U.S. patent application number 13/748984 was filed with the patent office on 2013-08-01 for cancer antigen peptides derived from wt1.
This patent application is currently assigned to International Institute of Cancer Immunology, Inc.. The applicant listed for this patent is International Institute of Cancer Immunology, Inc.. Invention is credited to Haruo Sugiyama.
Application Number | 20130196427 13/748984 |
Document ID | / |
Family ID | 35063778 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196427 |
Kind Code |
A1 |
Sugiyama; Haruo |
August 1, 2013 |
CANCER ANTIGEN PEPTIDES DERIVED FROM WT1
Abstract
The present invention relates to HLA-A26-binding cancer antigen
peptides derived from WT1, polynucleotides encoding said peptide,
CTL-inducers, and cancer vaccine comprising said peptide or
polynucleotide.
Inventors: |
Sugiyama; Haruo; (Minoo-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
International Institute of Cancer Immunology, Inc.; |
Osaka-fu |
|
JP |
|
|
Assignee: |
International Institute of Cancer
Immunology, Inc.
Osaka-fu
JP
|
Family ID: |
35063778 |
Appl. No.: |
13/748984 |
Filed: |
January 24, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12554151 |
Sep 4, 2009 |
8388975 |
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13748984 |
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10594507 |
Sep 28, 2006 |
7622119 |
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PCT/JP05/06113 |
Mar 30, 2005 |
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12554151 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
A61K 48/00 20130101;
G01N 33/56972 20130101; A61K 39/00 20130101; C07K 14/4748 20130101;
C12N 5/0638 20130101; C07K 16/30 20130101; A61K 38/00 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2004 |
JP |
2004-105219 |
Claims
1-18. (canceled)
19. A CTL, which recognizes a complex between an HLA-A26-binding
cancer antigen peptide derived from the amino acid sequence of
human WT1 set forth in SEQ ID NO: 1 and HLA-A26 antigen.
20. The CTL according to claim 19, wherein the peptide comprises
the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8, or
SEQ ID NO: 9.
21. The CTL according to claim 20, wherein the length of the
peptide is up to 11 amino acids.
22. The CTL according to claim 19, wherein the peptide comprises an
amino acid sequence identical to the amino acid sequence set forth
in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 9 except that one to
several amino acids are substituted, deleted and/or added.
23. The CTL according to claim 19, wherein the peptide comprises
the amino acid sequence set forth in SEQ ID NO: 2.
24. The CTL according to claim 19, wherein the peptide comprises
the amino acid sequence set forth in SEQ ID NO: 8.
25. The CTL according to claim 19, wherein the peptide comprises
the amino acid sequence set forth in SEQ ID NO: 9.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of allowed U.S.
application Ser. No. 10/594,507, filed on Sep. 28, 2006, which is a
371 of International Patent Application No. PCT/JP05/06113, filed
on Mar. 30, 2005, and claims priority to Japanese Patent
Application No. 2004-105219, filed on Mar. 31, 2004.
TECHNICAL FIELD
[0002] The present invention relates to cancer antigen peptides
derived from WT1 and their use.
BACKGROUND ART
[0003] WT1 gene (Wilms' tumor gene 1) has been identified as one of
causative genes of Wilms' tumor that is a childhood renal tumor
(Cell 60: 509, 1990, Nature 343: 774, 1990). WT1 gene encodes the
transcription factor WT1, and WT1 plays an important role in many
processes such as proliferation, differentiation and apoptosis of
cells, and development of tissues (Int. Rev. Cytol. 181: 151,
1998). The WT1 gene was originally defined as a tumor suppressor
gene. However, subsequent studies revealed that WT1 gene is
expressed in leukemia and various solid cancers including lung
cancer and breast cancer, indicating that WT 1 gene rather exerts
an oncogenic function promoting cancer growth. In addition, it was
demonstrated in vitro that, when peripheral blood mononuclear cells
positive for HLA-A*0201 or HLA-A*2402 are stimulated with
WT1-derived peptides, peptide-specific cytotoxic T-lymphocytes
(CTLs) are induced and kill leukemia or solid tumor cells which
endogenously express WT1. These results demonstrated that WT1 is a
promising target molecule of cancer immunotherapy (Int. J. Hematol
76: 127, 2002). However, it has been neither elucidated whether
said WT1 contains a peptide portion(s) capable of binding to
HLA-A26 antigen nor reported that there is such a peptide(s).
[0004] In addition, although binding sequences for a cancer antigen
peptide capable of binding to HLA-A*0201 (a member of HLA-A2
antigens) were deduced (WO 00/18795), only a few cancer antigen
peptides have been proved to be effective so far (WO 00/06602 and
WO 00/026249).
DISCLOSURE OF THE INVENTION
[0005] The purpose of the present invention is to provide cancer
antigen peptides derived from WT1, their use as a CTL-inducer, and
the like.
[0006] The present inventors have conducted intensive researches
aiming at identification of novel cancer antigen peptides derived
from WT1. As a result, it was revealed that peptides set forth in
SEQ ID NOS: 2, 8 and 9 can induce HLA-A26-restricted CTLs. That is,
the present inventors have for the first time found that WT1
contains a cancer antigen peptide portion(s) which binds to HLA-A26
antigen among a number of HLA antigen subclasses and is recognized
by CTLs. This finding led to the development of novel cancer
vaccine therapy which can induce WT1-specific CTLs in
HLA-A26-positive cancer patients.
[0007] Furthermore, the present inventors have for the first time
found that a peptide set forth in SEQ ID NO: 3 of which effects
have been unknown has an activity, as a cancer antigen, of binding
to HLA-A*0201 antigen and being recognized by CTLs.
[0008] The above-mentioned WT1-derived cancer antigen peptide and a
polynucleotide encoding the same and the like of the present
invention can be used effectively as a CTL inducer, namely cancer
vaccine. In addition, the cancer antigen peptides of the present
invention can be used effectively as an ingredient of a detecting
agent for WT1-specific CTLs. The present invention has been
established on the basis of these findings.
[0009] Thus, the present invention is related to:
[0010] (1) A peptide derived from the amino acid sequence of human
WT1 set forth in SEQ ID NO: 1 and having activity as an
HLA-A26-binding cancer antigen peptide;
[0011] (2) The peptide of (1) above, which comprises or consists of
8-11 contiguous amino acids in the amino acid sequence of human WT1
set forth in SEQ ID NO: 1;
[0012] (3) The peptide of (1) or (2) above, which comprises the
amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8 or SEQ
ID NO: 9;
[0013] (4) A tumor antigen peptide consisting of the amino acid
sequence set forth in SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO:
9;
[0014] (5) An epitope peptide comprising a peptide described in any
one of (1) to (4) above;
[0015] (6) A polynucleotide encoding a peptide described in any one
of (1) to (5) above;
[0016] (7) An expression vector containing the polynucleotide
described in (6) above;
[0017] (8) A cell containing the expression vector described in (7)
above;
[0018] (9) A process for producing a peptide described in any one
of (1) to (5) above, which comprises culturing the cell described
in (8) above under the condition where the peptide can be
expressed;
[0019] (10) An antibody which specifically binds to a peptide
described in any one of (1) to (4) above;
[0020] (11) An antigen-presenting cell on which a complex between
an HLA-A26-binding cancer antigen peptide derived from the amino
acid sequence of human WT1 set forth in SEQ ID NO: 1, preferably a
peptide described in any one of (2) to (4) above and an HLA-A26
antigen is presented;
[0021] (12) A CTL which recognizes a complex between an
HLA-A26-binding cancer antigen peptide derived from the amino acid
sequence of human WT1 set forth in SEQ ID NO: 1, preferably a
peptide described in any one of (2) to (4) above and an HLA-A26
antigen;
[0022] (13) A pharmaceutical composition which comprises a peptide
described in any one of (1) to (5) above, an expression vector
described in (7) above, a cell described in (8) above, an
antigen-presenting cell described in (11) above, or a CTL described
in (12) above, together with a pharmaceutically acceptable
carrier;
[0023] (14) The pharmaceutical composition of (13) above, which is
used as a CTL inducer;
[0024] (15) The pharmaceutical composition of (13) above, which is
used as cancer vaccine;
[0025] (16) An HLA monomer, dimer, tetramer or pentamer comprising
an HLA-A26-binding cancer antigen peptide derived from the amino
acid sequence of human WT1 set forth in SEQ ID NO: 1, preferably a
peptide described in any one of (2) to (4) above, together with an
HLA-A26 antigen;
[0026] (17) A reagent for the detection of CTLs specific for an
HLA-A26-binding cancer antigen peptide derived from WT1, which
reagent comprises an HLA monomer, dimer, tetramer or pentamer
described in (16) above as an ingredient;
[0027] (18) A pharmaceutical composition which comprises any one of
the following a) to f) together with a pharmaceutically acceptable
carrier: [0028] a) a peptide comprising the amino acid sequence set
forth in SEQ ID NO: 3 or SEQ ID NO: 4, [0029] b) an epitope peptide
comprising the peptide of a) above, [0030] c) an expression vector
containing a polynucleotide encoding the peptide of a) or b) above,
[0031] d) a cell containing the expression vector of c) above,
[0032] e) an antigen-presenting cell on which a complex between the
peptide of a) above and an HLA-A*0201 antigen is presented, and
[0033] f) a CTL which recognizes the complex between the peptide of
a) and an HLA-A*0201 antigen;
[0034] (19) The pharmaceutical composition of (18) above, which is
used as a CTL inducer;
[0035] (20) The pharmaceutical composition of (19) above, which is
used as cancer vaccine;
[0036] (21) An HLA monomer, dimer, tetramer or pentamer which
comprises a peptide comprising the amino acid sequence set forth in
SEQ ID NO: 3 or SEQ ID NO: 4 together with an HLA-A*0201 antigen;
or
[0037] (22) A reagent for the detection of CTLs specific for
HLA-A*0201-binding cancer antigen peptide derived from WT1, which
reagent comprises an HLA monomer, dimer, tetramer or pentamer
described in (21) above as an ingredient.
