0.01
mg/ml-100 mg/ml, more preferably 0.1 mg/ml-30 mg/ml, even more
preferably 0.1 mg/ml-10 mg/ml, for example, 0.5 mg/ml-5 mg/ml. In
this case, for example, 0.1 to 5 ml, preferably 0.5 ml to 2 ml of
the pharmaceutical composition of the present invention can be
administered to a subject by injection.
[0224] Further, the present invention provides methods of treating
and/or preventing cancer and/or preventing postoperative recurrence
thereof, which comprise administering to a subject a
therapeutically effective amount of a peptide of the present
invention or a pharmaceutical composition of the present invention.
As described above, the peptides of the present invention can be
administered to a subject in a single dose of usually 0.001 mg-1000
mg, for example, 0.01 mg-100 mg, for example, 0.1 mg-30 mg, for
example, 0.1 mg-10 mg, or for example, 0.5 mg-5 mg. In a preferred
embodiment, the peptides of the present invention are administered
to a subject together with an adjuvant. Further, the dosing
interval can be once every several days to several months,
preferably once every several days to every month, for example,
once every week or once every two weeks.
[0225] The CDCA1 peptides of the invention carry an
HLA-A01-restrictive CTL-inducing activity. Accordingly, their
therapeutic effect is also effective in HLA-A01-positive subjects.
Thus, in a preferred embodiment of the present invention, an
HLA-A01-positive subject can be selected in advance, before the
administration of a CDCA1 peptide of the present invention.
Furthermore, since the therapeutic effect of the CDCA1 of the
present invention is CDCA1-specific, it is a desirable condition
that cancer in a subject is expressing CDCA1. That is, the methods
of treating cancer of the present invention can comprise the step
of selecting an HLA-A01-positive subject and the step of selecting
a subject having cancer expressing CDCA1, before the administration
of the CDCA1 peptide.
(2) Pharmaceutical Compositions Containing Polynucleotides as the
Active Ingredient
[0226] The pharmaceutical compositions of the present invention can
also contain polynucleotides encoding the peptides of the present
invention in an expressible form. Herein, the phrase "in an
expressible form" means that the polynucleotide, when introduced
into a cell, will be expressed as a peptide of the present
invention. In an exemplified embodiment, the sequence of the
polynucleotide of the present invention includes regulatory
elements necessary for expression of the peptide of the present
invention. The polynucleotide(s) of the present invention can be
equipped with a sequence necessary to achieve stable insertion into
the genome of the target cell (see, e.g., Thomas K R & Capecchi
M R, Cell 1987, 51: 503-12 for a description of homologous
recombination cassette vectors). See, e.g., Wolff et al., Science
1990, 247: 1465-8; U.S. Pat. Nos. 5,580,859, 5,589,466, 5,804,566,
5,739,118, 5,736,524, 5,679,647; and WO98/04720. Examples of
DNA-based delivery technologies include "naked DNA", facilitated
(bupivacaine, polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0227] The peptides of the present invention can also be expressed
by viral or bacterial vectors. Examples of expression vectors
include attenuated viral hosts, such as vaccinia or fowlpox. For
example, as a vector to express the peptide of the present
invention, vaccinia virus can be used. Upon introduction into a
host, the recombinant vaccinia virus expresses the immunogenic
peptide, and thereby elicits an immune response. Vaccinia vectors
and methods useful in immunization protocols are described in,
e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille
Calmette Guerin). BCG vectors are described in Stover et al.,
Nature 1991, 351: 456-60. A wide variety of other vectors useful
for therapeutic administration or immunization, e.g., adeno and
adeno-associated virus vectors, retroviral vectors, Salmonella
typhi vectors, detoxified anthrax toxin vectors, and the like, will
be apparent. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71;
Shedlock et al., J Leukoc Biol 2000, 68: 793-806; Hipp et al., In
Vivo 2000, 14: 571-85.
[0228] Delivery of a polynucleotide of the present invention into a
patient can be either direct, in which case the patient can be
directly exposed to a vector harboring the polynucleotide of the
present invention, or indirect, in which case, cells are first
transformed with the vector harboring the polynucleotide of the
present invention in vitro, then the cells are transplanted into
the patient. These two approaches are known, respectively, as in
vivo and ex vivo gene therapies.
[0229] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 1993, 12: 488-505; Wu and Wu,
Biotherapy 1991, 3: 87-95; Tolstoshev, Ann Rev Pharmacol Toxicol
1993, 33: 573-96; Mulligan, Science 1993, 260: 926-32; Morgan &
Anderson, Ann Rev Biochem 1993, 62: 191-217; Trends in
Biotechnology 1993, 11(5): 155-215. Methods commonly known in the
art of recombinant DNA technology which can also be used for the
present invention are described in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, NY, 1993;
and Krieger, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, NY, 1990. Administration methods may be oral,
intradermal, subcutaneous, or intravenous injection, and such. A
systemic administration or a local administration to the vicinity
of the targeted sites is used. The administration can be performed
by single administration or boosted by multiple administrations.
The polynucleotides of the present invention can be administered to
a subject in a therapeutically or pharmaceutically effective dose
for inducing immunity (more specifically CTLs) against
CDCA1-expressing cancer cells, or in a therapeutically or
pharmaceutically effective dose for treating cancer. The dose of a
polynucleotide in a suitable carrier or the dose of a
polynucleotide in cells transformed with a polynucleotide encoding
a peptide of the present invention can be appropriately adjusted
according to the disease to be treated, the patient's age and
weight, the method of administration and such, and this may be
usually 0.001 mg-1000 mg, for example, 0.01 mg-100 mg, for example,
0.1 mg-30 mg, for example, 0.1 mg-10 mg, or for example, 0.5 mg-5
mg. The dosing interval can be once every several days to several
months, and for example, the dosing can be done in a once-per-week
interval. A skilled artisan can appropriately select a suitable
dosage.
X. Methods of Using Peptides, Exosomes, APCs and CTLs
[0230] The peptides and polynucleotides of the present invention
can be used to induce APCs and CTLs. CTLs can also be induced using
the exosomes and APCs of the present invention. The peptides,
polynucleotides, exosomes, and APCs can be used in combination with
any other compound(s) as long as their CTL-inducing ability is not
inhibited. Therefore, CTLs of the present invention can be induced
using a pharmaceutical composition comprising any of the peptides,
polynucleotides, APCs and exosomes of the present invention.
Further, APCs of the present invention can be induced using a
pharmaceutical composition comprising a peptide or polynucleotide
of the present invention.
(1) Methods of Inducing APCs
[0231] The present invention provides methods of inducing APCs
having CTL-inducing ability, using a peptide(s) or
polynucleotide(s) of the present invention.
[0232] The methods of the present invention comprise a step of
contacting an APC with a peptide of the present invention in vitro,
ex vivo, or in vivo. For example, a method of contacting APCs with
the peptide ex vivo may comprise the steps below: [0233] (a)
collecting APCs from a subject; and [0234] (b) contacting the APCs
of step (a) with a peptide of the present invention.
[0235] The above-described APCs are not limited to a particular
type of cell, and DCs, Langerhans cells, macrophages, B cells, and
activated T cells, which are known to present a proteinaceous
antigen on their cell surface to be recognized by lymphocytes, can
be used. DCs have the most potent CTL-inducing ability among APCs,
and thus it is preferable to use DCs. Any peptides of the present
invention can be used by themselves or in combination with other
peptides of the present invention. Further, peptides of the present
invention can be used in combination with other CTL-inducing
peptides (for example, other TAA-derived CTL-inducing
peptides).
[0236] Meanwhile, when a peptide of the present invention is
administered to a subject, APCs are contacted with the peptide in
vivo, and as a result, APCs having a high CTL-inducing ability are
induced in the body of the subject. Therefore, the methods of the
present invention may comprise a step of administering a peptide of
the present invention to a subject. Similarly, when a
polynucleotide of the present invention is administered to a
subject in an expressible form, a peptide of the present invention
is expressed in vivo, the expressed peptide is contacted with APCs
in vivo, and as a result APCs having a high CTL-inducing ability
are induced in the body of the subject. Therefore, the present
invention may also comprise a step of administering a
polynucleotide of the present invention to a subject.
[0237] In order to induce APCs having CTL-inducing ability, the
present invention may comprise a step of introducing a
polynucleotide of the present invention into APCs. For example, the
method may comprise the steps below: [0238] (a) collecting APCs
from a subject; and [0239] (b) introducing a polynucleotide
encoding a peptide of the present invention into the APCs of step
(a). Step (b) can be performed as described in the above "VI.
Antigen-presenting cells (APCs)" section.
[0240] Thus, in one embodiment, the present invention provides a
method of inducing APCs having CTL-inducing ability, which
comprises the step (a) or (b) below: [0241] (a) contacting APCs
with a peptide of the present invention; or [0242] (b) introducing
a polynucleotide encoding a peptide of the present invention into
APCs.
[0243] Furthermore, the present invention provides a method of
preparing APCs having CTL-inducing ability, which comprises the
step (a) or (b) below: [0244] (a) contacting APCs with a peptide of
the present invention; or [0245] (b) introducing a polynucleotide
encoding a peptide of the present invention into APCs.
[0246] The above-described methods can be performed in vitro, ex
vivo, or in vivo, and it is preferable to perform them in vitro or
ex vivo. APCs used in the above-described methods may be derived
from a subject scheduled for administration of the induced APCs, or
they may be derived from a different subject. When APCs derived
from a subject (donor) different from the subject scheduled for
administration are used, the subject of administration and the
donor must have the identical HLA type. In the methods of the
present invention, when a peptide having the amino acid sequence
selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37
or a modified peptide thereof is used as a peptide of the present
invention, the HLA type is preferably HLA-A01 (more preferably
HLA-A*01:01) in both the subject of administration and the donor.
Alternatively, APCs used in the above-described methods are
preferably APCs that express HLA-A01 (more preferably HLA-A*01:01).
The APCs can be prepared using known methods from PBMCs after PBMCs
are separated from blood collected from a donor by a specific
gravity centrifugal method or such.
[0247] In another embodiment, the present invention also provides
pharmaceutical compositions that comprise a peptide of the present
invention or a polynucleotide encoding the peptide for inducing an
APC(s) having CTL-inducing ability.
[0248] Alternatively, the present invention further provides use of
a peptide of the present invention or a polynucleotide encoding the
peptide in the manufacture of a pharmaceutical composition for
inducing an APC(s) having CTL-inducing ability.
[0249] Alternatively, the present invention further provides
peptides of the present invention or polynucleotides encoding the
peptides for use in the induction of an APC(s) having CTL-inducing
ability.
[0250] Alternatively, the present invention further provides
methods or processes of manufacturing a pharmaceutical composition
for inducing an APC(s), wherein the method or process comprises a
step of formulating a peptide of the present invention or a
polynucleotide encoding the peptide with a pharmaceutically or
physiologically acceptable carrier.
[0251] In another embodiment, the present invention further
provides methods or processes of manufacturing a pharmaceutical
composition for inducing an APC(s) having CTL-inducing ability,
wherein the method or process comprises a step of mixing a peptide
of the present invention or a polynucleotide encoding the peptide
with a pharmaceutically or physiologically acceptable carrier.
[0252] APCs induced by the methods of the present invention can
induce CTLs specific to CDCA1 (i.e., CTLs of the present
invention).
(2) Methods of Inducing CTLs
[0253] The present invention also provides methods of inducing CTLs
using peptides, polynucleotides, exosomes or APCs of the present
invention.
[0254] When a peptide(s), a polynucleotide(s), an exosome(s) or an
APC(s) of the present invention is administered to a subject, CTLs
are induced in the body of the subject and the strength of the
immune response targeting CDCA1-expressing cancer cells is
enhanced. Therefore, the methods of the present invention may
comprise a step of administering a peptide(s), a polynucleotide(s),
an APC(s) or an exosome(s) of the present invention to a
subject.
[0255] Alternatively, CTLs can be induced by using them in vitro or
ex vivo. For example, the methods of the present invention may
include the following steps: [0256] (a) collecting APCs from a
subject; [0257] (b) contacting the APCs of step (a) with a peptide
of the present invention; and [0258] (c) co-culturing the APCs of
step (b) with CD8-positive T cells.
[0259] The induced CTLs may be returned to the subject
afterwards.
[0260] The APCs to be co-cultured with the CD8-positive T cells in
step (c) above can also be prepared by introducing into APCs a
polynucleotide encoding a peptide of the present invention as
described above in the "VI. Antigen-presenting cells (APCs)"
section. However, the APCs to be used in the methods of the present
invention are not limited thereto, and any APCs that present on
their surface a complex of an HLA antigen and a peptide of the
present invention can be used.
[0261] In the methods of the present invention, instead of such
APCs, exosomes that present on their surface a complex of an HLA
antigen and a peptide of the present invention can also be used.
That is, the methods of the present invention can comprise a step
of co-culturing with exosomes that present on their surface a
complex of an HLA antigen and a peptide of the present invention.
Such exosomes can be prepared by the above-described methods in the
"V. Exosomes" section.
[0262] Further, CTLs can also be induced by introducing into a
CD8-positive T cell a vector comprising a polynucleotide encoding
each subunit of a TCR capable of binding to a peptide of the
present invention presented by an HLA antigen on the cell surface.
Such transformation can be carried out as described above in the
"VIII. T cell receptors (TCRs)" section.