[0038] The present invention provides cancer antigen peptides
derived from WT1, polynucleotides encoding the same, CTL-inducers
comprising the same, and the like. The CTL inducer of the present
invention is useful as cancer vaccine. The cancer vaccine of the
present invention is applicable to many cancer patients who are
positive for HLA-A26 or HLA-A*0201.
BRIEF DESCRIPTION OF DRAWINGS
[0039] FIG. 1 is a graph showing the results of experiments where
the cytotoxic activity of CTLs induced by stimulation with a
peptide of SEQ ID NO: 3 on T2 target cells either pulsed (filled
circle) or not-pulsed (open circle) with the peptide was examined.
In the figure, the vertical axis indicates the specific cytotoxic
activity (% specific lysis) and the abscissa axis the E/T ratio
which is a ratio of effector cell number (E) to target cell number
(T).
[0040] FIG. 2 is a graph showing the results of experiments where
the cytotoxic activity of CTLs induced by stimulation with a
peptide of SEQ ID NO: 3 on TF-1 cell line (filled circle), which is
positive for HLA-A*0201 and expresses WT1, or JY cell line (open
circle), which is positive for HLA-A*0201 and not expresses WT1,
was examined. In the figure, the vertical axis indicates the
specific cytotoxic activity (% specific lysis) and the abscissa
axis the E/T ratio.
[0041] FIG. 3 is a graph showing the results of experiments where
the cytotoxic activity of CTLs induced by stimulation with a
peptide of SEQ ID NO: 2 on B-LCL target cells either pulsed (solid
bar) or not-pulsed (open bar) with the peptide was examined. In the
figure, the vertical axis indicates the specific cytotoxic activity
(% specific lysis).
[0042] FIG. 4 is a graph showing the results of experiments where
the cytotoxic activity of CTLs induced by stimulation with a
peptide of SEQ ID NO: 2, 8 or 9 on B-LCL target cells either pulsed
(solid bar) or not-pulsed (open bar) with the peptide was examined.
In the figure, the vertical axis indicates the specific cytotoxic
activity (% specific lysis).
BEST MODE FOR CARRYING OUT THE INVENTION
[0043] The present invention provides a peptide which is derived
from the amino acid sequence of human WT1 set forth in SEQ ID NO: 1
and has activity as an HLA-A26-binding cancer antigen peptide.
[0044] The amino acid sequence of human WT1 set forth in SEQ ID NO:
1 is a known sequence (Cell, 60: 509, 1990, NCBI database Accession
No. XP.sub.--034418 and Accession No. P19544). The said amino acid
sequence of human WT1 is shown in SEQ ID NO: 1. As HLA-A26
antigens, HLA-A*2601, HLA-A*2602, HLA-A*2603 and the like are
known. For the present invention, preferred HLA-A26 antigen is
HLA-A*2601. In Japan, about 20% of population share HLA-A26 antigen
among HLA antigens.
[0045] It was for the first time found that the amino acid sequence
of WT1 contains cancer antigen peptide portions capable of binding
to HLA-A26 antigen and being recognized by CTLs, which led to the
establishment of the present invention.
[0046] In the context of the present invention, the term "having
(have) activity as an HLA-A26 binding cancer antigen peptide" means
that a peptide has an activity of binding to HLA-A26 antigen and
being recognized by cytotoxic T cells (CTLs), and is synonym for
"binding to HLA-A26 antigen and inducing CTLs (having a
CTL-inducing activity)", or "binding to HLA-A26 antigen and
activating CTLs (having a CTL-activating activity)".
[0047] Accordingly, the phrase "peptide derived from the amino acid
sequence of human WT1 set forth in SEQ ID NO: 1 and having activity
as an HLA-A26-binding cancer antigen peptide" refers to a peptide
which comprises a cancer antigen peptide consisting of a portion of
the amino acid sequence of WT1 set forth in SEQ ID NO: 1 and is
capable of binding to HLA-A26 antigen and being recognized by CTLs,
inclusive of the said cancer antigen peptide itself. The cancer
antigen peptide of the present invention can be any peptide so long
as it consists of a portion of amino acid sequence of human WT1 set
forth in SEQ ID NO: 1 and has activity as an HLA-A26 (HLA-A26
antigen)-binding cancer antigen peptide. The length of cancer
antigen peptides is preferably 8-11 amino acids, more preferably
9-10 amino acids. Peptides having activity as a cancer antigen
peptide of the present invention may have one or more additional
amino acids at the N- or C-terminus. When a peptide of the present
invention has an additional amino acid(s), the entire length of the
peptide is generally contiguous 8-100 amino acids, preferably
contiguous 8-50 amino acids, and more preferably contiguous 8-30
amino acids.
[0048] The cancer antigen peptide of the present invention can be
identified by synthesizing a partial peptide (candidate peptide)
consisting of 8-11 contiguous amino acids in the amino acid
sequence set forth in SEQ ID NO: 1, and assaying whether or not the
peptide has activity as an HLA-A26-binding cancer antigen
peptide.
[0049] The synthesis of peptides can be conducted according to a
method generally used in the field of peptide chemistry. Such a
method can be found in literatures including Peptide Synthesis,
Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press
Inc., New York, 1976; Peptide Synthesis, Maruzen, Inc., 1975;
Peptide-Gosei no Kiso to Jikken, Maruzen, Inc., 1985; and Iyakuhin
no Kaihatsu (Zoku), Vol. 14, Peptide Synthesis, Hirokawa-syoten,
1991.
[0050] It can be examined whether or not a candidate peptide is the
HLA-A26-binding cancer antigen peptide by a method described in,
for example, Tissue Antigen 61: 136. 2003, or a method described in
the working Examples below. It can also be examined whether or not
a candidate peptide is a peptide having activity as an
HLA-A26-binding cancer antigen peptide in a similar manner.
[0051] Specifically, peripheral blood mononuclear cells (PBMCs) are
firstly isolated from a human subject positive for HLA-A26 antigen
and cultured after adding (pulsing with) a candidate peptide. After
cultivation, the culture is repeatedly stimulated several times by
adding a peptide every few days to amplify CTLs specific for the
peptide. Then peptide-specific reaction of said CTLs is detected by
measuring cytokines such as IFN-y produced by CTLs or cytotoxic
activity of CTLs. The cytotoxic activity can be measured by, for
example, .sup.51Cr release assay (Int. J. Cancer, 58: p 317, 1994)
or the like. Examples of target cell usable in the assay include
.sup.51 Cr-labeled cells which are positive for WT1 and positive
for HLA-A26. Specific examples include .sup.51 Cr-labeled cells
which are obtained by introducing a gene encoding HLA-A26 gene
(e.g., Genbank Accession No. No. D14350) into a leukemia cell line
which is positive for WT1 and negative for HLA-A26.
[0052] When the results of assay above show that CTLs kill the
target cells or produce cytokines, the said candidate peptide is
identified to be "an HLA-A26-binding cancer antigen peptide" or "a
peptide having activity as an HLA-A26 binding cancer antigen
peptide".
[0053] Examples of specific embodiments of a peptide having
activity as an HLA-A26-binding cancer antigen peptide of the
present invention include a peptide comprising the amino acid
sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15,
and has activity as an HLA-A26-binding cancer antigen peptide.
Preferably, the present invention provides a peptide comprising the
amino acid sequence set forth in SEQ ID NO: 2, 8 or 9. There are no
limitations regarding the length of such a peptide on the condition
that said peptide has the activity of binding to HLA-A26 antigen
and being recognized by CTLs. However, a peptide (cancer antigen
peptide) having a binding activity to HLA antigen is known to
generally consist of 8 to 11 amino acids. Accordingly, the cancer
antigenic peptide portion in the peptide of the present invention
comprising the amino acid sequence set forth in SEQ ID NO: 2, 8, 9,
10, 11, 12, 13, 14 or 15 is a peptide preferably consisting of 9-11
amino acids, more preferably, 9-10 amino acids. Still more
preferably, it is a cancer antigen peptide consisting of the amino
acid sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14
or 15, and most preferably, a cancer antigen peptide consisting of
the amino acid sequence set forth in SEQ ID NO: 2, 8 or 9.
[0054] The peptides of the present invention may be altered as
appropriate within a range that the activity is maintained. As used
herein the "alteration" of amino acid residue means substitution,
deletion and/or addition of an amino acid residue(s) (the addition
is inclusive of addition of amino acid(s) at the N- and/or
C-terminus of peptide). The substitution of an amino acid
residue(s) is preferred. When the alteration involves substitution
of an amino acid residue(s), any number of amino acid residues at
any position can be replaced on the condition that the activity as
cancer antigen peptide is retained. However, since a peptide which
binds to HLA antigen is generally about 8-11 amino acid in length,
the alteration is preferably involves one to several amino
acids.
[0055] The present invention also provides a peptide (so-called an
epitope peptide) comprising a peptide of the present invention
together with a helper peptide or another cancer antigen
peptide.
[0056] Recently, a peptide ("epitope peptide") composed of multiple
(plural) CTL epitopes (antigen peptides) linked together has been
shown to have an activity of inducing CTLs efficiently. For
example, it has been reported that a peptide (about 30-mer) wherein
CTL epitopes each restricted to HLA-A2-, -A3,-A11 or B53 originated
from cancer antigen protein PSA are ligated together induced in
vivo CTLs specific for respective CTL epitopes (Journal of
Immunology 1998, 161: 3186-3194).
[0057] In addition, a peptide (epitope peptide) wherein a CTL
epitope(s) and a helper epitope(s) are ligated has been shown to
induce CTLs efficiently. In this context, "helper epitope" means a
peptide capable of activating a CD4-positive T cells (Immunity.,
1:751, 1994), and examples thereof include HBVc128-140 of hepatitis
B virus origin, TT947-967 of tetanus toxin origin, etc. CD4+ T
cells activated by said helper epitope exert various activities
including induction and maintenance of CTLs, differentiation, and
activation of effectors such as macrophages, etc, and hence are
considered to be important in the immunological anti-tumor
response. As a concrete example of a peptide wherein a helper
epitope(s) and CTL epitope(s) are linked together, it is reported
that a DNA (minigene) encoding a peptide composed of HBV-originated
HLA-A2-restricted antigen peptides (6 peptides), HLA-A11-restricted
antigen peptides (3 peptides) and a helper epitope induced in vivo
CTLs directed to the respective epitopes efficiently (Journal of
Immunology 1999, 162: 3915-3925). Practically, a peptide wherein a
CTL epitope (tumor antigen peptide corresponding to position
280-288 of melanoma antigen gp100) and a helper epitope (tetanus
toxin-originated T helper epitope) are ligated has been subjected
to clinical test (Clinical Cancer Res., 2001, 7:3012-3024).
[0058] Accordingly, as a specific embodiment, the peptides of the
present invention also include epitope peptides in which multiple
epitopes including the aforementioned peptides of the present
invention are ligated and which have a CTL-inducing activity.
[0059] When the epitope to be ligated to the cancer antigen peptide
of the present invention is a CTL epitope (cancer antigen peptide),
examples of CTL epitopes usable include WT1-derived CTL epitopes
capable of binding to
HLA-A*0201,-A*0204,-A*0205,-A*0206,-A*0207,-A11,-A24,-A31,-A*6801,-B7,-
-B8,-B*2705,-B37,-Cw'0401 or -Cw'0602 (Int. I Hematol 76: 127,
2002; Int. J. Hematol 78: 56, 2003, WO 00/06602, WO 00/18795).
Plural numbers of these CTL epitopes can be linked together, and
the length of one CTL epitope may be about 8-14 amino acids on the
basis of the analysis of antigen peptides bound to various HLA
molecules (Immunogenetics, 41: 178, 1995).
[0060] When the epitope to be ligated to the cancer antigen peptide
of the present invention is a helper epitope, examples of helper
epitopes usable include the aforementioned HBVc128-140 of hepatitis
B virus origin, TT947-967 of tetanus toxin origin, a WT1-derived
helper epitope (Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser
Arg Lys His, SEQ ID NO: 5), and the like. The helper epitope may be
about 13-30 amino acids, preferably, about 13-17 amino acids in
length.
[0061] Specifically, examples of epitope peptides of the present
invention include an epitope peptide which comprises a peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8,
9, 10, 11, 12, 13, 14 or 15 and a helper peptide, preferably an
epitope peptide which comprises a peptide consisting of the amino
acid sequence set forth in SEQ ID NO: 2, 8 or 9 and a helper
peptide.
[0062] More specifically, examples include an epitope peptide which
comprises a peptide consisting of the amino acid sequence set forth
in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably a
peptide consisting of the amino acid sequence set forth in SEQ ID
NO: 2, 8 or 9 and a peptide consisting of the amino acid sequence
set forth in SEQ ID NO: 5; an epitope peptide which comprises a
peptide consisting of the amino acid sequence set forth in SEQ ID
NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably a peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8
or 9 and a tetanus toxin-originated helper peptide (e.g., Phe Asn
Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His
Leu Glu, SEQ ID NO: 6); and an epitope peptide which comprises a
peptide consisting of the amino acid sequence set forth in SEQ ID
NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably a peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8
or 9 and a peptide (Ala Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly
Ile Thr Glu Leu, SEQ ID NO: 7; Clinical Cancer Res.,
2001,7:3012-3024).
[0063] The peptide (epitope peptide) wherein multiple epitopes are
ligated can be prepared by a usual method for peptide synthesis as
described above. It is also obtainable by a usual method for DNA
synthesis and genetic engineering on the basis of sequence
information of a polynucleotide encoding an epitope peptide wherein
multiple epitopes are ligated. Specifically, an epitope peptide
wherein a multiple epitopes are ligated can be prepared by
inserting said polynucleotide into a known expression vector,
transforming a host cell with the resultant recombinant expression
vector, culturing the resultant transformed cells, and recovering
the objective epitope peptide wherein multiple epitopes are ligated
from the culture. These processes can be conducted according to,
for example, a method described in a literature (Molecular Cloning,
T. Maniatis et al., CSH Laboratory (1983), DNA Cloning, D M.
Glover, IRL PRESS (1985)), or the method described hereinafter.
[0064] The CTL-inducing activity of the so produced epitope peptide
wherein multiple epitopes are ligated can be examined by subjecting
to, for example, the above-mentioned .sup.51 Cr release assay.
[0065] The above-described peptides (including epitope peptides) of
the present invention can be modified at the amino group of the
N-terminal amino acid and/or at the carboxyl group of the
C-terminal amino acid. Thus, peptides wherein the N-terminal and/or
the C-terminal amino acid residue is modified fall within the scope
of the peptide of the present invention.
[0066] Examples of a group for the modification of amino group of
the N-terminal amino acid include 1 to 3 groups selected from C1-6
alkyl group, phenyl group, cycloalkyl group and acyl group. Acyl
groups include C.sub.1-6 alkanoyl group, C.sub.1-6 alkanoyl group
substituted by phenyl group, carbonyl group substituted by
C.sub.5-7 cycloalkyl group, C.sub.1-6 alkylsulfonyl group,
phenylsulfonyl group, C.sub.2-6 alkoxycarbonyl group,
alkoxycarbonyl group substituted by phenyl group, carbonyl group
substituted by C.sub.5-7 cycloalkoxy group, phenoxycarbonyl group,
and the like.
[0067] Examples of peptides modified at the carboxyl group of the
C-terminal amino acid include esters and amides. Esters include
C.sub.1-6 alkyl esters, C.sub.0-6 alkyl esters substituted by
phenyl group, C.sub.5-7 cycloalkyl esters, and the like. Amides
specifically include amides, amides substituted by one or two
C.sub.1-6 alkyl groups, amides substituted by one or two C.sub.0-6
alkyl groups that are substituted by phenyl group, amides forming
5- to 7-membered azacycloalkane inclusive of nitrogen atom of amide
group, and the like.
[0068] The present invention also provides a polynucleotide
encoding the above-mentioned peptide of the present invention. The
polynucleotide encoding a peptide of the present invention may be
in the form of DNA or RNA. The polynucleotides of the present
invention can be easily prepared on the basis of information about
amino acid sequence of the present peptide or polynucleotide
sequence of DNA encoding the same. Specifically, synthesis can be
carried out using usual method of DNA synthesis or amplification by
PCR.
[0069] In particular, examples include a polynucleotide encoding an
epitope peptide which comprises a peptide consisting of the amino
acid sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14
or 15, preferably a peptide consisting of the amino acid sequence
set forth in SEQ ID NO: 2, 8 or 9 and a helper epitope.
[0070] Preferred examples include a polynucleotide encoding an
epitope peptide which comprises a peptide consisting of the amino
acid sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14
or 15, more preferably a peptide consisting of the amino acid
sequence set forth in SEQ ID NO: 2, 8 or 9 and a peptide consisting
of the amino acid sequence set forth in SEQ ID NO: 5; a
polynucleotide encoding an epitope peptide which comprises a
peptide consisting of the amino acid sequence set forth in SEQ ID
NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, more preferably a peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8
or 9 and a tetanus toxin-originated helper peptide (e.g., Phe Asn
Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His
Leu Glu, SEQ ID NO: 6); and a polynucleotide encoding an epitope
peptide which comprises a peptide consisting of the amino acid
sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15,
more preferably a peptide consisting of the amino acid sequence set
forth in SEQ ID NO: 2, 8 or 9 and a peptide (Ala Gln Tyr Ile Lys
Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu, SEQ ID NO: 7; Clinical
Cancer Res., 2001,7:3012-3024).
[0071] A recombinant expression vector for expressing the peptide
of the present invention can be constructed by incorporating a
polynucleotide prepared in the above into an expression vector.
[0072] An adequate expression vector can be selected depending on
the host to be used, purposes, and the like, and include plasmids,
phage vectors, virus vectors, and the like.
[0073] When the host is Escherichia coli, examples of vector
include plasmid vectors such as pUC118, pUC119, pBR322, pCR3, and
the like; and phage vectors such as .lamda.ZAPII, .lamda.gt11, and
the like. When the host is yeast, examples of vector include pYES2,
pYEUra3, and the like. When the host is insect cells, examples of
vector include pAcSGHisNT-A, and the like. When the host is animal
cells, examples of vector include plasmid vectors such as pKCR,
pCDM8, pGL2, pcDNA3.1, pRc/RSV, pRc/CMV, and the like; and virus
vectors such as retrovirus vector, adenovirus vector,
adeno-associated virus vector, and the like.
[0074] The expression vectors above may optionally contain a
factor(s) such as promoter capable of inducing expression
(expression-inducing promoter), a gene encoding a signal sequence,
a marker gene for selection, terminator, and the like.