[0263] Accordingly, in one embodiment, the present invention
provides methods of inducing CTLs, comprising a step selected from
below: [0264] (a) co-culturing CD8-positive T cells with APCs that
present on their surface a complex of an HLA antigen and a peptide
of present invention; [0265] (b) co-culturing CD8-positive T cells
with exosomes that present on their surface a complex of an HLA
antigen and a peptide of present invention; and [0266] (c)
introducing into CD8-positive T cells, a vector comprising a
polynucleotide encoding each subunit of a TCR capable of binding to
a peptide of the present invention presented by an HLA antigen on a
cell surface.
[0267] The above-described methods can be performed in vitro, ex
vivo, or in vivo, and it is preferable to perform them in vitro or
ex vivo. APCs or exosomes and CD8-positive T cells used in the
above-described methods may be derived from a subject scheduled for
administration of the induced CTLs, or they may be derived from a
different subject. When APCs or exosomes and CD8-positive T cells
derived from a subject (donor) different from the subject scheduled
for administration are used, the subject of administration and the
donor must have the identical HLA type. For example, when a peptide
having the amino acid sequence selected from among SEQ ID NOs: 1,
8, 10, 13, 25, 33 to 35 and 37 or a modified peptide thereof is
used as peptides of the present invention, the HLA type in both the
subject of administration and the donor is preferably HLA-A01 (more
preferably HLA-A*01:01). Alternatively, APCs or exosomes used in
the above-described methods are preferably APCs or exosomes that
present on their surface a complex of HLA-A01 (more preferably
HLA-A*01:01) and a peptide of the present invention (a peptide
having the amino acid sequence selected from among SEQ ID NOs: 1,
8, 10, 13, 25, 33 to 35 and 37 or a modified peptide thereof). In
this case, the induced CTLs show a specific cytotoxic activity
against cells that present a complex of HLA-A01 and a peptide of
the present invention (for example, CDCA1-expressing
HLA-A01-positive cells).
[0268] In another embodiment, the present invention also provides
compositions or pharmaceutical compositions for inducing CTLs,
comprising at least one active ingredient selected from below:
[0269] (a) a peptide of the present invention; [0270] (b) a
polynucleotide encoding a peptide of the present invention in an
expressible form; [0271] (c) an APC that presents on its surface a
peptide of the present invention; and [0272] (d) an exosome that
presents on its surface a peptide of the present invention.
[0273] In another embodiment, the present invention also provides
use of an active ingredient selected from below in the manufacture
of compositions or pharmaceutical compositions for inducing CTLs:
[0274] (a) a peptide of the present invention; [0275] (b) a
polynucleotide encoding a peptide of the present invention in an
expressible form; [0276] (c) an APC that presents on its surface a
peptide of the present invention; and [0277] (d) an exosome that
presents on its surface a peptide of the present invention.
[0278] Alternatively, the present invention further provides an
active ingredient selected from below for use in inducing CTLs:
[0279] (a) a peptide of the present invention; [0280] (b) a
polynucleotide encoding a peptide of the present invention in an
expressible form; [0281] (c) an APC that presents on its surface a
peptide of the present invention; and [0282] (d) an exosome that
presents on its surface a peptide of the present invention.
[0283] Alternatively, the present invention further provides a
method or process for manufacturing a composition or pharmaceutical
composition for inducing CTLs, which is a method or process that
comprises a step of formulating an active ingredient selected from
below with a pharmaceutically or physiologically acceptable
carrier: [0284] (a) a peptide of the present invention; [0285] (b)
a polynucleotide encoding a peptide of the present invention in an
expressible form; [0286] (c) an APC that presents on its surface a
peptide of the present invention; and [0287] (d) an exosome that
presents on its surface a peptide of the present invention.
[0288] In another embodiment, the present invention further
provides a method or process for manufacturing a composition or
pharmaceutical composition for inducing CTLs, which is a method or
process that comprises a step of mixing an active ingredient
selected from below with a pharmaceutically or physiologically
acceptable carrier: [0289] (a) a peptide of the present invention;
[0290] (b) a polynucleotide encoding a peptide of the present
invention in an expressible form; [0291] (c) an APC that presents
on its surface a peptide of the present invention; and [0292] (d)
an exosome that presents on its surface a peptide of the present
invention.
XI. Methods of Inducing an Immune Response
[0293] The present invention further provides methods of inducing
an immune response against CDCA1-expressing cancers. Applicable
cancers include bladder cancer, breast cancer, cervical cancer,
cholangiocellular cancer, chronic myeloid leukemia (CML), esophagus
cancer, gastric cancer, non-small-cell lung cancer, lymphoma,
osteosarcoma, prostate cancer, kidney cancer, small-cell lung
cancer, head and neck cancer, soft tissue tumor, colon cancer and
such, but are not limited thereto. It is preferable that the cancer
expresses HLA-A01.
[0294] The present invention further provides methods of inducing
an immune response against CDCA1-expressing cancer cells. CDCA1 is
recognized to be overexpressed in various types of cancers
described above. Thus, when an immune response against
CDCA1-expressing cancer cells is induced, proliferation of the
cancer cells is inhibited as a result. Accordingly, the present
invention further provides methods of inhibiting proliferation of
CDCA1-expressing cancer cells. The methods of the present invention
are suitable, in particular, for inhibiting proliferation of cancer
cells expressing CDCA1 and HLA-A01.
[0295] The methods of the present invention may comprise a step of
administering a composition comprising any of the peptides of the
present invention or a polynucleotide(s) encoding the peptide(s).
The methods of the present invention also contemplate
administration of APCs or exosomes presenting any of the peptides
of the present invention. The details can be referred to the "IX.
Pharmaceutical compositions" section, particularly portions
describing regarding use of the pharmaceutical compositions of the
present invention as vaccines. In addition, exosomes and APCs that
can be used in the methods of the present invention for inducing an
immune response are described in detail in "V. Exosomes", "VI.
Antigen-presenting cells (APCs)" and in Items (1) and (2) of "X.
Methods of using peptides, exosomes, APCs and CTLs" described
above.
[0296] In another embodiment, the present invention provides
pharmaceutical compositions or vaccines for inducing an immune
response against cancers expressing CDCA1 and HLA-A01, wherein the
pharmaceutical composition or vaccine comprises an active
ingredient selected from below: [0297] (a) a peptide of the present
invention; [0298] (b) a polynucleotide encoding a peptide of the
present invention in an expressible form; [0299] (c) an APC that
presents on its surface a peptide of the present invention; [0300]
(d) an exosome that presents on its surface a peptide of the
present invention; and [0301] (e) a CTL of the present
invention.
[0302] Alternatively, the present invention also provides
pharmaceutical compositions or vaccines for inducing an immune
response against cancer cells expressing CDCA1 and HLA-A01, wherein
the pharmaceutical composition or vaccine comprises an active
ingredient selected from below: [0303] (a) a peptide of the present
invention; [0304] (b) a polynucleotide encoding a peptide of the
present invention in an expressible form; [0305] (c) an APC that
presents on its surface a peptide of the present invention; [0306]
(d) an exosome that presents on its surface a peptide of the
present invention; and [0307] (e) a CTL of the present
invention.
[0308] Alternatively, the present invention further provides
pharmaceutical compositions or vaccines for inhibiting
proliferation of cancer cells expressing CDCA1 and HLA-A01, wherein
the pharmaceutical composition or vaccine comprises an active
ingredient selected from below: [0309] (a) a peptide of the present
invention; [0310] (b) a polynucleotide encoding a peptide of the
present invention in an expressible form; [0311] (c) an APC that
presents on its surface a peptide of the present invention; [0312]
(d) an exosome that presents on its surface a peptide of the
present invention; and [0313] (e) a CTL of the present
invention.
[0314] In another embodiment, the present invention provides use of
an active ingredient selected from below in the manufacture of
pharmaceutical compositions or vaccines for inducing an immune
response against cancers expressing CDCA1 and HLA-A01: [0315] (a) a
peptide of the present invention; [0316] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0317] (c) an APC that presents on its surface a peptide of the
present invention; [0318] (d) an exosome that presents on its
surface a peptide of the present invention; and [0319] (e) a CTL of
the present invention.
[0320] Alternatively, the present invention also provides use of an
active ingredient selected from below in the manufacture of
pharmaceutical compositions or vaccines for inducing an immune
response against cancer cells expressing CDCA1 and HLA-A01: [0321]
(a) a peptide of the present invention; [0322] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0323] (c) an APC that presents on its surface a peptide of the
present invention; [0324] (d) an exosome that presents on its
surface a peptide of the present invention; and [0325] (e) a CTL of
the present invention.
[0326] Alternatively, the present invention further provides use of
an active ingredient selected from below in the manufacture of
pharmaceutical compositions or vaccines for inhibiting
proliferation of cancer cells expressing CDCA1 and HLA-A01: [0327]
(a) a peptide of the present invention; [0328] (b) a polynucleotide
encoding a peptide of the present invention in an expressible form;
[0329] (c) an APC that presents on its surface a peptide of the
present invention; [0330] (d) an exosome that presents on its
surface a peptide of the present invention; and [0331] (e) a CTL of
the present invention.
[0332] The present invention further provides methods or processes
for manufacturing pharmaceutical compositions that induce an immune
response against cancers expressing CDCA1 and HLA-A01, which is a
method that may comprise a step of mixing or formulating a peptide
of the present invention with a pharmaceutically acceptable
carrier.
[0333] Alternatively, the present invention provides methods for
inhibiting proliferation of cancer cells expressing CDCA1 and
HLA-A01 in diseased sites of diseases mediated by angiogenesis or
methods of inducing an immune response against cancers, which
comprises a step of administering to a subject vaccines or
pharmaceutical compositions comprising an active ingredient
selected from below: [0334] (a) a peptide of the present invention;
[0335] (b) a polynucleotide encoding a peptide of the present
invention in an expressible form; [0336] (c) an APC that presents a
peptide of the present invention on its surface; [0337] (d) an
exosome that presents a peptide of the present invention on its
surface; and [0338] (e) a CTL of the present invention.
[0339] In the context of the present invention, cancers expressing
CDCA1 and HLA-A01 can be treated by administering a peptide, a
polynucleotide, an APC, an exosome and/or a CTL of the present
invention. Alternatively, an immune response against
CDCA1-expressing cancers can be induced by administering a peptide,
a polynucleotide, an APC, an exosome and/or a CTL of the present
invention. Examples of such cancers include bladder cancer, breast
cancer, cervical cancer, cholangiocellular cancer, chronic myeloid
leukemia (CML), esophagus cancer, gastric cancer, non-small-cell
lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney
cancer, small-cell lung cancer, head and neck cancer, soft tissue
tumor, colon cancer and such, but are not limited thereto. Further,
an immune response against cancer cells expressing CDCA1 and
HLA-A01 can be induced by administering a peptide, a
polynucleotide, an APC, an exosome and/or a CTL of the present
invention. Therefore, before administering a vaccine or
pharmaceutical composition comprising an active ingredient
described above, it is preferable to confirm whether the level of
CDCA1 expression at a diseased site in the subject to be treated is
augmented or not. Similarly, it is also desirable to confirm that
the subject to be treated is HLA-A01-positive. That is, in a
preferred embodiment, the present invention can select a subject
who is HLA-A01-positive and who has cancer expressing CDCA1 and
administer the vaccine or the pharmaceutical composition of the
present invention to the selected subject.
[0340] Thus, in one embodiment, the present invention provides a
method of treating a cancer expressing CDCA1 and HLA-A01 in a
patient in need of the cancer treatment, wherein the method
comprises the steps below: [0341] (i) measuring the level of CDCA1
expression in a biological sample collected from the diseased site
of an HLA-A01-positive subject with cancer; [0342] (ii) identifying
a subject with CDCA1-expressing cancer based on the CDCA1
expression level measured in (i); and [0343] (iii) administering to
the subject with CDCA1-overexpressing cancer as compared to normal
control at least one ingredient selected from the group consisting
of (a) to (e) above.
[0344] Alternatively, the present invention further provides
vaccines or pharmaceutical compositions comprising at least one
active ingredient selected from the group consisting of (a) to (e)
above for administration to an HLA-A01-positive subject with
CDCA1-expressing cancer. The present invention further provides a
method of identifying or selecting a subject to be treated with at
least one active ingredient selected from the group consisting of
(a) to (e) above, wherein the method comprises the steps below:
[0345] (i) measuring the level of CDCA1 expression in a biological
sample collected from the diseased site of an HLA-A01-positive
subject with cancer; [0346] (ii) identifying a subject with
CDCA1-expressing cancer based on the CDCA1 expression level
measured in (i); and [0347] (iii) identifying or selecting the
subject identified in (ii) as a subject who may be treated with at
least one active ingredient selected from the group consisting of
(a) to (e) above.
[0348] Biological samples collected from a subject for measuring
the CDCA1 expression level in the above-described methods are not
particularly limited, and for example, tissue samples containing
cancer cells collected by biopsy or such can be preferably used.