[0075] Furthermore, the expression vector may contain an additional
sequence for expressing the peptide as a fusion protein with
thioredoxin, His tag, GST (glutathione S-transferase), or the like,
so as to facilitate the isolation and purification. Vectors usable
in such a case include GST fusion protein vectors containing an
appropriate promoter (lac, tac, trc, trp, CMV, SV40 early promoter,
etc) that functions in host cells, such as pGEX4T; vectors
containing Tag sequence (Myc, His, etc) such as pcDNA3.1/Myc-His;
and vectors capable of expressing a fusion protein with thioredoxin
and His tag such as pET32a.
[0076] Transformed cells containing the vector of the present
invention can be prepared by transforming host cells with an
expression vector obtained in the above.
[0077] Host cells usable herein include Escherichia coli, yeast,
insect cells and animal cells. Examples of Escherichia coli include
strains of E. coli K-12 such as HB101, C600, JM109, DH5a and AD494
(DE3). Examples of yeast include Saccharomyces cerevisiae. Examples
of animal cells include L929, BALB/c3T3, C127, CHO, COS, Vero and
Hela cells. Examples of insect cells include sf9.
[0078] Introduction of an expression vector into host cells can be
conducted using a conventional method suited for the respective
host cells above. Specifically, introduction can be conducted using
calcium phosphate method, DEAE-dextran method, electroporation
method, and a method using lipid for gene transfer (Lipofectamine,
Lipofectin; Gibco-BRL). Transformed cells containing the expression
vector can be selected by culturing the host cells after
introduction in a conventional medium containing a selection
marker.
[0079] The peptide of the present invention can be produced by
culturing the transformed cells under appropriate conditions
(conditions under which peptides can be expressed). The resultant
peptide may be further isolated and purified according to standard
biochemical purification procedures. The purification procedures
include salting out, ion exchange chromatography, absorption
chromatography, affinity chromatography, gel filtration
chromatography, and the like. When the polypeptide of the present
invention has been expressed as a fusion peptide with thioredoxin,
His tag, GST, or the like, as mentioned above, the peptide can be
isolated and purified by appropriate purification procedures making
use of the characteristics of the fusion protein or tags.
[0080] The present invention provides an antibody which
specifically binds to a peptide of the present invention. The
antibody of the present invention is not restricted to any form and
may be polyclonal or monoclonal antibody raised against a peptide
of the present invention as an antigen.
[0081] As mentioned above, there is no limitation regarding the
antibody of the present invention on the condition that it
specifically binds to a peptide of the present invention. Examples
of antibody include those specifically bind to a peptide consisting
of the amino acid sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11,
12, 13, 14 or 15, preferably to a peptide consisting of the amino
acid sequence set forth in SEQ ID NO: 2, 8 or 9.
[0082] Methods of preparation of antibodies are well known in the
art and the antibodies of the present invention can be prepared by
such conventional methods (Current protocols in Molecular Biology
edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section
11.12-11.13, Antibodies; A Laboratory Manual, Lane, H, D. et al.,
ed., Cold Spring Harber Laboratory Press, New York 1989).
[0083] Specifically, antibodies can be prepared by immunizing a
non-human animal such as rabbit with a peptide of the present
invention as an antigen, and recovering antibodies from serum of
the immunized animal in a conventional manner. On the other hand,
monoclonal antibodies can be obtained by immunizing a non-human
animal such as mouse with a peptide of the present invention,
subjecting the resultant splenocytes to cell fusion with myeloma
cells, and recovering antibodies from the resultant hybridoma cells
(Current protocols in Molecular Biology edit. Ausubel et al. (1987)
Publish. John Wiley and Sons. Section 11.4-11.11).
[0084] The antibodies against the peptide of the present invention
can also be produced while enhancing the immunological response
using different adjuvants depending on the host. Examples of
adjuvants include Freund adjuvants; mineral gels such as aluminium
hydroxide; surfactants such as lysolecithin, Pluronic polyol,
polyanion, peptide, oil emulsion, keyhole limpet hemocyanin and
dinitorophenol; human adjuvants such as BCG (Bacille de
Calmette-Guerin) or Corynebacterium, and the like.
[0085] As mentioned above, antibodies that recognize a peptide of
the present invention and antibodies that neutralize the activity
thereof can be easily prepared by immunizing an animal with a
peptide of the present invention appropriately in a conventional
manner. The antibodies may be used in affinity chromatography,
immunological diagnostic method, and the like. Immunological
diagnostic method may be selected as appropriate from
immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), a fluorescent or luminescent assay, and the like.
The immunological diagnostic method is effective in the diagnosis
of cancer accompanied by elevated expression of WT1, for example,
blood cancers such as leukemia, myelodysplastic syndrome, multiple
myeloma and malignant lymphoma, and solid cancers such as gastric
cancer, colon cancer, lung cancer, breast cancer, embryonal cancer,
hepatic cancer, skin cancer, bladder cancer, prostate cancer,
uterine cancer, cervical cancer, and ovarian cancer.
[0086] The present invention provides an antigen-presenting cell
which presents a complex between a peptide of the present invention
and an HLA-A26 antigen.
[0087] As shown in the Examples below, stimulation with a peptide
of the present invention induced CTLs, which indicates that there
existed antigen-presenting cells presenting a complex between a
peptide of the present invention and an HLA-A26 antigen and that
CTLs specifically recognizing said antigen-presenting cells were
induced. Such antigen-presenting cells presenting a complex between
a peptide of the present invention and an HLA-A26 antigen are used
effectively in the cell therapy (DC therapy) as hereinafter
described.
[0088] The antigen-presenting cells of the present invention
include any cells presenting a complex of an HLA-A26 antigen and a
cancer antigen peptide of the present invention, and specific
examples include antigen-presenting cells wherein a complex between
a peptide consisting of the amino acid sequence set forth in SEQ ID
NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably a peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8
or 9 and an HLA-A26 antigen is presented on the cell surface of
dendritic cells.
[0089] The antigen-presenting cells used in the cell therapy (DC
therapy) can be prepared by isolating cells having
antigen-presenting ability from a cancer patient, pulsing the cells
in vitro with a peptide of the present invention, or introducing
into the cells a polynucleotide or an expression vector containing
the same of the present invention, and allowing the cells to
present a complex between an HLA-A26 antigen and a cancer antigen
peptide of the present invention on the cell surface. The "cells
having antigen-presenting ability" are not limited to particular
cells and may be any cells expressing HLA-A26 antigen capable of
presenting a peptide of the present invention on the cell surface;
however, dendritic cells known to have especially high
antigen-presenting ability are preferred.
[0090] Further, the substance with which cells having
antigen-presenting ability above are pulsed may be a peptide of the
present invention, or a polynucleotide encoding the peptide of the
present invention or an expression vector containing the same.
[0091] The antigen-presenting cells of the present invention can be
prepared by, for example, isolating cells having antigen-presenting
ability from a cancer patient, pulsing the cells in vitro with a
peptide of the present invention (e.g., a cancer antigen peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8,
9, 10, 11, 12, 13, 14 or 15, preferably a cancer antigen peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8
or 9), thereby producing a complex between an HLA-A26 antigen and
the peptide of the present invention (Cancer Immunol.
Immunother.,46:82, 1998, J. Immunol., 158: p 1796,1997, Cancer
Res., 59: p 1184, 1999). When dendritic cells are used,
antigen-presenting cells of the present invention can be prepared
by, for example, isolating lymphocytes from peripheral blood of a
cancer patient by Ficoll method, removing non-adherent cells,
incubating the adherent cells in the presence of GM-CSF and IL-4 to
induce dendritic cells, and pulsing the dendritic cells by
incubating with a peptide of the present invention.
[0092] In the case where antigen-presenting cells of the present
invention are prepared by introducing a polynucleotide encoding a
peptide of the present invention (e.g., a polynucleotide encoding
an epitope peptide which comprises the amino acid sequence set
forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably
the amino acid sequence set forth in SEQ ID NO: 2, 8 or 9) or an
expression vector containing the same into the above-mentioned
cells having an antigen-presenting ability, the preparation can be
carried out referring to a method described in Cancer Res., 56: p
5672, 1996 or J. Immunol., 161: p 5607, 1998, or the like, when the
polynucleotide is DNA. The preparation of antigen-presenting cells
can be effected using RNA as well as DNA in a similar manner,
referring to J. Exp. Med., 184: p 465, 1996, or the like.
[0093] The present invention also provides CTLs which recognize a
complex between a cancer antigen peptide of the present invention
and an HLA-A26 antigen.
[0094] As shown in the Examples below, stimulation with a peptide
of the present invention resulted in the CTL-inducing activity.
This indicates that there existed antigen-presenting cells
presenting a complex between a peptide of the present invention and
an HLA-A26 antigen and that CTLs specifically recognizing said
antigen-presenting cells were induced. Such CTLs which specifically
recognize a complex between an HLA-A26 antigen and a peptide of the
present invention can be used effectively in the adoptive
immunotherapy as hereinafter described.
[0095] CTLs of the present invention may be in any form on the
condition that they recognize specifically a complex of a peptide
of the present invention with an HLA-A26 antigen, and include a
monoclonal CTL and CTL mixture (population) comprising different
kinds of clones. In particular, examples include CTLs specifically
recognize a complex of a peptide consisting of the amino acid
sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15,
preferably a peptide consisting of the amino acid sequence set
forth in SEQ ID NO: 2, 8 or 9 with an HLA-A26 antigen.
[0096] CTLs used in the adoptive immunotherapy can be prepared by
isolating peripheral blood lymphocytes from a patient, stimulating
the cells in vitro with a peptide of the present invention (e.g., a
cancer antigen peptide consisting of the amino acid sequence set
forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably a
cancer antigen peptide consisting of the amino acid sequence set
forth in SEQ ID NO: 2, 8 or 9), or a polynucleotide encoding a
peptide of the present invention (e.g., a polynucleotide encoding
an epitope peptide comprising the amino acid sequence set forth in
SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15, preferably the amino
acid sequence set forth in SEQ ID NO: 2, 8 or 9), or an expression
vector containing the same (Journal of Experimental Medicine 1999,
190: 1669).
[0097] The above-described peptides, expression vectors, cells,
antigen-presenting cells and CTLs of the present invention can be
used as an active ingredient of a CTL-inducer, i.e., cancer
vaccine, by formulating into an appropriate form depending on the
respective substances, as hereinafter described in more detail.
(1) Cancer Vaccine Comprising as an Active Ingredient a Peptide of
the Present Invention
[0098] The peptide of the present invention has a CTL-inducing
ability and the so induced CTLs can exert the anti-cancer activity
through cytotoxic action or production of lymphokines. The peptide
of the present invention therefore can be used as an active
ingredient of cancer vaccine for treating or preventing cancer.
Thus, the present invention provides cancer vaccine (pharmaceutical
composition as cancer vaccine) comprising as an active ingredient a
peptide of the present invention. When cancer vaccine of the
present invention is administered to an HLA-A26-positive and WT
1-positive patient, the peptide (e.g., a cancer antigen peptide
consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8,
9, 10, 11, 12, 13, 14 or 15, preferably the amino acid sequence set
forth in SEQ ID NO: 2, 8 or 9) is presented to HLA-A26 antigen of
antigen-presenting cells. Then, CTLs specifically recognizing the
presented HLA-A26 antigen complex can proliferate and destroy the
cancer cells, whereby the treatment or prevention of cancer becomes
possible. The cancer vaccine of the present invention can be used
in the prevention or treatment of cancer accompanied by elevated
expression level of WT1 gene, for example, blood cancers such as
leukemia, myelodysplastic syndrome, multiple myeloma and malignant
lymphoma, and solid cancers such as gastric cancer, colon cancer,
lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin
cancer, bladder cancer, prostate cancer, uterine cancer, cervical
cancer, and ovarian cancer.
[0099] Thus, in another embodiment, the present invention provides
a method for treatment or prevention of cancer, which comprises
administering an effective amount of cancer vaccine of the present
invention to an HLA-A26-positive and WT1-positive patient.
[0100] The cancer vaccine comprising as an active ingredient a
peptide of the present invention may contain a single CTL epitope
(e.g., a tumor antigen peptide consisting of the amino acid
sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15,
preferably the amino acid sequence set forth in SEQ ID NO: 2, 8 or
9) or an epitope peptide wherein a peptide is ligated with other
peptide(s) (CTL epitope, helper epitope, etc.) as an active
ingredient. Recently, it has been shown that an epitope peptide
wherein multiple CTL epitopes (antigen peptides) are linked has an
activity of inducing in vivo CTLs efficiently. For example, it was
reported that about 30-mer epitope peptide wherein CTL epitopes
(antigen peptides) restricted to HLA-A2-, -A3,-Al 1 or B53
originated from cancer antigen protein PSA are ligated induced in
vivo CTLs specific for respective CTL epitopes (Journal of
Immunology 1998, 161: 3186-3194). It has also been reported that an
epitope peptide wherein a CTL epitope(s) and a helper epitope(s)
are ligated can induce CTLs efficiently. When an epitope peptide in
these forms is administered, said peptide is incorporated into
antigen-presenting cells; the respective antigen peptides generated
by intracellular degradation bind to HLA antigens to form
complexes; the complexes are presented on the surface of
antigen-presenting cells in high density; and CTLs specific for the
complexes efficiently proliferate in the body and destroy cancer
cells. In this way, the treatment or prevention of cancer is
achieved.
[0101] The cancer vaccine comprising as an active ingredient a
peptide of the present invention may be administered together with
a pharmaceutically acceptable carrier, for example, an appropriate
adjuvant, or in the form of particles so that the cellular immunity
can be established effectively. As an adjuvant, those described in
a literature (Clin. Microbiol. Rev., 7:277-289, 1994), and the like
are applicable. Concrete examples include microorganism-derived
components, cytokines, plant-derived components, marine
organism-derived components, mineral gels such as aluminium
hydroxide, surfactants such as lysolecithin and Pluronic polyols,
polyanions, peptides, oil emulsion (emulsion preparations) and the
like. Liposomal preparations, particulate preparations in which the
ingredient is bound to beads having a diameter of several pm,
preparations in which the ingredient is attached to lipids, and the
like, are also contemplated.
[0102] Administration may be achieved, for example, intradermally,
subcutaneously, intramuscularly, or intravenously. Although the
dosage of the peptide of the present invention in the formulation
may be adjusted as appropriate depending on the disease to be
treated, the age and the body weight of patient, it is usually
within the range of 0.0001 mg-1000 mg, preferably 0.001 mg-1000 mg,
more preferably 0.1 mg-10 mg, which can be preferably administered
once in every several days to every several months.
(2) DNA Vaccine Comprising as an Active Ingredient an Expression
Vector Containing a Polynucleotide Encoding a Peptide of the
Present Invention
[0103] Not only the above-mentioned peptide of the present
invention but also an expression vector containing a polynucleotide
encoding a peptide of the present invention can be an active
ingredient of DNA vaccine for treating or preventing cancer. Thus,
the present invention provides cancer vaccine (pharmaceutical
composition as cancer vaccine) comprising as an active ingredient
an expression vector containing a polynucleotide encoding a peptide
of the present invention. In another embodiment, the present
invention provides a method of treating or preventing cancer, which
comprises administering an effective amount of DNA vaccine of the
present invention to an HLA-A26-positive and WT1-positive
patient.
[0104] Recently, a polynucleotide encoding an epitope peptide
wherein multiple (plural) CTL epitopes (antigen peptides) are
ligated or wherein a CTL epitope(s) and a helper epitope(s) are
ligated has been shown to have an activity of inducing CTLs in vivo
efficiently. For example, it is reported that a DNA (minigene)
encoding an epitope peptide wherein HBV-originated
HLA-A2-restricted antigen peptides (6 peptides), HLA-A11-restricted
antigen peptides (3 peptides) and a helper epitope are ligated
induced in vivo CTLs directed to the respective epitopes
efficiently (Journal of Immunology 1999, 162: 3915-3925).
[0105] Accordingly, an active ingredient of cancer vaccine can be
obtained by incorporating a polynucleotide encoding an epitope
peptide of the present invention into an appropriate expression
vector.
[0106] When administering an expression vector containing a
polynucleotide of the present invention as an active ingredient of
cancer vaccine (DNA vaccine), the following methods can be
used.
[0107] As a method for introducing a polynucleotide of the present
invention into cells, any means including those utilizing viral
vectors or other methods are applicable (Nikkei-Science, April,
1994, 20-45; Gekkan-Yakuji, 36(1), 23-48 (1994);
Jikken-Igaku-Zokan, 12(15), 1994, and references cited
therein).
[0108] Examples of a method utilizing a viral vector include those
wherein a DNA of the present invention is incorporated into DNA or
RNA virus such as retrovirus, adenovirus, adeno-associated virus,
herpes virus, vaccinia virus, poxvirus, poliovirus, or Sindbis
virus, and then introduced into cells. Above all, a method using
retrovirus, adenovirus, adeno-associated virus, or vaccinia virus,
or the like, is particularly preferred.
[0109] Examples of other methods include a method wherein an
expression plasmid is directly injected intramuscularly (DNA
vaccination), liposome method, Lipofectin method, microinjection,
calcium phosphate method and electroporation. DNA vaccination and
liposome method are preferred.
[0110] Regarding a method for allowing the polynucleotide of the
present invention to act as a medicament in practice, there are an
in vivo method wherein the polynucleotide is directly introduced
into the body and an ex vivo method wherein the DNA is introduced
extracorporeally into a certain cells removed from human, and the
cells are reintroduced into the body (Nikkei-Science, April, 1994,
20-45; Gekkan-Yakuji, 36(1), 23-48 (1994); Jikkenn-Igaku-Zokan,
12(15), 1994; and references cited therein). The in vivo method is
more preferred.
[0111] In the case of in vivo method, the administration can be
effected through any appropriate routes depending on the disease
and symptoms to be treated. For example, it may be administered via
intravenous, intraarterial, subcutaneous, intracutaneous,
intramuscular route, or the like. When the administration is
carried out by in vivo method, the composition may be administered
in various forms such as solution though, it is typically
formulated, for example, into an injection which contains as an
active ingredient an expression vector containing a polynucleotide
of the present invention. The injection may contain a conventional
carriers, if necessary. As for the liposomes or membrane-fused
liposomes (such as Sendai virus (HVJ)-liposomes) which contains an
expression vector containing a polynucleotide of the present
invention, they may be in the form of liposomal formulation such as
suspension, frozen drug, centrifugally-concentrated frozen drug, or
the like.
[0112] Although the content of an expression vector containing a
polynucleotide in a formulation may be adjusted as appropriate
depending on, for example, the disease to be treated, age and body
weight of a patient, usually 0.0001 mg-100 mg, preferably 0.001
mg-10 mg of an expression vector containing a polynucleotide of the
present invention can be administered once in every several days to
every several months.
[0113] When the above-mentioned expression vector containing a
polynucleotide of the present invention is administered to a cancer
patient, a polypeptide corresponding to the said polynucleotide is
highly expressed in antigen-presenting cells. Thereafter, cancer
antigen peptides generated by intracellular degradation form
complexes with HLA antigens; the complexes are then presented on
the surface of antigen-presenting cells in high density; and CTLs
specifically recognize said complexes are efficiently proliferate
in the body, and destroy cancer cells. In this way, treatment or
prevention of cancer is achieved. The cancer vaccine comprising as
an active ingredient an expression vector containing a
polynucleotide of the present invention can be used in the
prevention or treatment of cancer accompanied by elevated
expression level of WT1 gene, for example, blood cancers such as
leukemia, myelodysplastic syndrome, multiple myeloma and malignant
lymphoma, and solid cancers such as gastric cancer, colon cancer,
lung cancer, breast cancer, embryonal cancer, hepatic cancer, skin
cancer, bladder cancer, prostate cancer, uterine cancer, cervical
cancer, and ovarian cancer.