The CDCA1 expression level in a biological sample can be measured
by known methods, and for example, methods that detect
transcription products of the CDCA1 gene by probes or PCR methods
(for example, cDNA microarray method, Northern blot method, RT-PCR
method or such), methods that detect translation products of the
CDCA1 gene by antibodies or such (for example, Western blot method,
immunostaining method or such), and such can be used. Further,
biological samples may be blood samples, and in this case, the
blood level of an antibody against CDCA1 is measured, and the CDCA1
expression level at a diseased site may be assessed based on the
blood level. The blood level of an antibody against CDCA1 can be
measured using known methods, and for example, enzyme immunoassay
(EIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RLA) and such using the CDCA1 protein or a peptide of the present
invention as an antigen can be used. Alternatively, the CDCA1
expression level at a diseased site may be assessed by detecting
CTLs specific to a peptide of the present invention. The CTL levels
specific to a peptide of the present invention can be measured, for
example, by separating PBMCs from the blood collected from a
subject and measuring the cytotoxic activity against target cells
pulsed with the peptide of the present invention. The cytotoxic
activity can be measured, for example, by the amount of
interferon-gamma release. Furthermore, complexes of the peptides of
the present invention and HLA described below can also be used for
measuring the CTL levels. Whether a subject's cancer expresses
CDCA1 or not may be determined by comparing with the measurement
results in a biological material of the same type collected from a
subject without cancer. That is, if the level of a substance to be
measured in a biological sample collected from a subject having
cancer is elevated relative to the level in a biological material
of the same type collected from another subject without cancer
(normal control level), it can be determined that the cancer of the
subject having cancer expresses CDCA1.
[0349] In a preferred embodiment, it is preferable to confirm the
HLA type of a subject before administering at least one active
ingredient selected from the group consisting of (a) to (e) above.
For example, it is preferable to select an HLA-A01-positive subject
as a subject to be administered with an active ingredient which is
related to a peptide having the amino acid sequence selected from
among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37. The HLA of a
subject can be confirmed by immunologically detecting an HLA on the
surface of cells collected from the subject with an
HLA-A01-specific antibody. Alternatively, the HLA of a subject can
be determined by analyzing the genetic information of genomic DNA
or mRNA obtained from cells collected from the subject. If a
subject is HLA-A01-positive, then effective treatment can be
expected in the present invention. In other words, a subject having
homozygous or heterozygous HLA-A01 can be a target of
administration.
[0350] The present invention further provides complexes of a
peptide of the present invention and HLA. The complexes of the
present invention described above may be monomers or multimers.
When a complex of the present invention is a multimer, the number
of polymerization is not particularly limited, and it can be a
multimer of any number of polymerization. Examples include a
tetramer, pentamer, hexamer and such, but are not limited thereto.
The multimers of the present invention also encompass dextramers
(WO2002/072631) and streptamers (Knabel M et al., Nat Med. 2002;
8(6): 631-7.). Complexes of a peptide of the present invention and
HLA can be prepared according to known methods (for example, Altman
J D et al., Science. 1996, 274(5284): 94-6; WO2002/072631;
WO2009/003492; Knabel M et al., Nat Med. 2002; 8(6): 631-7, and
such). The complexes of the present invention, for example, can be
used in the quantification of CTLs specific to a peptide of the
present invention. For example, a blood sample is collected from a
subject administered with a pharmaceutical composition of the
present invention, and CD4-negative cells are adjusted after
separation of PBMCs and contacted with a fluorescent dye-conjugated
complex of the present invention. Then, the percentage of CTLs
specific to a peptide of the present invention can be measured by
flow cytometry analysis. For example, immune response-inducing
effects by a pharmaceutical composition of the present invention
can be monitored by measuring the specific CTLs against a peptide
of the present invention before, during and/or after administration
of the pharmaceutical composition of the present invention.
XII. Antibodies
[0351] The present invention further provides antibodies that bind
to the peptide of the present invention. Preferable antibodies bind
specifically to a peptide of the present invention, but do not bind
(or weakly bind) to one that is not the peptide of the present
invention. The binding specificity of an antibody can be confirmed
by inhibition assay. That is, if the binding between an antibody to
be analyzed and a full-length CDCA1 polypeptide is inhibited in the
presence of a peptide of the present invention, this antibody is
shown to specifically bind to the peptide of the present invention.
Antibodies against peptides of the present invention can be used in
assays of disease diagnosis and prognosis, as well as subject
selection for administration of the pharmaceutical compositions of
the present invention and monitoring of the pharmaceutical
compositions of the present invention.
[0352] The present invention also provides various immunological
assays for detecting and/or quantifying peptides of the present
invention or fragments thereof. Such immunological assays include
radioimmunoassay, immunochromatography, enzyme-linked immunosorbent
assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA) and
such, without being limited thereto, and are performed within the
scope of the various immunological assay formats well known in the
art.
[0353] The antibodies of the present invention can be used in
immunological imaging methods that can detect CDCA1-epxressing
diseases, and examples thereof include radioactive scintigraphic
imaging using a labelled antibody of the present invention, without
being limited thereto. Such assay methods are used clinically in
the detection, monitoring, and prognosis of CDCA1-expressing
cancers; and examples of such cancer include bladder cancer, breast
cancer, cervical cancer, cholangiocellular cancer, chronic myeloid
leukemia (CML), esophagus cancer, gastric cancer, non-small-cell
lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney
cancer, small-cell lung cancer, head and neck cancer, soft tissue
tumor, colon cancer and such, without being limited thereto.
[0354] The antibodies of the present invention can be used in any
arbitrary form such as monoclonal antibodies or polyclonal
antibodies, and may further include anti-sera obtained by
immunizing an animal such as a rabbit with a peptide of the present
invention, all classes of polyclonal antibodies and monoclonal
antibodies, human antibodies, as well as chimeric antibodies and
humanized antibodies generated through gene recombination.
[0355] The peptide of the present invention or a fragment thereof
used as an antigen for obtaining antibodies can be obtained by
chemical synthesis or genetic engineering techniques based on the
amino acid sequences disclosed herein.
[0356] The peptide used as an immunizing antigen may be a peptide
of the present invention or a fragment of a peptide of the present
invention. Further, the peptide may be bound to or conjugated with
a carrier for increasing immunogenicity. Keyhole limpet hemocyanin
(KLH) is well-known as a cancer. Methods for binding KLH to a
peptide are also well known in the art.
[0357] Any mammal can be immunized with an antigen described above,
and it is preferable to consider the compatibility with the parent
cell used in cell fusion when generating a monoclonal antibody.
Generally, animals of the order Rodentia, Lagomorpha or Primate can
be used. Animals of the order Rodentia include, for example, mice,
rats and hamsters. Animals of the order Lagomorpha include, for
example, rabbits. Animals of the order Primate include, for
example, Catarrhini monkeys (old world monkeys) such as cynomolgus
monkey (Macaca fascicularis), rhesus monkeys, hamadryas, and
chimpanzee.
[0358] Methods of immunizing animals with an antigen are known in
the art. Intraperitoneal injection and subcutaneous injection of an
antigen are standard methods for immunizing mammals. More
specifically, an antigen is diluted and suspended in an appropriate
amount of phosphate buffered saline (PBS), physiological saline, or
such. As needed, an antigen suspension solution can be administered
to mammals after being mixed with an appropriate amount of a
standard adjuvant such as Freund's complete adjuvant and
emulsified. Then, it is preferable to administer the antigen mixed
with an appropriate amount of a Freund's incomplete adjuvant
several times every 4 to 21 days. A suitable carrier may be used
for immunization. After the above immunization, the serum can be
examined by standard method with respect to increase in the
quantity of the desired antibody.
[0359] Polyclonal antibodies against a peptide of the present
invention can be prepared by collecting blood from mammals that
have been confirmed with an increase in the serum level of the
desired antibody after immunization, and separating the serum from
blood by any conventional method. A polyclonal antibody may be a
polyclonal antibody-containing serum, or a polyclonal
antibody-containing fraction may be isolated from the serum.
Immunoglobulin G or M can be prepared from fractions that recognize
only a peptide of the present invention by, for example, using an
affinity column conjugated with the peptide of the present
invention, and then further purifying the fractions using a protein
A or protein G column
[0360] In order to prepare monoclonal antibodies, upon confirming
an increase in the serum level of the desired antibody after
immunization, immune cells are collected from the mammals and
subjected to cell fusion. Immune cells used for cell fusion may be
preferably obtained from the spleen. For the other parent cells
fused with the above immune cells, for example, a mammalian myeloma
cell, preferably a myeloma cell that has acquired a property for
drug selection of fusion cells can be used.
[0361] The above immune cells can be fused with myeloma cells by
following known methods, for example, the method of Milstein et al.
(Galfre and Milstein, Methods Enzymol 73: 1981, 3-46).
[0362] Hybridomas obtained by cell fusion can be selected by
culturing them in a standard selection medium such as the HAT
medium (a medium containing hypoxanthine, aminopterin and
thymidine). Cell culturing is typically continued in the HAT medium
for a sufficient period of time (for example, several days to
several weeks) to allow death of all other cells (non-fused cells)
besides the desired hybridomas. Then, hybridoma cells producing the
desired antibody can be screened and cloned by performing a
standard limiting dilution.
[0363] In addition to the above methods of immunizing a non-human
animal with an antigen for hybridoma preparation, human lymphocytes
such as EB vines-infected lymphocytes can be immunized in vitro
with a peptide, cells expressing the peptide, or lysates thereof.
Then, the immunized lymphocytes can be fused with immortalized
human-derived myeloma cells such as U266 to obtain hybridomas
producing a desired human antibody capable of binding to the
peptide (JPS63-17688).
[0364] Next, the obtained hybridoma is transplanted into the
abdominal cavity of a mouse, and the ascites is extracted. The
obtained monoclonal antibody can be purified by, for example,
ammonium sulfate precipitation, protein A or protein G column, DEAE
ion-exchange chromatography, or affinity column conjugated with the
peptide of the present invention.
[0365] Alternatively, antibody-producing immune cells such as the
immunized lymphocytes can be immortalized by a cancer gene and used
for the preparation of monoclonal antibodies.
[0366] The monoclonal antibodies obtained as such can also be
prepared by recombination using genetic engineering techniques
(see, e.g., Borrebaeck and Larrick, Therapeutic Monoclonal
Antibodies published in United Kingdom by MacMillan Publishers LTD
(1990)). For example, an antibody-encoding DNA can be cloned from
immune cells such as antibody-producing hybridoma or immunized
lymphocytes and inserted into a suitable vector, and then this is
introduced into host cells to prepare a recombinant antibody. The
present invention also provides recombinant antibodies prepared as
described above.
[0367] Further, the antibodies of the present invention may be
antibody fragments or modified antibodies, as long as they bind to
the peptides of the present invention. For example, the antibody
fragments may be Fab, F(ab').sub.2, Fv, or a single chain Fv (scFv)
in which Fv fragments derived from an H chain and an L chain are
linked with a suitable linker (Huston et al., Proc Natl Acad Sci
USA 1988, 85: 5879-83). More specifically, antibody fragments may
be generated by treating an antibody with an enzyme such as papain
or pepsin. Alternatively, a gene encoding an antibody fragment may
be constructed, inserted into an expression vector, and expressed
in an appropriate host cell (see, e.g., Co et al., J Immunol 1994,
152: 2968-76; Better and Horwitz,
[0368] Methods Enzymol 1989, 178: 476-96; Pluckthun and Skerra,
Methods Enzymol 1989, 178: 497-515; Lamoyi, Methods Enzymol 1986,
121: 652-63; Rousseaux et al., Methods Enzymol 1986, 121: 663-9;
Bird and Walker, Trends Biotechnol 1991, 9: 132-7).
[0369] Antibodies may be modified by conjugation with various
molecules such as polyethyleneglycol (PEG). The present invention
provides such modified antibodies. Modified antibodies can be
obtained by chemically modifying the antibodies. These modification
methods are conventional in the art.
[0370] Alternatively, the antibodies of the present invention can
be obtained as chimeric antibodies of a non-human antibody-derived
variable region and a human antibody-derived constant region, or as
humanized antibodies comprising a non-human antibody-derived
complementarity determining region (CDR) and a human
antibody-derived framework region (FR) and constant region. Such
antibodies can be prepared according to known techniques.
Humanization can be carried out by substituting a human antibody
sequence(s) with a corresponding non-human antibody CDR sequence(s)
(see, e.g., Verhoeyen et al., Science 1988, 239: 1534-6). Thus,
such humanized antibodies are chimeric antibodies in which the
substantially less than an intact human variable domain has been
substituted with a corresponding sequence from a non-human
species.
[0371] Intact human antibodies comprising a human variable region
in addition to the human framework and constant regions can also be
used. Such antibodies can be generated using various techniques
known in the art. For example, in vitro methods include use of
recombinant libraries of human antibody fragments presented on
bacteriophages (for example, Hoogenboom & Winter, J. Mol. Biol.
1991, 227: 381). Similarly, human antibodies can also be generated
by introducing human immunoglobulin gene loci into transgenic
animals, for example, mice, in which the endogenous immunoglobulin
genes have been partially or completely inactivated. This approach
is described in, for example, U.S. Pat. Nos. 6,150,584, 5,545,807,
5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.
[0372] Antibodies obtained as described above may be purified to
homogeneity. For example, antibody separation and purification can
be performed according to separation methods and purification
methods used for general proteins. For example, an antibody can be
separated and isolated by appropriately selecting and combining use
of column chromatographies such as affinity chromatography, filter,
ultrafiltration, salting-out, dialysis, SDS-polyacrylamide gel
electrophoresis and isoelectric focusing electrophoresis
(Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold
Spring Harbor Laboratory (1988)), but are not limited thereto.
Protein A column and protein G column can be used as the affinity
column. Exemplary protein A columns to be used include, for
example, Hyper D, POROS and Sepharose F. F. (Pharmacia).
[0373] Besides affinity chromatography, exemplary chromatography
includes, for example, ion-exchange chromatography, hydrophobic
chromatography, gel filtration, reversed-phase chromatography,
adsorption chromatography and such (Strategies for Protein
Purification and Characterization: A Laboratory Course Manual. Ed
Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press
(1996)). The chromatography procedures can be carried out by
liquid-phase chromatography such as HPLC and FPLC.
[0374] The antigen-binding activity of an antibody of the present
invention can be measured, for example, by using absorbance
measurement, enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), radioimmunoassay (RIA), and/or
immunofluorescence (IF). In the case of ELISA, an antibody of the
present invention is immobilized onto a plate, a peptide of the
present invention is applied to the plate, and then a sample
containing the desired antibody, such as culture supernatant of
antibody-producing cells or purified antibodies, is applied. Next,
a secondary antibody that recognizes the primary antibody and is
labelled with an enzyme such as alkaline phosphatase is applied and
the plate is incubated. Then, after washing, an enzyme substrate
such as p-nitrophenyl phosphate is applied to the plate, and the
antigen-binding activity of the sample is evaluated by measuring
absorbance. To assess the binding activity of an antibody, peptide
fragments such as C-terminal or N-terminal fragments may be used as
an antigen. BIAcore (Pharmacia) may be used to evaluate the
activity of an antibody of the present invention.
[0375] It is possible to detect or measure a peptide of the present
invention using the above methods, by exposing an antibody of the
present invention to a sample assumed to contain the peptide of the
present invention, and detecting or measuring an immune complex
formed between the antibody and the peptide.
[0376] For example, an antibody of the present invention can be
used to detect a peptide of the present invention present in the
blood sample (for example, serum sample) of a subject.
Alternatively, an antibody of the present invention present in the
blood sample (for example, serum sample) of a subject can also be
detected using a peptide of the present invention. The result of
measuring a peptide of the present invention or an antibody of the
present invention in the blood sample of a subject can be utilized
to the subject selection for administration of the pharmaceutical
compositions of the present invention or monitoring of the efficacy
of the pharmaceutical compositions of the present invention.
XIII. Vectors and Host Cells
[0377] The present invention provides vectors comprising a
polynucleotide encoding a peptide of the present invention and host
cells introduced with the vectors. A vector of the present
invention may be used to keep a polynucleotide of the present
invention in a host cell, to express a peptide of the present
invention in a host cell, or to administer a polynucleotide of the
present invention for gene therapy.
[0378] When E. coli is a host cell and a vector is amplified and
produced in a large amount in E. coli (for example, JM109,
DH5-alpha, HB101 or XL1-Blue), the vector needs to have a
"replication origin" for amplification in E. coli and a marker gene
for selection of transformed E. coli (for example, a drug
resistance gene selected by a drug such as ampicillin,
tetracycline, kanamycin, chloramphenicol). For example, the
M13-series vectors, pUC-series vectors, pBR322, pBluescript,
pCR-Script and such can be used. In addition, pGEM-T, pDIRECT and
pT7 can be used for cloning as well as the above vectors. When a
vector is used in the production of a peptide of the present
invention, an expression vector can be used. For example, an
expression vector for expression in E. coli needs to have the above
features for amplification in E. coli. When E. coli such as JM109,
DHS-alpha, HB101 or XL1-Blue are used as a host cell, the vector
needs to have a promoter, for example, lacZ promoter (Ward et al.,
Nature 1989, 341: 544-6; FASEB J. 1989, 6: 2422-7), araB promoter
(Better et al., Science 1988, 240: 1041-3), T7 promoter or the
like, that can efficiently express the desired gene in E. coli. In
that respect, pGEX-5X-1 (Pharmacia), "QIAexpress system" (Qiagen),
pEGFP and pET (in this case, the host is preferably BL21 which
expresses T7 RNA polymerase), for example, can be used instead of
the above vectors. Additionally, the vector may contain a signal
sequence for peptide secretion. An exemplary signal sequence that
directs the peptide to be secreted to the periplasm of the E. coli
is the pelB signal sequence (Lei et al., J Bacteriol. 1987, 169:
4379). Means for introducing the vectors into the target host cells
include, for example, the calcium chloride method and the
electroporation method.
[0379] In addition to E. coli, for example, expression vectors
derived from mammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS
(Nucleic Acids Res 1990, 18(17): 5322), pEF, pCDM8), expression
vectors derived from insect cells (for example, "Bac-to-BAC
baculovirus expression system" (GIBCO BRL), pBacPAK8), expression
vectors derived from plants (e.g., pMH1, pMH2), expression vectors
derived from animal viruses (e.g., pHSV, pMV, pAdexLcw), expression
vectors derived from retroviruses (e.g., pZIpneo), expression
vectors derived from yeast (e.g., "Pichia Expression Kit"
(Invitrogen), pNV11, SP-Q01) and expression vectors derived from
Bacillus subtilis (e.g., pPL608, pKTH50) can be used for producing
the polypeptide of the present invention.
[0380] In order to express the vector in animal cells such as CHO,
COS or NIH3T3 cells, the vector needs to carry a promoter necessary
for expression in such cells, for example, the SV40 promoter
(Mulligan et al., Nature 1979, 277: 108), the MMLV-LTR promoter,
the EF1-alpha promoter (Mizushima et al., Nucleic Acids Res. 1990,
18: 5322), the CMV promoter and the like, and preferably a marker
gene for selecting transformants (for example, a drug resistance
gene selected by a drug (e.g., neomycin, G418)). Examples of known
vectors with these characteristics include, for example, pMAM,
pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.
[0381] The embodiments of the present invention are exemplified
below based on the above explanation; however, the present
invention is not limited to these embodiments. [0382] [1] A peptide
of less than 15 amino acids having cytotoxic T cell (CTL)-inducing
ability, which comprises the amino acid sequence selected from the
group of: [0383] (a) the amino acid sequence selected from the
group consisting of SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37;
and [0384] (b) the amino acid sequence in which one, two, three or
several amino acids are substituted, deleted, inserted and/or added
to the amino acid sequence selected from the group consisting of
SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37. [0385] [2] The
peptide of [1], comprising the amino acid sequence in which one or
more (i.e., one, two or three) substitution(s) selected from (a) to
(c) below is introduced into the amino acid sequence selected from
the group consisting of SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and
37: [0386] (a) the second amino acid from the N terminus is
substituted with an amino acid selected from the group consisting
of threonine and serine; [0387] (b) the third amino acid from the N
terminus is substituted with an amino acid selected from the group
consisting of aspartic acid and glutamic acid; and [0388] (c) the
C-terminal amino acid is substituted with tyrosine. [0389] [3] The
peptide of [1], which consists of the amino acid sequence selected
from the group consisting of SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35
and 37. [0390] [4] A polynucleotide, which encodes the peptide of
any one of [1] to [3]. [0391] [5] A composition comprising a
pharmaceutically acceptable carrier and at least one ingredient
selected from the group consisting of (a) to (e) below: [0392] (a)
one or more types of peptides of any one of [1] to [3]; [0393] (b)
one or more types of polynucleotides encoding the peptide(s) of any
one of [1] to [3] in an expressible form; [0394] (c) an
antigen-presenting cell (APC) that presents on its cell surface a
complex of the peptide of any one of [1] to [3] and an HLA antigen;
[0395] (d) an exosome that presents on its cell surface a complex
of the peptide of any one of [1] to [0396] [3] and an HLA antigen;
and [0397] (e) a CTL that targets the peptide of any one of [1] to
[3]. [0398] [6] The composition of [5], which is a composition for
inducing a CTL(s), wherein the ingredient is at least one
ingredient selected from the group consisting of (a) to (d) below:
[0399] (a) one or more types of peptides of any one of [1] to [3];
[0400] (b) one or more types of polynucleotides encoding the
peptide(s) of any one of [1] to [3] in an expressible form; [0401]
(c) an antigen-presenting cell (APC) that presents on its cell
surface a complex of the peptide of any one of [1] to [3] and an
HLA antigen; and [0402] (d) an exosome that presents on its cell
surface a complex of the peptide of any one of [1] to [0403] [3]
and an HLA antigen. [0404] [7] The composition of [5], which is a
pharmaceutical composition. [0405] [8] The composition of [7],
which is a pharmaceutical composition for one or more uses selected
from the group consisting of (i) cancer treatment, (ii) cancer
prevention (prophylaxis) and (iii) prevention (prophylaxis) of
postoperative cancer recurrence. [0406] [9] The composition of [7],
which is for inducing an immune response against cancer. [0407]
[10] The composition of [8] or [9], wherein the cancer is selected
from the group consisting of bladder cancer, breast cancer,
cervical cancer, cholangiocellular cancer, chronic myeloid leukemia
(CML), esophagus cancer, gastric cancer, non-small-cell lung
cancer, lymphoma, osteosarcoma, prostate cancer, kidney cancer,
small-cell lung cancer, head and neck cancer, soft tissue tumor and
colon cancer. [0408] [11] The composition of any one of [5] to
[10], which is formulated for administration to a subject positive
for HLA-A01. [0409] [12] A method of inducing an APC(s) having
CTL-inducing ability, which comprises a step selected from the
group consisting of: [0410] (a) contacting an APC(s) with the
peptide of any one of [1] to [3] in vitro, ex vivo or in vivo; and
[0411] (b) introducing a polynucleotide encoding the peptide of any
one of [1] to [3] into an APC(s). [0412] [13] A method of inducing
a CTL(s), which comprises a step selected from the group consisting
of (a) to (c) below: [0413] (a) co-culturing a CD8-positive T
cell(s) with an APC(s) that presents on its surface a complex of an
HLA antigen and the peptide of any one of [1] to [3]; [0414] (b)
co-culturing a CDR-positive T cell(s) with an exosome(s) that
presents on its surface a complex of an HLA antigen and the peptide
of any one of [1] to [3]; and [0415] (c) introducing into a
CD8-positive T cell(s) a polynucleotide encoding each subunit of a
T cell receptor (TCR) capable of binding to the peptide of any one
of [1] to [3] presented by an HLA antigen on a cell surface. [0416]
[14] An APC that presents on its surface a complex of an HLA
antigen and the peptide of any one of [1] to [3]. [0417] [15] The
APC of [14], which is induced by the method of [12]. [0418] [16] A
CTL that targets the peptide of any one of [1] to [3]. [0419] [17]
The CTL of [16], which is induced by the method of [13]. [0420]
[18] A method of inducing an immune response against cancer, which
comprises administering to a subject at least one ingredient
selected from the group consisting of (a) to (e) below: [0421] (a)
one or more types of peptides of any one of [1] to [3]; [0422] (b)
one or more types of polynucleotides encoding the peptide(s) of any
one of [1] to [3] in an expressible form; [0423] (c) an
antigen-presenting cell (APC) that presents on its cell surface a
complex of the peptide of any one of [1] to [3] and an HLA antigen;
[0424] (d) an exosome that presents on its cell surface a complex
of the peptide of any one of [1] to [0425] [3] and an HLA antigen;
and [0426] (e) a CTL that targets the peptide of any one of [1] to
[3]. [0427] [19] A method of treating and/or preventing cancer,
and/or preventing postoperative recurrence thereof, which comprises
administering to a subject at least one ingredient selected from
the group consisting of (a) to (e) below: [0428] (a) one or more
types of peptides of any one of [1] to [3];
[0429] (b) one or more types of polynucleotides encoding the
peptide(s) of any one of [1] to [3] in an expressible form; [0430]
(c) an antigen-presenting cell (APC) that presents on its cell
surface a complex of the peptide of any one of [1] to [3] and an
HLA antigen; [0431] (d) an exosome that presents on its cell
surface a complex of the peptide of any one of [1] to [0432] [3]
and an HLA antigen; and [0433] (e) a CTL that targets the peptide
of any one of [1] to [3]. [0434] [20] An antibody that binds to the
peptide of any one of [1] to [3]. [0435] [21] A method of screening
for a peptide having CTL-inducing ability, which comprises the
steps of: [0436] (a) generating candidate sequences consisting of
an amino acid sequence in which one, two or several amino acid
residues are substituted, deleted, inserted and/or added to an
original amino acid sequence consisting of the amino acid sequence
selected from among SEQ ID NOs: 1, 8, 10, 13, 25, 33 to 35 and 37;
[0437] (b) selecting from among the candidate sequences generated
in (a), a candidate sequence that does not have significant
homology (sequence identity) with any known human gene product
other than CDCA1; [0438] (c) contacting an APC(s) with a peptide
consisting of the candidate sequence selected in (b); [0439] (d)
contacting the APC(s) of (c) with a CD8-positive T cell(s); and
[0440] (e) selecting a peptide having an equal to or higher
CTL-inducing ability than that of a peptide consisting of the
original amino acid sequence. [0441] [22] Use of at least one
active ingredient selected from the group consisting of (a) to (e)
below in the manufacture of a composition for inducing an immune
response against cancer: [0442] (a) one or more types of peptides
of any one of [1] to [3]; [0443] (b) one or more types of
polynucleotides encoding the peptide(s) of any one of [1] to [3] in
an expressible form; [0444] (c) an antigen-presenting cell (APC)
that presents on its cell surface a complex of the peptide of any
one of [1] to [3] and an HLA antigen; [0445] (d) an exosome that
presents on its cell surface a complex of the peptide of any one of
[1] to [0446] [3] and an HLA antigen; and [0447] (e) a CTL that
targets the peptide of any one of [1] to [3]. [0448] [23] Use of at
least one ingredient selected from the group consisting of (a) to
(e) below in the manufacture of a pharmaceutical composition for
treating and/or preventing cancer, and/or preventing postoperative
recurrence thereof: [0449] (a) one or more types of peptides of any
one of [1] to [3]; [0450] (b) one or more types of polynucleotides
encoding the peptide(s) of any one of [1] to [3] in an expressible
form; [0451] (c) an antigen-presenting cell (APC) that presents on
its cell surface a complex of the peptide of any one of [1] to [3]
and an HLA antigen; [0452] (d) an exosome that presents on its cell
surface a complex of the peptide of any one of [1] to [3] and an
HLA antigen; and [0453] (e) a CTL that targets the peptide of any
one of [1] to [3]. [0454] [24] Use of at least one ingredient
selected from the group consisting of (a) to (e) below for inducing
an immune response against cancer: [0455] (a) one or more types of
peptides of any one of [1] to [3]; [0456] (b) one or more types of
polynucleotides encoding the peptide(s) of any one of [1] to [3] in
an expressible form; [0457] (c) an antigen-presenting cell (APC)
that presents on its cell surface a complex of the peptide of any
one of [1] to [3] and an HLA antigen; [0458] (d) an exosome that
presents on its cell surface a complex of the peptide of any one of
[1] to [3] and an HLA antigen; and [0459] (e) a CTL that targets
the peptide of any one of [1] to [3]. [0460] [25] Use of at least
one ingredient selected from the group consisting of (a) to (e)
below for treating and/or preventing cancer and/or preventing
postoperative recurrence thereof: [0461] (a) one or more types of
peptides of any one of [1] to [3]; [0462] (b) one or more types of
polynucleotides encoding the peptide(s) of any one of [1] to [3] in
an expressible form; [0463] (c) an antigen-presenting cell (APC)
that presents on its cell surface a complex of the peptide of any
one of [1] to [3] and an HLA antigen; [0464] (d) an exosome that
presents on its cell surface a complex of the peptide of any one of
[1] to [3] and an HLA antigen; and [0465] (e) a CTL that targets
the peptide of any one of [1] to [4]. [0466] [26] A method of
inducing cytotoxic activity against a CDCA1-epxressing cell(s),
which comprises a step of administering to a subject at least one
ingredient selected from the group consisting of (a) to (e) below:
[0467] (a) one or more types of peptides of any one of [1] to [3];
[0468] (b) one or more types of polynucleotides encoding the
peptide(s) of any one of [1] to [3] in an expressible form; [0469]
(c) an antigen-presenting cell (APC) that presents on its cell
surface a complex of the peptide of any one of [1] to [3] and an
HLA antigen; [0470] (d) an exosome that presents on its cell
surface a complex of the peptide of any one of [1] to [3] and an
HLA antigen; and [0471] (e) a CTL that targets the peptide of any
one of [1] to [3]. [0472] [27] A freeze-dried formulation
comprising one or more types of peptides of any one of [1] to [3].