(3) Cancer Vaccine Comprising as an Active Ingredient
Antigen-Presenting Cells of the Present Invention
[0114] The present invention provides cancer vaccine comprising as
an active ingredient antigen-presenting cells of the present
invention.
[0115] Recently, a cell therapy (DC therapy) which comprises
isolating lymphocytes from peripheral blood of a cancer patient,
inducing dendritic cells from the lymphocytes, pulsing the
dendritic cells with a peptide or the like in vitro, and
reintroducing the resultant antigen-presenting cells into the
patient by subcutaneous injection or the like has been reported
(Cancer Immunol. Immunother., 46:82, 1998, J. Immunol., 158: p
1796, 1997, Cancer Res., 59: p 1184, 1999, Cancer Res., 56: p 5672,
1996, J. Immunol., 161: p 5607, 1998, J. Exp. Med., 184: p 465,
1996). The above-described antigen-presenting cells of the present
invention therefore can be used as an active ingredient of cancer
vaccine to be used in the cell therapy.
[0116] The cancer vaccine comprising as an active ingredient
antigen-presenting cells of the present invention preferably
contains physiological saline, phosphate buffered saline (PBS),
medium, or the like, to stably maintain the antigen-presenting
cells. It may be administered, for example, intravenously,
subcutaneously, or intradermally. The dose is similar to that
described in the references cited above.
[0117] When the cancer vaccine above is reintroduced into a
patient's body, specific CTLs are efficiently induced in the body
of a patient positive for HLA-A26 and positive for WT1, whereby
treatment or prevention of cancer can be effected. The cancer
vaccine comprising as an active ingredient antigen-presenting cells
of the present invention can be used in the prevention or treatment
of cancer accompanied by elevated expression level of WT1 gene, for
example, blood cancers such as leukemia, myelodysplastic syndrome,
multiple myeloma and malignant lymphoma, and solid cancers such as
gastric cancer, colon cancer, lung cancer, breast cancer, embryonal
cancer, hepatic cancer, skin cancer, bladder cancer, prostate
cancer, uterine cancer, cervical cancer, and ovarian cancer.
(4) Cancer Vaccine Comprising as an Active Ingredient CTLs of the
Present Invention
[0118] The present invention provides cancer vaccine
(pharmaceutical composition as cancer vaccine) comprising as an
active ingredient CTLs of the present invention. CTLs of the
present invention can be used effectively in the adoptive
immunotherapy as hereinafter described.
[0119] In melanomas, therapeutic effect has been observed in
adoptive immunotherapy wherein tumor-infiltrating T cells are
removed from a patient and cultured ex vivo in large quantities,
and returned into the same patient (J. Natl. Cancer. Inst., 86:
1159, 1994). Further, in mouse melanoma, suppression of metastasis
has been observed when splenocytes were stimulated with cancer
antigen peptide TRP-2 in vitro to amplify CTLs specific for the
cancer antigen peptide, and the CTLs are administered to a
melanoma-grafted mouse (J. Exp. Med., 185:453, 1997). This resulted
from in vitro proliferation of CTLs that specifically recognize a
complex between an HLA antigen of antigen-presenting cells and a
cancer antigen peptide. Accordingly, a therapeutic method
comprising stimulating in vitro peripheral blood lymphocytes from a
patient with a peptide, or a polynucleotide or an expression vector
of the present invention to proliferate cancer-specific CTLs, and
returning the CTLs into the patient is believed to be effective.
Thus, the CTLs of the present invention can be used as an active
ingredient of cancer vaccine in the adoptive immunotherapy.
[0120] The cancer vaccine comprising as an active ingredient CTLs
of the present invention preferably contains physiological saline,
phosphate buffered saline (PBS), medium, or the like to stably
maintain CTLs. It may be administered, for example, intravenously,
subcutaneously or intradermally. The dose is similar to that
described in the references cited above.
[0121] When the cancer vaccine is returned to a patient positive
for HLA-A26 and positive for WT1, the cytotoxic action of CTLs on
cancer cells is enhanced in the body of a patient, and the cancer
cells are destroyed. In this way, cancer can be treated. The cancer
vaccine comprising as an active ingredient CTLs of the present
invention can be used in the prevention or treatment of cancer
accompanied by elevated expression level of WT1 gene, for example,
blood cancers such as leukemia, myelodysplastic syndrome, multiple
myeloma and malignant lymphoma, and solid cancers such as gastric
cancer, colon cancer, lung cancer, breast cancer, embryonal cancer,
hepatic cancer, skin cancer, bladder cancer, prostate cancer,
uterine cancer, cervical cancer, and ovarian cancer.
[0122] The present invention also provides an HLA monomer, an HLA
dimer, an HLA tetramer or an HLA pentamer comprising a cancer
antigen peptide of the present invention and HLA-A26 antigen.
[0123] In the cancer immunotherapy, examination of the frequency or
amount of CTL precursor cells directed to a cancer antigen (cancer
antigen peptide) in a patient before treatment, or examination of
the frequency or amount of CTLs in a patient during treatment with
a cancer antigen (cancer antigen peptide) can provide a significant
indicator in the selection of patients highly responsive to the
cancer antigen (cancer antigen peptide), the monitoring of
therapeutic effects, or the evaluation of suitability of treatment.
An HLA monomer, an HLA dimer, an HLA tetramer and an HLA pentamer
each comprising a tumor antigen peptide together with an HLA
antigen are useful as a reagent in the detection of CTLs specific
for antigen (antigen peptide), specifically, in the measurement of
frequency or amount of CTLs.
[0124] As used herein, the HLA tetramer refers to a tetramer
prepared by biotinylating a complex (HLA monomer) obtained by
association of an HLA antigen .alpha.-chain and
.beta.2-microglobulin with a peptide (antigen peptide), and
allowing to bind to avidin for tetramerization (Science 279:
2103-2106 (1998); and Science 274: 94-96 (1996)).
[0125] The HLA monomer is a monomer that is used in the preparation
of the above-mentioned HLA tetramer and is formed by biotinylating
an association of HLA antigen .alpha.-chain, .beta.2-microglobulin
and antigen peptide.
[0126] The HLA dimer is a dimer prepared by fusing HLA antigen
.alpha.-chain and Ig (immunoglobulin, for example, IgG1), and
binding the resultant fusion to .beta.2-microglobulin and antigen
peptide (Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993)). The
antigen peptide-specific CTLs bound to HLA dimer can be detected
by, for example, allowing labeled anti-IgG1 antibody to bind to IgG
1.
[0127] The HLA pentamer is a recently developed technique and
refers to a pentamer wherein five molecules of a complex comprising
HLA antigen and antigen peptide are polymerized through Coiled-Coil
domain. Since the HLA antigen-antigen peptide complex can be
labeled with fluorescence or the like, the analysis can be carried
out by flow cytometry or the like similarly to HLA tetramer.
[0128] The aforementioned HLA-monomer, dimer, tetramer and pentamer
are all available by custom production from a manufacture such as
ProImmune or BD Biosciences. At present, HLA tetramers and the like
which comprise different antigen peptides are commercially
available (Medical & Biological Laboratories Co., Ltd.,
etc.)
[0129] Examples of the HLA monomer, dimer, tetramer and pentamer of
the present invention, specifically, include HLA monomers, dimers,
tetramers and pentamers each comprising a peptide consisting of the
amino acid sequence set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12,
13, 14 or 15, preferably the amino acid sequence set forth in SEQ
ID NO: 2, 8 or 9 and an HLA-A26 antigen. Preferably, an HLA
tetramer or HLA pentamer and more preferably, an HLA tetramer is
used in the detection of CTLs.
[0130] The HLA monomer, HLA tetramer and HLA pentamer are
preferably labeled with fluorescence so that the bound CTLs can be
easily sorted out or detected by a known detection measure such as
flow cytometry, fluorescent microscopy, and the like. Examples
include HLA monomers, tetramers and pentamers labeled with
phycoerythrin(PE), fluorescein isothiocyanate (FITC), peridinyl
chlorophyll protein (PerCP), allophycocyanin (APC), or the
like.
[0131] The HLA-A26 antigen (HLA-A26 antigen .alpha.-chain) which is
a component of the HLA monomer, dimer, tetramer and pentamer of the
present invention can be cloned easily by a conventional method
such as PCR on the basis of information about known base sequence
of HLA-A26 and the like disclosed in Genbank Accession No.
D14350.
[0132] The .beta.2-microglobulin which is a component of the HLA
monomer, dimer, tetramer and pentamer of the present invention is
preferably originated from human. The human .beta.2-microglobulin
can be cloned easily by a conventional method such as PCR on the
basis of information about known base sequence of human
.beta.2-microglobulin disclosed in GenBank Acc.No. AB021288.
[0133] The process for the preparation of HLA monomer, dimer,
tetramer and pentamer is well known from the respective literatures
mentioned above; however, the preparation will be hereinafter
described briefly regarding HLA tetramer.
[0134] First, an appropriate host cell such as E. coli or mammalian
cells capable of expressing a protein is transformed with an
HLA-A26 .alpha.-chain expression vector and a .beta.2-microglobulin
expression vector, and allowed to express. E. coli (e.g., BL21) is
preferably used here. The resultant monomer HLA-A26 complex and a
peptide of the present invention are then mixed to form a soluble
HLA-peptide complex. The C-terminal sequence of HLA-A26
.alpha.-chain of the resultant HLA-peptide complex is biotinylated
with BirA enzyme. When a biotinylated HLA-peptide complex and a
fluorescently labeled avidin are mixed at the molar ratio of 4:1,
an HLA tetramer is formed. It is preferred to purify the resulting
protein by gel filtration or the like in each step above.
[0135] The HLA monomer, dimer, tetramer and pentamer described
above are used effectively as a detecting agent for CTLs which are
specific for HLA-A26-binding cancer antigen peptides.
[0136] The CTL-detecting agent of the present invention can be used
for the following purposes, for example. [0137] 1) It can be used
to examine the frequency or amount of CTL precursors directed to a
cancer antigen peptide of the present invention before treatment
with the cancer antigen peptide. This makes it possible to evaluate
the responsiveness of a patient to the cancer antigen peptide.
[0138] 2) It can be used to examine the frequency or amount of CTLs
in a patient during treatment with a cancer antigen peptide of the
present invention. This makes it possible to monitor the
therapeutic effects, evaluate the suitability of treatment, and
confirm that treatment proceeds favorably, and the like.
[0139] Detection of CTLs can be carried out by, specifically,
isolating a biological sample (e.g., PBMC) containing CTLs from a
subject patient, bringing an HLA tetramer or the like of the
present invention into contact with the biological sample, and
measuring the existing frequency or amount of CTLs specific for the
peptide of the present invention bound to the HLA tetramer or the
like by flow cytometry, or the like.
[0140] The present inventors also found for the first time that a
peptide comprising the amino acid sequence of Asp Leu Asn Ala Leu
Leu Pro Ala Val (SEQ ID NO: 3) is an HLA-A*0201-binding cancer
antigen peptide. Although the peptide sequence set forth in SEQ ID
NO: 3 has been disclosed in WO 00/18795, the following findings
have been for the first time disclosed by the present invention:
said peptide has an activity as an HLA-A*0201-binding cancer
antigen peptide, said peptide is generated by intracellular
processing of WT 1 protein, a complex of said peptide with an
HLA-A*0201 antigen is presented on the cell surface and recognized
by CTLs, and said peptide is a therapeutically effective cancer
antigen peptide.
[0141] Thus, the present invention provides a pharmaceutical
composition comprising any one of the following a) to f):
[0142] a) a peptide comprising the amino acid sequence set forth in
SEQ ID NO: 3,
[0143] b) an epitope peptide comprising the peptide of a)
above,
[0144] c) an expression vector containing a polynucleotide encoding
the peptide of a) or b) above,
[0145] d) a cell containing the expression vector of c) above,
[0146] e) an antigen-presenting cell on which a complex between the
peptide of a) above and an HLA-A*0201 antigen is presented, and
[0147] f) a CTL which recognizes a complex between the peptide of
a) above and an HLA-A*0201 antigen, together with a
pharmaceutically acceptable carrier.
[0148] Regarding an HLA-A2 antigen such as HLA-A*0201 antigen, a
certain rules (binding motifs) in the sequence of an antigen
peptide capable of binding to said HLA antigen and being presented
are known. That is, the binding motif for HLA-A2-binding peptide is
known that, in the peptides consisting of 8-11 amino acid, the
amino acid residue at position 2 is leucine, methionine, valine,
isoleucine or glutamine, and the amino acid at the C-terminus is
valine or leucine (Immunogenetics, 41, p 178, 1995, J. Immunol.,
155: p 4749, 1995). Accordingly, the amino acid residue at the
position 2 and/or the C-terminus of the above-mentioned peptide
shown in SEQ ID NO: 3 can be substituted by another amino acid
residue available in view of the above-mentioned motifs. Thus, not
only the peptide shown in SEQ ID NO: 3 but also a variant set forth
in SEQ ID NO: 4 which is under the rule of the above-mentioned
motifs (with the proviso the a peptide shown in SEQ ID NO: 3 is
excluded) can be used in a pharmaceutical composition similar to
the above as long as said variant has an activity of cancer antigen
peptide.
[0149] Thus, the present invention provides a pharmaceutical
composition comprising any one of the following a)' to f)':
[0150] a)' a cancer antigen peptide comprising the amino acid
sequence set forth in SEQ ID NO: 4,
[0151] b)' an epitope peptide comprising the peptide of a)'
above,
[0152] c)' an expression vector containing a polynucleotide
encoding the peptide of a)' or b)' above,
[0153] d)' a cell containing the expression vector of c)'
above,
[0154] e)' an antigen-presenting cell on which a complex between
the peptide of a)' above and an HLA-A*0201 antigen is presented,
and
[0155] f)' a CTL which recognizes a complex between the peptide of
a)' above and an HLA-A*0201 antigen,
together with a pharmaceutically acceptable carrier.
[0156] The method for preparing or measuring activity of the
respective substances described in a)-f) and a)'-f)', and their use
as a CTL inducer or cancer vaccine are the same as those described
in regard to the peptide of the present invention which has
activity as an HLA-A26-bindng cancer antigen peptide. However, the
peptide set forth in SEQ ID NO: 3 or 4 is used in place the peptide
set forth in SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15 and that
HLA-A*0201 antigen is used in place of HLA-A26 antigen. The
HLA-A*0201 antigen is known in Genbank Accession No. M84379.
[0157] The present invention also provides an HLA monomer, an HLA
dimer, an HLA tetramer or an HLA pentamer comprising a peptide
having the amino acid sequence of Asp Leu Asn Ala Leu Leu Pro Ala
Val set forth in SEQ ID NO: 3, and an HLA-A*0201 antigen; and a
detecting agent for CTLs which are specific for WT 1-derived
HLA-A*0201-binding cancer antigen peptide comprising the same as an
ingredient. The description provided above in regard to a peptide
having activity as an HLA-A26-binding cancer antigen peptide is
applicable to these HLA monomer, HLA dimer, HLA tetramer and HLA
pentamer, and CTL-detecting agent. However, the peptide set forth
in SEQ ID NO: 3 or 4 is used in place of the peptide set forth in
SEQ ID NO: 2, 8, 9, 10, 11, 12, 13, 14 or 15 and that HLA-A*0201
antigen is used in place of HLA-A26 antigen.
EXAMPLES
[0158] The present invention is further illustrated by the
following examples, but is not limited by these examples in any
respect.
Example 1
Identification of HLA-A*0201-Binding Antigen Peptides
[0159] A peptide having an amino acid sequence set forth in SEQ ID
NO: 3 which corresponds to the one at position 7-15 of human WT1
amino acid sequence (Cell 60: 509, 1990, SEQ ID NO: 1) was
synthesized by solid-phase synthesis.
[0160] After obtaining informed-consent, blood was collected from
HLA-A*0201-positive healthy subjects and peripheral blood
mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque
separation solution. HLA-A*0201-positive T2 cell lines incapable of
presenting endogenous peptides to HLA due to deficit in TAP gene
(J. Immunol. 150: 1763, 1993) were pulsed with the peptide of SEQ
ID NO: 3 at 20 .mu.M for 2 hours, then radiated (7500 cGy), and
co-cultured with PBMCs at the cell number ratio of 1:1. As the
culture solution, a complete medium composed of 45% AIM-V medium,
45% RPMI1640 medium, 10% inactivated human serum, 0.1 mM MEM
non-essential amino acids, 100 ng/mL streptomycin, 100 IU/mL
penicillin and 25 ng/mL 2-mercaptoethanol was used. Seven days
later, the second-time stimulation was conducted in a similar
manner, and thereto was added 25 IU/mL of IL-2 (Shionogi & Co.,
Ltd) on the next day. Stimulation was conducted five times in
total, and effector cells obtained after 5 days from the last
stimulation were used in the measurement of cytotoxicity.
[0161] The cytotoxic activity of CTLs on target cells was measured
by 51 Cr-release assay. The .sup.51 Cr-labeled target cells
(1.times.10.sup.4 cells/100 .mu.L in total) were cocultured with
different numbers of effector cells (100 .mu.L) in a round-bottom
96-well plate. After culturing at 37.degree. C. for 3-5 hours, the
plate was centrifuged to separate the supernatant (100 .mu.L) which
was subjected to the measurement of .gamma. ray. Specific
cytotoxicity (% specific lysis) was calculated as follows:
% specific lysis=(cpm experimental release-cpm spontaneous
release)/(cpm maximal release-cpm spontaneous
release).times.100
[0162] The "spontaneous release" and the "maximal release" were
measured in the supernatant obtained from the culture of target
cells and that obtained from the culture of target cells treated
with 1% Triton X-100 solution, respectively. The significant
difference test was conducted by Student's t-test. The cytotoxic
activity was measured using, as target cells, T2 cells pulsed with
the same peptide as used in the stimulation or T2 cells not pulsed
with the peptide. The results are shown in FIG. 1. CTLs induced by
stimulation with T2 cells pulsed with the peptide of SEQ ID NO: 3
exerted more potent cytotoxic activity on T2 cells pulsed with the
peptide than on T2 cells not-pulsed with the peptide (p<0.05).
These results demonstrated that CTLs specifically recognize the
WT1-derived peptide of SEQ ID NO: 3 are induced from
HLA-A*0201-positive human PBMCs by stimulation with the peptide of
SEQ ID NO: 3.
[0163] Then, the cytotoxic activity of CTLs induced by the peptide
of SEQ ID NO: 3 on TF-1 and JY cell lines was examined, wherein the
TF-1 cell line is positive for HLA-A*0201 and expresses WT1 (J.