[0473] [28] A pharmaceutical composition, which is prepared by a
method that comprises dissolving one or more types of peptides of
any one of [1] to [3] in a water-soluble carrier, and performing
filtration sterilization. [0474] [29] A filtration-sterilized
aqueous solution, which is an aqueous solution that comprises one
or more types of peptides of any one of [1] to [3] and a
water-soluble carrier. [0475] [30] An emulsion comprising one or
more types of peptides of any one of [1] to [3], a water-soluble
carrier and an oil adjuvant. [0476] [31] A kit comprising a
container that houses the composition of any one of [5] to [11] and
a container that stores an adjuvant. [0477] [32] A kit comprising a
container that stores a freeze-dried formulation comprising the
peptide of any one of [1] to [3], a container that stores an
adjuvant, and a container that stores a re-dissolving solution for
the freeze-dried formulation.
[0478] Preferably, the kit of the present invention further
comprises an HLA-A01 detection reagent additionally.
[0479] The present invention is explained herein in detail with
reference to its specific embodiments. However, it should be
understood that the above explanation is in fact an illustrative
and explanatory explanation, and is intended to explain the present
invention and preferred embodiments thereof. Through routine
experimentation, one skilled in the art will readily recognize that
various changes and modifications can be made therein without
departing from the spirit and scope of the present invention. Thus,
the present invention is not confined to the above explanation, but
is intended to be defined by the appended claims and equivalents
thereto.
[0480] Hereinbelow, the present invention is described in more
detail with reference to the Examples. Nevertheless, while the
following materials, method and Examples may serve to assist one of
ordinary skill in making and using certain embodiments of the
present invention, there are only intended to illustrate aspects of
the present invention and thus in no way to limit the scope of the
present invention. One of ordinary skill in the art can use methods
and materials similar or equivalent to those described herein in
the practice or testing of the present invention.
Examples
Materials and Methods
Cell Lines
[0481] C1R, an HLA-A- and HLA-B-negative human B lymphoblastoid
cell line, and COS7, an African green monkey kidney cell line, were
purchased from ATCC.
Generation of Target Cells with Constitutive HLA-A*01:01
Expression
[0482] C1R cells (C1R-A01) that constitutively express HLA-A*01:01
was used as cells for stimulating CTLs. A cDNA encoding the
HLA-A*01:01 gene was amplified by PCR and introduced into an
expression vector. C1R cells incorporated with HLA-A*01:01 gene
expression vector were cultured for two weeks under drug selection
with a medium containing G418 (Invitrogen). After diluting
G418-resistant C1R cell suspension, the cells were seeded into a
96-well plate, and subjected to selective culture for another 30
days with a medium containing G418. The HLA-A*01:01 expression in
C1R cells was verified by flow cytometric analysis.
Selection of CDCA1-Derived Peptides
[0483] CDCA1-derived 8 mer, 9 mer and 10 mer peptides that are
expected to bind to HLA-A*01:01 were determined using the binding
prediction server "NetMHC 3.2" (www.cbs.dtu.dk/services/NetMHC-3.20
(Buus et al., Tissue Antigens. 2003, 62(5): 378-84; Nielsen et al.,
Protein Sci. 2003, 12(5): 1007-17; Lundegaard C et al.,
Bioinformatics. 2008, 24(11): 1397-98), "BIMAS"
(http://www-bimas.cit.nih.gov/molbio/hla_bind/) (Parker K C et al.,
J Immunol 1994, 152(1): 163-75) and
"SYFPEITHI"(http://www.syfpeithi.de/bin/MHCServer.d11/EpitopePrediction.h-
tm) (Rammensee H G et al., Immunogenetics 1999, 50(3-4):
213-9).
Peptide Synthesis
[0484] The peptides were chemically synthesized by American Peptide
Company (Sunnyvale, Calif.) according to a standard solid-phase
synthesis method, and purified by reversed phase high-performance
liquid chromatography (HPLC). The quality (purity of 90% or higher)
of the peptides was guaranteed by HPLC and mass spectrometry. The
peptides were dissolved in dimethylsulfoxide (final concentration:
20 mg/ml) and stored at -80 degrees C.
In Vitro CTL Induction
[0485] Monocyte-derived dendritic cells (DCs) were used as the
antigen-presenting cell to induce a cytotoxic T lymphocyte (CTL)
specific to peptides presented on human leukocyte antigens (HLAs).
DCs were prepared in vitro as already reported in the literature
(Nakahara S et al., Cancer Res 2003, 63(14): 4112-8). Specifically,
peripheral-blood mononuclear cells
[0486] (PBMCs) collected from healthy volunteers
(HLA-A*01:01-positive) with the Ficoll-Paque plus solution
(Pharmacia) were seeded into plastic tissue culture dishes (Coming)
to allow attachment of monocytes in PBMCs to the dishes. Then, the
cells were cultured in the presence of 1000 IU/ml granulocyte
macrophage colony-stimulating factor (R&D System) and 1000
IU/ml interleukin(IL)-4 (R&D System) for seven days. As medium,
an AIM-V medium (Invitrogen) containing inactivated AB type serum
(MP Biomedicals) was used (2% ABS/AIM-V medium). DCs induced to
differentiate from monocytes by cytokines were pulsed with 20
micro-g/ml each of the synthesized peptides (37 degrees C., for
three hours). Peptide pulsing was performed in the AIM-V medium.
These peptide-pulsed DCs were inactivated by X-ray irradiation (20
Gy), and mixed in a 1:20 ratio with autologous CD8-positive T cells
obtained by using the CD8 Positive Isolation Kit (Invitrogen)
(1.5.times.10.sup.4 DCs and 3.times.10.sup.5 CD8-positive T cells),
and cultured in a 48-well plate (Coming). The vokmie of 2%
ABS/AIM-V medium was 0.5 ml per 1 well, and IL-7 (R&D System)
was added (final concentration: 10 ng/ml) to the well. Two days
after the start of culture, IL-2 (Novartis) was added thereto
(final concentration: 20 IU/ml). On 7.sup.th day of culture and
14.sup.th day of culture, CD8-positive T cells were further
stimulated with peptide-pulsed DCs. The DCs were prepared before
use by the same method as above. On 21.sup.st day or thereafter
(after the third DC stimulation), IFN-gamma production of
CD8-positive T cells against peptide-pulsed C1R-A01 was confirmed
by enzyme-linked immunospot (ELISPOT) assay (Tanaka H et al., Br J
Cancer 2001, 84(1): 94-9; Umano Y et al., Br J Cancer 2001, 84(8):
1052-7; Uchida N et al., Clin Cancer Res 2004, 10(24): 8577-86;
Suda T et al., Cancer Sci 2006, 97(5): 411-9; Watanabe T et al.,
Cancer Sci 2005, 96(8): 498-506).
CTL Proliferation Procedure
[0487] CTLs were propagated using a method similar to that reported
by Riddell et al. (Walter E A et al., N Engl J Med 1995, 333(16):
1038-44; Riddell SR et al., Nat Med 1996, 2(2): 216-23). In a flask
for tissue culture (FALCON), the CTLs were cultured in 5% ABS/AIM-V
medium (culture solution volume: 25 ml/flask) with two types of
human B lymphoblastoid cell lines (5.times.10.sup.6 cells each)
treated with mitomycin C and anti-CD3 antibody (BD biosciences,
final concentration: 40 ng/ml). On the next day after the culturing
was started, IL-2 was added to the culture (IL-2 final
concentration: 120 IU/ml). On days 5, 8, and 11, the medium was
changed to 5% ABS/AIM-V medium containing 60 IU/ml IL-2 (IL-2 final
concentration: 30 IU/ml) (Tanaka H et al., Br J Cancer 2001, 84(1):
94-9; Umano Y et al., Br J Cancer 2001, 84(8): 1052-7; Uchida N et
al., Clin Cancer Res 2004, 10(24): 8577-86; Suda T et al., Cancer
Sci 2006, 97(5): 411-9; Watanabe T et al., Cancer Sci 2005, 96(8):
498-506).
Establishment of CTL Clones (Limiting Dilution Method)
[0488] After CTLs were induced in vitro, the CTLs were seeded at 1
cell/well or 10 cells/well in a 96-well round-bottomed microplate
(Corning). The CTLs were cultured in 5% ABS/AIM-V medium (culture
solution volume: 150 .mu.l/well) with two types of human B
lymphoblastoid cell lines (1.times.10.sup.4 cells each) treated
with mitomycin C, anti-CD3 antibody (final concentration: 30
ng/ml), and IL-2 (final concentration: 125 Mimi). After 10 days, 50
.mu.l of 5% ABS/AIM-V medium containing 500 IU/ml IL-2 was added to
the culture. After 14.sup.th day, CTLs that showed peptide-specific
IFN-gamma production in the ELISPOT assay were propagated by the
method mentioned above (Uchida N et al., Clin Cancer Res 2004,
10(24): 8577-86; Suda T el al., Cancer Sci 2006, 97(5): 411-9;
Watanabe T et al., Cancer Sci 2005, 96(8): 498-506).
Confirmation of IFN-Gamma Production
[0489] The IFN-gamma ELISPOT assay and IFN-gamma ELISA were
performed to confirm peptide-specific IFN-gamma production of CTLs
induced by a peptide. C1R-A01 pulsed with a peptide was prepared as
target cells. The IFN-gamma ELISPOT assay and IFN-gamma ELISA were
performed according to the procedures recommended by the assay kit
manufacturer.
Establishment of Target Cells in which CDCA1 and HLA-A*01:01 were
Forcibly Expressed
[0490] The cDNA encoding the CDCA1 or HLA-A*01:01 gene were
amplified by PCR. Each PCR amplified product was incorporated into
an expression vector. Using Lipofectamine 2000 (Invitrogen), either
or both of the CDCA1 gene expression vector and the HLA-A*01:01
gene expression vector were introduced into COST cells, an
HLA-negative cell line, according to the procedure recommended by
the manufacturer. On the next day of the gene introduction, the
COST cells were separated using Versene (Invitrogen) and used as
target cells.
Results
[0491] Selection of HLA-A*01:01 binding peptides derived from
CDCA1
[0492] Table 1a, Table 1 b, and Table 1c show 8 mer, 9 mer, and 10
mer peptides derived from CDCA1 that were predicted to bind to
HLA-A*01:01 by "NetMHC 3.2" in descending order of binding
affinity, respectively. Table 2a and Table 2b show 9 mer and 10 mer
peptides derived from CDCA1 that were predicted to bind to
HLA-A*01:01 by "BIMAS" in descending order of binding score. Table
3a and Table 3b show 9 mer and 10 mer peptides derived from CDCA1
that were predicted to bind to HLA-A*01:01 by "SYFPEITHI" in
descending order of binding score. The number of Start Position in
each table indicates the position of the first amino acid of the
peptide when counted from the N-terminus of the CDCA1 protein.