Cell. Physiol. 140: 323, 1989), while the JY cell line is positive
for HLA-A*0201 but does not express WT1 (J. Biol. Chem. 252, 1997).
The results are shown in FIG. 2. CTLs induced by stimulation with
the peptide of SEQ ID NO: 3 killed TF-1 cells and not JY cells
(p<0.05). These results demonstrated that a peptide of SEQ ID
NO: 3 is produced through the processing of intracellularly
expressed endogenous WT1 protein, presented as an antigen together
with an HLA-A*0201 molecule, and recognized by CTLs.
Example 2
Identification of Antigen Peptides Binding to HLA-A26 (1)
[0164] A peptide shown in SEQ ID NO: 2 which corresponds to the
position 368-376 of amino acid sequence of human WT1 (Cell, 60:509,
1990, SEQ ID NO: 1) was synthesized by solid state synthesis.
[0165] After obtaining informed consent, PBMCs were prepared from
HLA-A26-positive healthy subject in a similar manner to Example 1,
and stimulated by adding the peptide of SEQ ID NO: 2. One week
later, PBMCs were pulsed with the peptide of SEQ ID NO: 2, and used
as stimulators in the stimulation. The stimulation was conducted
every one week 4 times in total. One week later, B cells (B-LCL)
transformed with EB virus were pulsed with the peptide of SEQ ID
NO: 2, and used as stimulators in the stimulation. After 5 days
from the final stimulation, the cytotoxic activity was measured by
.sup.5 1 Cr-release assay in a similar manner to Example 1 using,
as target cells, B-LCL cells pulsed with the peptide of SEQ ID NO:
2 and B-LCL cells not-pulsed with the peptide. The results are
shown in FIG. 3. CTLs induced by stimulation with the peptide of
SEQ ID NO: 2 exerted more potent cytotoxic activity on B-LCL cells
pulsed with the peptide than on B-LCL cells not-pulsed with the
peptide. These results demonstrated that CTLs specifically
recognize the WT1-derived peptide of SEQ ID NO: 2 are induced from
HLA-A26-positive human PBMCs by stimulation with the peptide of SEQ
ID NO: 2.
[0166] The genotype of HLA-A26-positive healthy subject used herein
is known be HLA-A*2601.
Example 3
Identification of Antigen Peptides Binding to HLA-A26 (2)
[0167] Peptides shown in SEQ ID NOS: 8, 9 and 2 which correspond to
the position 152-160, 185-193 and 368-376, respectively, of amino
acid sequence of human WT1 (Cell, 60: 509, 1990, SEQ ID NO: 1) were
synthesized by solid state synthesis.
[0168] After obtaining informed consent, PBMCs were prepared from
HLA-A26-positive healthy subject in a similar manner to Example 1,
and stimulated by adding a peptide of SEQ ID NO: 8. One week later,
PBMCs were pulsed with the peptide of SEQ ID NO: 8, and used as
stimulators in the stimulation. The stimulation was conducted every
one week 3 times in total. After 3 times of stimulation,
CD8-positive cells were enriched by negative selection method.
Further, stimulation with the peptide of SEQ ID NO: 8 was conducted
2 times. After 5 days from the final stimulation, the cytotoxic
activity was measured by .sup.51 Cr-release assay in a similar
manner to Example 1 using, as target cells, B-LCL cells pulsed with
the peptide of SEQ ID NO: 8 and B-LCL cells not-pulsed with the
peptide. Experiments were carried out in the same manner as above
using a peptide of SEQ ID NO: 9 and a peptide of SEQ ID NO: 2. The
results are shown in FIG. 4. CTLs induced by stimulation with the
peptide of SEQ ID NO: 8, the peptide of SEQ ID NO: 9 or the peptide
of SEQ ID NO: 2 exerted more potent cytotoxic activity on B-LCL
cells pulsed with the peptide than on B-LCL cells not-pulsed with
the peptide. These results demonstrated that CTLs specifically
recognize the WT1-derived peptide of SEQ ID NO: 8, 9 and 2 are
induced from HLA-A26-positive human PBMCs by stimulation with the
peptide of SEQ ID NO: 8, 9 and 2, respectively.
INDUSTRIAL APPLICABILITY
[0169] Cancer antigen peptides derived from WT1, polynucleotides
encoding the same, CTL-inducers comprising the same, and the like
are provided by the present invention. The CTL inducer of the
present invention is useful as cancer vaccine. The cancer vaccine
of the present invention is applicable to many cancer patients who
are positive for HLA-A26 or HLA-A*0201.
Sequence Listing Free Text
[0170] The amino acid sequence set forth in SEQ ID NO: 2 is a
synthetic peptide. [0171] The amino acid sequence set forth in SEQ
ID NO: 3 is a synthetic peptide. [0172] In the amino acid sequence
set forth in SEQ ID NO: 4, the second amino acid is leucine,
methionine, valine, isoleucine or glutamine and the ninth amino
acid is valine or leucine. [0173] The amino acid sequence set forth
in SEQ ID NO: 5 is a synthetic peptide. [0174] The amino acid
sequence set forth in SEQ ID NO: 6 is a synthetic peptide. [0175]
The amino acid sequence set forth in SEQ ID NO: 7 is a synthetic
peptide. [0176] The amino acid sequence set forth in SEQ ID NO: 8
is a synthetic peptide. [0177] The amino acid sequence set forth in
SEQ ID NO: 9 is a synthetic peptide. [0178] The amino acid sequence
set forth in SEQ ID NO: 10 is a synthetic peptide. [0179] The amino
acid sequence set forth in SEQ ID NO: 11 is a synthetic peptide.
[0180] The amino acid sequence set forth in SEQ ID NO: 12 is a
synthetic peptide. [0181] The amino acid sequence set forth in SEQ
ID NO: 13 is a synthetic peptide. [0182] The amino acid sequence
set forth in SEQ ID NO: 14 is a synthetic peptide. [0183] The amino
acid sequence set forth in SEQ ID NO: 15 is a synthetic peptide.
Sequence CWU 1
1
151449PRTHomosapiens 1Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu
Leu Pro Ala Val Pro 1 5 10 15 Ser Leu Gly Gly Gly Gly Gly Cys Ala
Leu Pro Val Ser Gly Ala Ala 20 25 30 Gln Trp Ala Pro Val Leu Asp
Phe Ala Pro Pro Gly Ala Ser Ala Tyr 35 40 45 Gly Ser Leu Gly Gly
Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro 50 55 60 Pro Pro Pro
Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly 65 70 75 80 Ala
Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe 85 90
95 Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe
100 105 110 Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg
Met Phe 115 120 125 Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser
Gln Pro Ala Ile 130 135 140 Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe
Asp Gly Thr Pro Ser Tyr 145 150 155 160 Gly His Thr Pro Ser His His
Ala Ala Gln Phe Pro Asn His Ser Phe 165 170 175 Lys His Glu Asp Pro
Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln 180 185 190 Tyr Ser Val
Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser 195 200 205 Cys
Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp 210 215
220 Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln
225 230 235 240 Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly
Ser Ser Ser 245 250 255 Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His
Ser Thr Gly Tyr Glu 260 265 270 Ser Asp Asn His Thr Thr Pro Ile Leu
Cys Gly Ala Gln Tyr Arg Ile 275 280 285 His Thr His Gly Val Phe Arg
Gly Ile Gln Asp Val Arg Arg Val Pro 290 295 300 Gly Val Ala Pro Thr
Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys 305 310 315 320 Arg Pro
Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys 325 330 335
Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro 340
345 350 Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser
Asp 355 360 365 Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys
Pro Phe Gln 370 375 380 Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser
Asp His Leu Lys Thr 385 390 395 400 His Thr Arg Thr His Thr Gly Lys
Thr Ser Glu Lys Pro Phe Ser Cys 405 410 415 Arg Trp Pro Ser Cys Gln
Lys Lys Phe Ala Arg Ser Asp Glu Leu Val 420 425 430 Arg His His Asn
Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala 435 440 445 Leu
29PRTArtificialSynthetic peptide 2Asp Gln Leu Lys Arg His Gln Arg
Arg 1 5 39PRTArtificialSynthetic peptide 3Asp Leu Asn Ala Leu Leu
Pro Ala Val 1 5 49PRTArtificialSynthetic peptide 4Asp Xaa Asn Ala
Leu Leu Pro Ala Xaa 1 5 516PRTArtificialSynthetic peptide 5Lys Arg
Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His 1 5 10 15
621PRTArtificialSynthetic peptide 6Phe Asn Asn Phe Thr Val Ser Phe
Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala Ser His Leu Glu 20
716PRTArtificialSynthetic peptide 7Ala Gln Tyr Ile Lys Ala Asn Ser
Lys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 89PRTArtificialSynthetic
peptide 8Val Thr Phe Asp Gly Thr Pro Ser Tyr 1 5
99PRTArtificialSynthetic peptide 9Gln Gly Ser Leu Gly Glu Gln Gln
Tyr 1 5 109PRTArtificialSynthetic peptide 10Phe Ala Pro Pro Gly Ala
Ser Ala Tyr 1 5 119PRTArtificialSynthetic peptide 11Pro Ile Leu Cys
Gly Ala Gln Tyr Arg 1 5 1210PRTArtificialSynthetic peptide 12Gly
Val Phe Arg Gly Ile Gln Asp Val Arg 1 5 10
139PRTArtificialSynthetic peptide 13Cys Leu Glu Ser Gln Pro Ala Ile
Arg 1 5 149PRTArtificialSynthetic peptide 14Lys Arg Tyr Phe Lys Leu
Ser His Leu 1 5 159PRTArtificialSynthetic peptide 15Ala Leu Leu Pro
Ala Val Pro Ser Leu 1 5
* * * * *