Thirty-nine peptides in total potentially having HLA-A*01:01
binding ability became epitope peptide candidates.
TABLE-US-00001 TABLE 1a CDCA1-derived 8mer peptide predicted to
bind to HLA-A*01:01 by NetMHC3.2 Start Amino Acid Affinity Position
Sequence (nM) SEQ ID NO 138 YMEFLWQY 50 1
TABLE-US-00002 TABLE 1b CDCA1-derived 9mer peptides predicted to
bind to HLA-A*01:01 by NetMHC3.2 Start Amino Acid Affinity Position
Sequence (nM) SEQ ID NO 183 LSDGIQELQ 2486 2 49 YMRALQIVY 2570 3
325 ESDESELKK 3358 4 303 LSDNREKLA 6648 5 57 YGIRLEHFY 6700 6 380
VQEKRGAVY 8200 7
TABLE-US-00003 TABLE 1c CDCA1-derived 10mer peptides predicted to
bind to HLA-A*01:01 by NetMHC3.2 Start Amino Acid Affinity Position
Sequence (nM) SEQ ID NO 136 ETYMEFLWQY 92 8 66 MMPVNSEVMY 455 9 56
VYGIRLEHFY 1040 10 121 LSGIINHIHF 2410 11 287 PSCQLEVQLY 2630 12 48
IYMRALQIVY 2933 13 325 ESDESELKKL 4331 14 436 GIEKAAEDSY 4636 15
379 KVQEKRGAVY 4867 16 129 HFREACRETY 7569 17 335 KTEENSFKRL 8751
18 424 LNLKTALEKY 9039 19 451 KTAELKRKMF 9261 20 146 KSSADKMQQL
9961 21
TABLE-US-00004 TABLE 2a CDCA1-derived 9mer peptides predicted to
bind to HLA-A*01:01 by BIMAS Start Amino Acid Position Sequence
Score SEQ ID NO 335 KTEENSFKR 112.5 22 41 KPEVLHMIY 56.25 23 25
GADGKNLTK 50 24 290 QLEVQLYQK 36 25 244 IVDSPEKLK 10 26
TABLE-US-00005 TABLE 2b CDCA1-derived 10mer peptides prediced to
bind to HLA-A*01:01 by BIMAS Start Amino Acid Position Sequence
Score SEQ ID NO 452 TAELKRKMFK 90 27 91 HLDSFLPICR 25 28 323
QIESDESELK 18 29 290 QLEVQLYQKK 18 30 447 KIDEKTAELK 10 31 106
TADILCPKAK 10 32
TABLE-US-00006 TABLE 3a CDCA1-derived 9mer peptides predicted to
bind to HLA-A*01:01 by SYFPEITHI Start Amino Acid Position Sequence
Score SEQ ID NO 130 FREACRETY 24 33 246 DSPEKLKNY 19 34 268
ARQEVVEKY 18 35 1 METLSFPRY 18 36 288 SCQLEVQLY 18 37
TABLE-US-00007 TABLE 3b CDCA1-derived 10mer peptides predicted to
bind to HLA-A*01:01 by SYFPEITHI Start Amino Acid Position Sequence
Score SEQ ID NO 303 LSDNREKLAS 18 38 245 VDSPEKLKNY 18 39
CTL Induction by HLA-A*01:01-Restricted CDCA1-Derived Peptides
[0493] CTLs specific to peptides derived from CDCA1 were induced
according to the protocol described in "Materials and Methods."
Peptide-specific IFN-gamma production was measured by the IFN-gamma
ELISPOT assay (FIG. 1). Peptide-specific IFN-gamma production
relative to the control well was observed in: [0494] Well #1 with
CDCA1-A01-8-138 (SEQ ID NO: 1) (a), [0495] Well #7 with
CDCA1-A01-9-290 (SEQ ID NO: 25) (b), [0496] Well #5 with
CDCA1-A01-9-130 (SEQ ID NO: 33) (c), [0497] Well #7 with
CDCA1-A01-9-246 (SEQ ID NO: 34) (d), [0498] Well #1 with
CDCA1-A01-9-268 (SEQ ID NO: 35) (e), [0499] Well #3 with
CDCA1-A01-9-288 (SEQ ID NO: 37) (f), [0500] Well #2 with
CDCA1-A01-10-136 (SEQ ID NO: 8) (g), [0501] Well #6 with
CDCA1-A01-10-56 (SEQ ID NO: 10) (h), and [0502] Well #6 with
CDCA1-A01-10-48 (SEQ ID NO: 13) (i).
[0503] Meanwhile, no IFN-gamma production specific to other
peptides shown in Table 1b, Table 1c, Table 2a, Table 2b, Table 3a,
and Table 3b was observed. No IFN-gamma production specific to
CDCA1-A01-10-66 (SEQ ID NO: 9), an example of typical negative
data, was observed (j). Every peptide was likely to bind to
HLA-A*01:01, but the nine peptides derived from CDCA1 were selected
as peptides having strong CTL-inducing ability consequently.
Establishment of CTL Clones Specific to CDCA1-Derived
HLA-A*01:01-Restricted Peptides
[0504] By limiting dilution method, CTL clones were established
from cells in Well #2 which showed IFN-gamma production specific to
CDCA1-A01-10-136 (SEQ ID NO: 8) (FIG. 1g), Well #6 which showed
IFN-gamma production specific to CDCA1-A01-10-56 (SEQ ID NO: 10)
(FIG. 1h), and Well #6 which showed IFN-gamma production specific
to CDCA1-A01-10-48 (SEQ ID NO: 13) (FIG. 1i) in the IFN-gamma
ELISPOT assay. As a result of IFN-gamma measurement by ELISA, CTL
clones stimulated by CDCA1-A01-10-136 (SEQ ID NO: 8),
CDCA1-A01-10-56 (SEQ ID NO: 10), or CDCA1-A01-10-48 (SEQ ID NO: 13)
showed peptide-specific IFN-gamma production (FIG. 2).
IFN-Gamma Production of CTLs Against Target Cells Expressing CDCA1
and HLA-A*01:01
[0505] IFN-gamma production of CTL clones specific to
CDCA1-A01-10-136 (SEQ ID NO: 8) against target cells expressing
CDCA1 and HLA-A*01:01 was verified. COS7 cells expressing both
CDCA1 and HLA-A*01:01 were prepared as target cells. COS7 cells
expressing either CDCA1 or HLA-A*01:01 were prepared as negative
control cells. CTL clones specific to CDCA1-A01-10-136 (SEQ ID NO:
8) showed IFN-gamma production against COS7 cells expressing both
CDCA1 and HLA-A*01:01 (FIG. 3). By contrast, no significant
IFN-gamma production was observed against negative control cells.
From the above, it was clearly demonstrated that CDCA1-A01-10-136
(SEQ ID NO: 8) is a peptide produced by antigen processing and is
presented on the cell surface together with the HLA-A01 molecule
and recognized by CTLs. The result suggests that CDCA1-A01-10-136
(SEQ ID NO: 8) is useful as a cancer vaccine targeting cancer
patients with elevated CDCA1 expression.
Homology Analysis of Antigen Peptides
[0506] It was confirmed that CDCA1-A01-8-138 (SEQ ID NO: 1),
CDCA1-A01-9-290 (SEQ ID NO: 25), CDCA1-A01-9-130 (SEQ ID NO: 33),
CDCA1-A01-9-246 (SEQ ID NO: 34), CDCA1-A01-9-268 (SEQ ID NO: 35),
CDCA1-A01-9-288 (SEQ ID NO: 37), CDCA1-A01-10-136 (SEQ ID NO: 8),
CDCA1-A01-10-56 (SEQ ID NO: 10), and CDCA1-A01-10-48 (SEQ ID NO:
13) were able to induce CTLs that showed peptide-specific IFN-gamma
production. Thus, in order to confirm that the sequences of
CDCA1-A01-8-138 (SEQ ID NO: 1), CDCA1-A01-9-290 (SEQ ID NO: 25),
CDCA1-A01-9-130 (SEQ ID NO: 33), CDCA1-A01-9-246 (SEQ ID NO: 34),
CDCA1-A01-9-268 (SEQ ID NO: 35), CDCA1-A01-9-288 (SEQ ID NO: 37),
CDCA1-A01-10-136 (SEQ ID NO: 8), CDCA1-A01-10-56 (SEQ ID NO: 10),
and CDCA1-A01-10-48 (SEQ ID NO: 13) are derived only from CDCA1,
the homology analysis of the peptide sequences was performed using
the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The
result shows that the sequences of CDCA1-A01-8-138 (SEQ ID NO: 1),
CDCA1-A01-9-290 (SEQ ID NO: 25), CDCA1 A01-9-130 (SEQ ID NO: 33),
CDCA1-A01-9-246 (SEQ ID NO: 34), CDCA1-A01-9-268 (SEQ ID NO: 35),
CDCA1-A01-9-288 (SEQ ID NO: 37), CDCA1-A01-10-136 (SEQ ID NO: 8),
CDCA1 A01-10-56 (SEQ ID NO: 10), and CDCA1-A01-10-48 (SEQ ID NO:
13) were observed only in CDCA1. Therefore, to the best of the
present inventors' knowledge, these peptides are unique to CDCA1
and considered to have little possibility of eliciting an
unintended immune response against molecules other than CDCA1 that
are already known to sensitize the human immune system. In
conclusion, novel HLA-A*01:01-restrictive epitope peptides derived
from CDCA1 were identified and shown to be applicable to cancer
immunotherapy.
INDUSTRIAL APPLICABILITY
[0507] The present invention provides CDCA 1-derived novel
HLA-A01-restricted epitope peptides that induce a potent and
specific anti-tumor immune response and thus have applicability for
a wide range of cancer types. The peptides, compositions, APCs. and
CTLs in the present invention can be used as a peptide vaccine for
cancer expressing CDCA1, for example, bladder cancer, breast
cancer, cervical cancer, cholangiocellular cancer, chronic myeloid
leukemia (CML), esophagus cancer, gastric cancer, non-small-cell
lung cancer, lymphoma, osteosarcoma, prostate cancer, kidney
cancer, small-cell lung cancer, head and neck cancer, soft tissue
tumor, and colon cancer.
[0508] While the present invention is herein described in detail
and with respect to specific embodiments thereof, it is to be
understood that the foregoing description is exemplary and
explanatory in nature and is intended to illustrate the present
invention and its preferred embodiments. Through routine
experimentation, one skilled in the art will readily recognize that
various changes and modifications can be made therein without
departing from the spirit and scope of the present invention, the
metes and bounds of which are defined by the appended claims.
Sequence CWU 1
1
4818PRTArtificial Sequencea peptide derived from CDCA1 1Tyr Met Glu
Phe Leu Trp Gln Tyr1 529PRTArtificial Sequencea peptide derived
from CDCA1 2Leu Ser Asp Gly Ile Gln Glu Leu Gln1 539PRTArtificial
Sequencea peptide derived from CDCA1 3Tyr Met Arg Ala Leu Gln Ile
Val Tyr1 549PRTArtificial Sequencea peptide derived from CDCA1 4Glu
Ser Asp Glu Ser Glu Leu Lys Lys1 559PRTArtificial Sequencea peptide
derived from CDCA1 5Leu Ser Asp Asn Arg Glu Lys Leu Ala1
569PRTArtificial Sequencea peptide derived from CDCA1 6Tyr Gly Ile
Arg Leu Glu His Phe Tyr1 579PRTArtificial Sequencea peptide derived
from CDCA1 7Val Gln Glu Lys Arg Gly Ala Val Tyr1 5810PRTArtificial
Sequencea peptide derived from CDCA1 8Glu Thr Tyr Met Glu Phe Leu
Trp Gln Tyr1 5 10910PRTArtificial Sequencea peptide derived from
CDCA1 9Met Met Pro Val Asn Ser Glu Val Met Tyr1 5
101010PRTArtificial Sequencea peptide derived from CDCA1 10Val Tyr
Gly Ile Arg Leu Glu His Phe Tyr1 5 101110PRTArtificial Sequencea
peptide derived from CDCA1 11Leu Ser Gly Ile Ile Asn Phe Ile His
Phe1 5 101210PRTArtificial Sequencea peptide derived from CDCA1
12Pro Ser Cys Gln Leu Glu Val Gln Leu Tyr1 5 101310PRTArtificial
Sequencea peptide derived from CDCA1 13Ile Tyr Met Arg Ala Leu Gln
Ile Val Tyr1 5 101410PRTArtificial Sequencea peptide derived from
CDCA1 14Glu Ser Asp Glu Ser Glu Leu Lys Lys Leu1 5
101510PRTArtificial Sequencea peptide derived from CDCA1 15Gly Ile
Glu Lys Ala Ala Glu Asp Ser Tyr1 5 101610PRTArtificial Sequencea
peptide derived from CDCA1 16Lys Val Gln Glu Lys Arg Gly Ala Val
Tyr1 5 101710PRTArtificial Sequencea peptide derived from CDCA1
17His Phe Arg Glu Ala Cys Arg Glu Thr Tyr1 5 101810PRTArtificial
Sequencea peptide derived from CDCA1 18Lys Thr Glu Glu Asn Ser Phe
Lys Arg Leu1 5 101910PRTArtificial Sequencea peptide derived from
CDCA1 19Leu Asn Leu Lys Thr Ala Leu Glu Lys Tyr1 5
102010PRTArtificial Sequencea peptide derived from CDCA1 20Lys Thr
Ala Glu Leu Lys Arg Lys Met Phe1 5 102110PRTArtificial Sequencea
peptide derived from CDCA1 21Lys Ser Ser Ala Asp Lys Met Gln Gln
Leu1 5 10229PRTArtificial Sequencea peptide derived from CDCA1
22Lys Thr Glu Glu Asn Ser Phe Lys Arg1 5239PRTArtificial Sequencea
peptide derived from CDCA1 23Lys Pro Glu Val Leu His Met Ile Tyr1
5249PRTArtificial Sequencea peptide derived from CDCA1 24Gly Ala
Asp Gly Lys Asn Leu Thr Lys1 5259PRTArtificial Sequencea peptide
derived from CDCA1 25Gln Leu Glu Val Gln Leu Tyr Gln Lys1
5269PRTArtificial Sequencea peptide derived from CDCA1 26Ile Val
Asp Ser Pro Glu Lys Leu Lys1 52710PRTArtificial Sequencea peptide
derived from CDCA1 27Thr Ala Glu Leu Lys Arg Lys Met Phe Lys1 5
102810PRTArtificial Sequencea peptide derived from CDCA1 28His Leu
Asp Ser Phe Leu Pro Ile Cys Arg1 5 102910PRTArtificial Sequencea
peptide derived from CDCA1 29Gln Ile Glu Ser Asp Glu Ser Glu Leu
Lys1 5 103010PRTArtificial Sequencea peptide derived from CDCA1
30Gln Leu Glu Val Gln Leu Tyr Gln Lys Lys1 5 103110PRTArtificial
Sequencea peptide derived from CDCA1 31Lys Ile Asp Glu Lys Thr Ala
Glu Leu Lys1 5 103210PRTArtificial Sequencea peptide derived from
CDCA1 32Thr Ala Asp Ile Leu Cys Pro Lys Ala Lys1 5
10339PRTArtificial Sequencea peptide derived from CDCA1 33Phe Arg
Glu Ala Cys Arg Glu Thr Tyr1 5349PRTArtificial Sequencea peptide
derived from CDCA1 34Asp Ser Pro Glu Lys Leu Lys Asn Tyr1
5359PRTArtificial Sequencea peptide derived from CDCA1 35Ala Arg
Gln Glu Val Val Glu Lys Tyr1 5369PRTArtificial Sequencea peptide
derived from CDCA1 36Met Glu Thr Leu Ser Phe Pro Arg Tyr1
5379PRTArtificial Sequencea peptide derived from CDCA1 37Ser Cys
Gln Leu Glu Val Gln Leu Tyr1 53810PRTArtificial Sequencea peptide
derived from CDCA1 38Leu Ser Asp Asn Arg Glu Lys Leu Ala Ser1 5
103910PRTArtificial Sequencea peptide derived from CDCA1 39Val Asp
Ser Pro Glu Lys Leu Lys Asn Tyr1 5 104022DNAArtificial Sequencea
PCR primer for the TCR analysis 40gtctaccagg cattcgcttc at
224124DNAArtificial Sequencea PCR primer for the TCR analysis
41tcagctggac cacagccgca gcgt 244221DNAArtificial Sequencea PCR
primer for the TCR analysis 42tcagaaatcc tttctcttga c
214324DNAArtificial Sequencea PCR primer for the TCR analysis
43ctagcctctg gaatcctttc tctt 24441989DNAHomo
sapiensCDS(301)..(1695) 44gcggaatggg gcgggacttc cagtaggagg
cggcaagttt gaaaagtgat gacggttgac 60gtttgctgat ttttgacttt gcttgtagct
gctccccgaa ctcgccgtct tcctgtcggc 120ggccggcact gtaggtgagc
gcgagaggac ggaggaagga agcctgcaga cagacgcctt 180ctccatccca
aggcgcgggc aggtgccggg acgctgggcc tggcggtgtt ttcgtcgtgc
240tcagcggtgg gaggaggcgg aagaaaccag agcctgggag attaacagga
aacttccaag 300atg gaa act ttg tct ttc ccc aga tat aat gta gct gag
att gtg att 348Met Glu Thr Leu Ser Phe Pro Arg Tyr Asn Val Ala Glu
Ile Val Ile1 5 10 15cat att cgc aat aag atc tta aca gga gct gat ggt
aaa aac ctc acc 396His Ile Arg Asn Lys Ile Leu Thr Gly Ala Asp Gly
Lys Asn Leu Thr 20 25 30aag aat gat ctt tat cca aat cca aag cct gaa
gtc ttg cac atg atc 444Lys Asn Asp Leu Tyr Pro Asn Pro Lys Pro Glu
Val Leu His Met Ile 35 40 45tac atg aga gcc tta caa ata gta tat gga
att cga ctg gaa cat ttt 492Tyr Met Arg Ala Leu Gln Ile Val Tyr Gly
Ile Arg Leu Glu His Phe 50 55 60tac atg atg cca gtg aac tct gaa gtc
atg tat cca cat tta atg gaa 540Tyr Met Met Pro Val Asn Ser Glu Val
Met Tyr Pro His Leu Met Glu65 70 75 80ggc ttc tta cca ttc agc aat
tta gtt act cat ctg gac tca ttt ttg 588Gly Phe Leu Pro Phe Ser Asn
Leu Val Thr His Leu Asp Ser Phe Leu 85 90 95cct atc tgc cgg gtg aat
gac ttt gag act gct gat att cta tgt cca 636Pro Ile Cys Arg Val Asn
Asp Phe Glu Thr Ala Asp Ile Leu Cys Pro 100 105 110aaa gca aaa cgg
aca agt cgg ttt tta agt ggc att atc aac ttt att 684Lys Ala Lys Arg
Thr Ser Arg Phe Leu Ser Gly Ile Ile Asn Phe Ile 115 120 125cac ttc
aga gaa gca tgc cgt gaa acg tat atg gaa ttt ctt tgg caa 732His Phe
Arg Glu Ala Cys Arg Glu Thr Tyr Met Glu Phe Leu Trp Gln 130 135
140tat aaa tcc tct gcg gac aaa atg caa cag tta aac gcc gca cac cag
780Tyr Lys Ser Ser Ala Asp Lys Met Gln Gln Leu Asn Ala Ala His
Gln145 150 155 160gag gca tta atg aaa ctg gag aga ctt gat tct gtt
cca gtt gaa gag 828Glu Ala Leu Met Lys Leu Glu Arg Leu Asp Ser Val
Pro Val Glu Glu 165 170 175caa gaa gag ttc aag cag ctt tca gat gga
att cag gag cta caa caa 876Gln Glu Glu Phe Lys Gln Leu Ser Asp Gly
Ile Gln Glu Leu Gln Gln 180 185 190tca cta aat cag gat ttt cat caa
aaa acg ata gtg ctg caa gag gga 924Ser Leu Asn Gln Asp Phe His Gln
Lys Thr Ile Val Leu Gln Glu Gly 195 200 205aat tcc caa aag aag tca
aat att tca gag aaa acc aag cgt ttg aat 972Asn Ser Gln Lys Lys Ser
Asn Ile Ser Glu Lys Thr Lys Arg Leu Asn 210 215 220gaa cta aaa ttg
tcg gtg gtt tct ttg aaa gaa ata caa gag agt ttg 1020Glu Leu Lys Leu
Ser Val Val Ser Leu Lys Glu Ile Gln Glu Ser Leu225 230 235 240aaa
aca aaa att gtg gat tct cca gag aag tta aag aat tat aaa gaa 1068Lys
Thr Lys Ile Val Asp Ser Pro Glu Lys Leu Lys Asn Tyr Lys Glu 245 250
255aaa atg aaa gat acg gtc cag aag ctt aaa aat gcc aga caa gaa gtg
1116Lys Met Lys Asp Thr Val Gln Lys Leu Lys Asn Ala Arg Gln Glu Val
260 265 270gtg gag aaa tat gaa atc tat gga gac tca gtt gac tgc ctg
cct tca 1164Val Glu Lys Tyr Glu Ile Tyr Gly Asp Ser Val Asp Cys Leu
Pro Ser 275 280 285tgt cag ttg gaa gtg cag tta tat caa aag aaa ata
cag gac ctt tca 1212Cys Gln Leu Glu Val Gln Leu Tyr Gln Lys Lys Ile
Gln Asp Leu Ser 290 295 300gat aat agg gaa aaa tta gcc agt atc tta
aag gag agc ctg aac ttg 1260Asp Asn Arg Glu Lys Leu Ala Ser Ile Leu
Lys Glu Ser Leu Asn Leu305 310 315 320gag gac caa att gag agt gat
gag tca gaa ctg aag aaa ttg aag act 1308Glu Asp Gln Ile Glu Ser Asp
Glu Ser Glu Leu Lys Lys Leu Lys Thr 325 330 335gaa gaa aat tcg ttc
aaa aga ctg atg att gtg aag aag gaa aaa ctt 1356Glu Glu Asn Ser Phe
Lys Arg Leu Met Ile Val Lys Lys Glu Lys Leu 340 345 350gcc aca gca
caa ttc aaa ata aat aag aag cat gaa gat gtt aag caa 1404Ala Thr Ala
Gln Phe Lys Ile Asn Lys Lys His Glu Asp Val Lys Gln 355 360 365tac
aaa cgc aca gta att gag gat tgc aat aaa gtt caa gaa aaa aga 1452Tyr
Lys Arg Thr Val Ile Glu Asp Cys Asn Lys Val Gln Glu Lys Arg 370 375
380ggt gct gtc tat gaa cga gta acc aca att aat caa gaa atc caa aaa
1500Gly Ala Val Tyr Glu Arg Val Thr Thr Ile Asn Gln Glu Ile Gln
Lys385 390 395 400att aaa ctt gga att caa caa cta aaa gat gct gct
gaa agg gag aaa 1548Ile Lys Leu Gly Ile Gln Gln Leu Lys Asp Ala Ala
Glu Arg Glu Lys 405 410 415ctg aag tcc cag gaa ata ttt cta aac ttg
aaa act gct ttg gag aaa 1596Leu Lys Ser Gln Glu Ile Phe Leu Asn Leu
Lys Thr Ala Leu Glu Lys 420 425 430tac cac gac ggt att gaa aag gca
gca gag gac tcc tat gct aag ata 1644Tyr His Asp Gly Ile Glu Lys Ala
Ala Glu Asp Ser Tyr Ala Lys Ile 435 440 445gat gag aag aca gct gaa
ctg aag agg aag atg ttc aaa atg tca acc 1692Asp Glu Lys Thr Ala Glu
Leu Lys Arg Lys Met Phe Lys Met Ser Thr 450 455 460tga ttaacaaaat
tacatgtctt tttgtaaatg gcttgccatc ttttaatttt 1745ctatttagaa
agaaaagttg aagcgaatgg aagtatcaga agtaccaaat aatgttggct
1805tcatcagttt ttatacactc tcataagtag ttaataagat gaatttaatg
taggctttta 1865ttaatttata attaaaataa cttgtgcagc tattcatgtc
tctactctgc cccttgttgt 1925aaatagtttg agtaaaacaa aactagttac
ctttgaaata tatatatttt tttctgttac 1985tatc 198945464PRTHomo sapiens
45Met Glu Thr Leu Ser Phe Pro Arg Tyr Asn Val Ala Glu Ile Val Ile1
5 10 15His Ile Arg Asn Lys Ile Leu Thr Gly Ala Asp Gly Lys Asn Leu
Thr 20 25 30Lys Asn Asp Leu Tyr Pro Asn Pro Lys Pro Glu Val Leu His
Met Ile 35 40 45Tyr Met Arg Ala Leu Gln Ile Val Tyr Gly Ile Arg Leu
Glu His Phe 50 55 60Tyr Met Met Pro Val Asn Ser Glu Val Met Tyr Pro
His Leu Met Glu65 70 75 80Gly Phe Leu Pro Phe Ser Asn Leu Val Thr
His Leu Asp Ser Phe Leu 85 90 95Pro Ile Cys Arg Val Asn Asp Phe Glu
Thr Ala Asp Ile Leu Cys Pro 100 105 110Lys Ala Lys Arg Thr Ser Arg
Phe Leu Ser Gly Ile Ile Asn Phe Ile 115 120 125His Phe Arg Glu Ala
Cys Arg Glu Thr Tyr Met Glu Phe Leu Trp Gln 130 135 140Tyr Lys Ser
Ser Ala Asp Lys Met Gln Gln Leu Asn Ala Ala His Gln145 150 155
160Glu Ala Leu Met Lys Leu Glu Arg Leu Asp Ser Val Pro Val Glu Glu
165 170 175Gln Glu Glu Phe Lys Gln Leu Ser Asp Gly Ile Gln Glu Leu
Gln Gln 180 185 190Ser Leu Asn Gln Asp Phe His Gln Lys Thr Ile Val
Leu Gln Glu Gly 195 200 205Asn Ser Gln Lys Lys Ser Asn Ile Ser Glu
Lys Thr Lys Arg Leu Asn 210 215 220Glu Leu Lys Leu Ser Val Val Ser
Leu Lys Glu Ile Gln Glu Ser Leu225 230 235 240Lys Thr Lys Ile Val
Asp Ser Pro Glu Lys Leu Lys Asn Tyr Lys Glu 245 250 255Lys Met Lys
Asp Thr Val Gln Lys Leu Lys Asn Ala Arg Gln Glu Val 260 265 270Val
Glu Lys Tyr Glu Ile Tyr Gly Asp Ser Val Asp Cys Leu Pro Ser 275 280
285Cys Gln Leu Glu Val Gln Leu Tyr Gln Lys Lys Ile Gln Asp Leu Ser
290 295 300Asp Asn Arg Glu Lys Leu Ala Ser Ile Leu Lys Glu Ser Leu
Asn Leu305 310 315 320Glu Asp Gln Ile Glu Ser Asp Glu Ser Glu Leu
Lys Lys Leu Lys Thr 325 330 335Glu Glu Asn Ser Phe Lys Arg Leu Met
Ile Val Lys Lys Glu Lys Leu 340 345 350Ala Thr Ala Gln Phe Lys Ile
Asn Lys Lys His Glu Asp Val Lys Gln 355 360 365Tyr Lys Arg Thr Val
Ile Glu Asp Cys Asn Lys Val Gln Glu Lys Arg 370 375 380Gly Ala Val
Tyr Glu Arg Val Thr Thr Ile Asn Gln Glu Ile Gln Lys385 390 395
400Ile Lys Leu Gly Ile Gln Gln Leu Lys Asp Ala Ala Glu Arg Glu Lys
405 410 415Leu Lys Ser Gln Glu Ile Phe Leu Asn Leu Lys Thr Ala Leu
Glu Lys 420 425 430Tyr His Asp Gly Ile Glu Lys Ala Ala Glu Asp Ser
Tyr Ala Lys Ile 435 440 445Asp Glu Lys Thr Ala Glu Leu Lys Arg Lys
Met Phe Lys Met Ser Thr 450 455 460461843DNAHomo
sapiensCDS(155)..(1549) 46gcggaatggg gcgggacttc cagtaggagg
cggcaagttt gaaaagtgat gacggttgac 60gtttgctgat ttttgacttt gcttgtagct
gctccccgaa ctcgccgtct tcctgtcggc 120ggccggcact gtagattaac
aggaaacttc caag atg gaa act ttg tct ttc ccc 175 Met Glu Thr Leu Ser
Phe Pro 1 5aga tat aat gta gct gag att gtg att cat att cgc aat aag
atc tta 223Arg Tyr Asn Val Ala Glu Ile Val Ile His Ile Arg Asn Lys
Ile Leu 10 15 20aca gga gct gat ggt aaa aac ctc acc aag aat gat ctt
tat cca aat 271Thr Gly Ala Asp Gly Lys Asn Leu Thr Lys Asn Asp Leu
Tyr Pro Asn 25 30 35cca aag cct gaa gtc ttg cac atg atc tac atg aga
gcc tta caa ata 319Pro Lys Pro Glu Val Leu His Met Ile Tyr Met Arg
Ala Leu Gln Ile40 45 50 55gta tat gga att cga ctg gaa cat ttt tac
atg atg cca gtg aac tct 367Val Tyr Gly Ile Arg Leu Glu His Phe Tyr
Met Met Pro Val Asn Ser 60 65 70gaa gtc atg tat cca cat tta atg gaa
ggc ttc tta cca ttc agc aat 415Glu Val Met Tyr Pro His Leu Met Glu
Gly Phe Leu Pro Phe Ser Asn 75 80 85tta gtt act cat ctg gac tca ttt
ttg cct atc tgc cgg gtg aat gac 463Leu Val Thr His Leu Asp Ser Phe
Leu Pro Ile Cys Arg Val Asn Asp 90 95 100ttt gag act gct gat att
cta tgt cca aaa gca aaa cgg aca agt cgg 511Phe Glu Thr Ala Asp Ile
Leu Cys Pro Lys Ala Lys Arg Thr Ser Arg 105 110 115ttt tta agt ggc
att atc aac ttt att cac ttc aga gaa gca tgc cgt 559Phe Leu Ser Gly
Ile Ile Asn Phe Ile His Phe Arg Glu Ala Cys Arg120 125 130 135gaa
acg tat atg gaa ttt ctt tgg caa tat aaa tcc tct gcg gac aaa 607Glu
Thr Tyr Met Glu Phe Leu Trp Gln Tyr Lys Ser Ser Ala Asp Lys 140 145
150atg caa cag tta aac gcc gca cac cag gag gca tta atg aaa ctg gag
655Met Gln Gln Leu Asn Ala Ala His Gln Glu Ala Leu Met Lys Leu Glu
155 160 165aga ctt gat tct gtt cca gtt gaa gag caa gaa gag ttc aag
cag ctt 703Arg Leu Asp Ser Val Pro Val Glu Glu Gln Glu Glu Phe Lys
Gln Leu 170 175 180tca gat gga att cag gag cta caa caa tca cta aat
cag gat ttt cat 751Ser Asp Gly Ile Gln Glu Leu Gln Gln Ser Leu Asn
Gln Asp Phe His 185 190 195caa aaa acg ata gtg ctg caa gag gga aat
tcc caa aag aag tca aat 799Gln Lys Thr Ile Val Leu Gln Glu Gly Asn
Ser Gln Lys Lys Ser Asn200 205 210 215att tca gag aaa acc aag cgt
ttg aat gaa cta aaa ttg tcg gtg gtt 847Ile Ser Glu Lys Thr Lys Arg
Leu Asn Glu
Leu Lys Leu Ser Val Val 220 225 230tct ttg aaa gaa ata caa gag agt
ttg aaa aca aaa att gtg gat tct 895Ser Leu Lys Glu Ile Gln Glu Ser
Leu Lys Thr Lys Ile Val Asp Ser 235 240 245cca gag aag tta aag aat
tat aaa gaa aaa atg aaa gat acg gtc cag 943Pro Glu Lys Leu Lys Asn
Tyr Lys Glu Lys Met Lys Asp Thr Val Gln 250 255 260aag ctt aaa aat
gcc aga caa gaa gtg gtg gag aaa tat gaa atc tat 991Lys Leu Lys Asn
Ala Arg Gln Glu Val Val Glu Lys Tyr Glu Ile Tyr 265 270 275gga gac
tca gtt gac tgc ctg cct tca tgt cag ttg gaa gtg cag tta 1039Gly Asp
Ser Val Asp Cys Leu Pro Ser Cys Gln Leu Glu Val Gln Leu280 285 290
295tat caa aag aaa ata cag gac ctt tca gat aat agg gaa aaa tta gcc
1087Tyr Gln Lys Lys Ile Gln Asp Leu Ser Asp Asn Arg Glu Lys Leu Ala
300 305 310agt atc tta aag gag agc ctg aac ttg gag gac caa att gag
agt gat 1135Ser Ile Leu Lys Glu Ser Leu Asn Leu Glu Asp Gln Ile Glu
Ser Asp 315 320 325gag tca gaa ctg aag aaa ttg aag act gaa gaa aat
tcg ttc aaa aga 1183Glu Ser Glu Leu Lys Lys Leu Lys Thr Glu Glu Asn
Ser Phe Lys Arg 330 335 340ctg atg att gtg aag aag gaa aaa ctt gcc
aca gca caa ttc aaa ata 1231Leu Met Ile Val Lys Lys Glu Lys Leu Ala
Thr Ala Gln Phe Lys Ile 345 350 355aat aag aag cat gaa gat gtt aag
caa tac aaa cgc aca gta att gag 1279Asn Lys Lys His Glu Asp Val Lys
Gln Tyr Lys Arg Thr Val Ile Glu360 365 370 375gat tgc aat aaa gtt
caa gaa aaa aga ggt gct gtc tat gaa cga gta 1327Asp Cys Asn Lys Val
Gln Glu Lys Arg Gly Ala Val Tyr Glu Arg Val 380 385 390acc aca att
aat caa gaa atc caa aaa att aaa ctt gga att caa caa 1375Thr Thr Ile
Asn Gln Glu Ile Gln Lys Ile Lys Leu Gly Ile Gln Gln 395 400 405cta
aaa gat gct gct gaa agg gag aaa ctg aag tcc cag gaa ata ttt 1423Leu
Lys Asp Ala Ala Glu Arg Glu Lys Leu Lys Ser Gln Glu Ile Phe 410 415
420cta aac ttg aaa act gct ttg gag aaa tac cac gac ggt att gaa aag
1471Leu Asn Leu Lys Thr Ala Leu Glu Lys Tyr His Asp Gly Ile Glu Lys
425 430 435gca gca gag gac tcc tat gct aag ata gat gag aag aca gct
gaa ctg 1519Ala Ala Glu Asp Ser Tyr Ala Lys Ile Asp Glu Lys Thr Ala
Glu Leu440 445 450 455aag agg aag atg ttc aaa atg tca acc tga
ttaacaaaat tacatgtctt 1569Lys Arg Lys Met Phe Lys Met Ser Thr
460tttgtaaatg gcttgccatc ttttaatttt ctatttagaa agaaaagttg
aagcgaatgg 1629aagtatcaga agtaccaaat aatgttggct tcatcagttt
ttatacactc tcataagtag 1689ttaataagat gaatttaatg taggctttta
ttaatttata attaaaataa cttgtgcagc 1749tattcatgtc tctactctgc
cccttgttgt aaatagtttg agtaaaacaa aactagttac 1809ctttgaaata
tatatatttt tttctgttac tatc 184347464PRTHomo sapiens 47Met Glu Thr
Leu Ser Phe Pro Arg Tyr Asn Val Ala Glu Ile Val Ile1 5 10 15His Ile
Arg Asn Lys Ile Leu Thr Gly Ala Asp Gly Lys Asn Leu Thr 20 25 30Lys
Asn Asp Leu Tyr Pro Asn Pro Lys Pro Glu Val Leu His Met Ile 35 40
45Tyr Met Arg Ala Leu Gln Ile Val Tyr Gly Ile Arg Leu Glu His Phe
50 55 60Tyr Met Met Pro Val Asn Ser Glu Val Met Tyr Pro His Leu Met
Glu65 70 75 80Gly Phe Leu Pro Phe Ser Asn Leu Val Thr His Leu Asp
Ser Phe Leu 85 90 95Pro Ile Cys Arg Val Asn Asp Phe Glu Thr Ala Asp
Ile Leu Cys Pro 100 105 110Lys Ala Lys Arg Thr Ser Arg Phe Leu Ser
Gly Ile Ile Asn Phe Ile 115 120 125His Phe Arg Glu Ala Cys Arg Glu
Thr Tyr Met Glu Phe Leu Trp Gln 130 135 140Tyr Lys Ser Ser Ala Asp
Lys Met Gln Gln Leu Asn Ala Ala His Gln145 150 155 160Glu Ala Leu
Met Lys Leu Glu Arg Leu Asp Ser Val Pro Val Glu Glu 165 170 175Gln
Glu Glu Phe Lys Gln Leu Ser Asp Gly Ile Gln Glu Leu Gln Gln 180 185
190Ser Leu Asn Gln Asp Phe His Gln Lys Thr Ile Val Leu Gln Glu Gly
195 200 205Asn Ser Gln Lys Lys Ser Asn Ile Ser Glu Lys Thr Lys Arg
Leu Asn 210 215 220Glu Leu Lys Leu Ser Val Val Ser Leu Lys Glu Ile
Gln Glu Ser Leu225 230 235 240Lys Thr Lys Ile Val Asp Ser Pro Glu
Lys Leu Lys Asn Tyr Lys Glu 245 250 255Lys Met Lys Asp Thr Val Gln
Lys Leu Lys Asn Ala Arg Gln Glu Val 260 265 270Val Glu Lys Tyr Glu
Ile Tyr Gly Asp Ser Val Asp Cys Leu Pro Ser 275 280 285Cys Gln Leu
Glu Val Gln Leu Tyr Gln Lys Lys Ile Gln Asp Leu Ser 290 295 300Asp
Asn Arg Glu Lys Leu Ala Ser Ile Leu Lys Glu Ser Leu Asn Leu305 310
315 320Glu Asp Gln Ile Glu Ser Asp Glu Ser Glu Leu Lys Lys Leu Lys
Thr 325 330 335Glu Glu Asn Ser Phe Lys Arg Leu Met Ile Val Lys Lys
Glu Lys Leu 340 345 350Ala Thr Ala Gln Phe Lys Ile Asn Lys Lys His
Glu Asp Val Lys Gln 355 360 365Tyr Lys Arg Thr Val Ile Glu Asp Cys
Asn Lys Val Gln Glu Lys Arg 370 375 380Gly Ala Val Tyr Glu Arg Val
Thr Thr Ile Asn Gln Glu Ile Gln Lys385 390 395 400Ile Lys Leu Gly
Ile Gln Gln Leu Lys Asp Ala Ala Glu Arg Glu Lys 405 410 415Leu Lys
Ser Gln Glu Ile Phe Leu Asn Leu Lys Thr Ala Leu Glu Lys 420 425
430Tyr His Asp Gly Ile Glu Lys Ala Ala Glu Asp Ser Tyr Ala Lys Ile
435 440 445Asp Glu Lys Thr Ala Glu Leu Lys Arg Lys Met Phe Lys Met
Ser Thr 450 455 460484PRTArtificial Sequencea linker peptide 48Asn
Lys Arg Lys1
* * * * *
References