DESCRIPTION
Immunity-inducing Agent
TECHNICAL FIELD
[0001]
The present invention relates to a novel immunity-inducing agent useful as a
therapeutic and/or prophylactic agent for cancer.
BACKGROUND ART
[0002]
Cancer is the commonest cause for death among all of the causes for death,
and therapies carried out therefor at present are mainly surgical treatment, which may
be carried out in combination with radiotherapy and/or chemotherapy. In spite of
the developments of new surgical methods and discovery of new anti-cancer agents
in recent years, treatment results of cancers have not been improved very much at
present except for some cancers. In recent years, by virtue of the development in
molecular biology and cancer immunology, cancer antigens recognized by cytotoxic
T cells reactive with cancers, as well as the genes encoding the cancer antigens, were
identified, and expectations for antigen-specific immunotherapies have been raised.
[0003]
In immunotherapy, in order to reduce side effects, it is necessary that the
peptide or protein to be recognized as the antigen exist hardly in normal cells and
exist specifically in cancer cells. In 1991, Boon et al. of Ludwig Institute in
Belgium isolated a human melanoma antigen MAGE 1, which is recognized by CD8-
positive T cells, by a cDNA-expression cloning method using an autologous cancer
cell line and cancer-reactive T cells (Non-patent Document 1). Thereafter, the
SEREX (serological identifications of antigens by recombinant expression cloning)
method, wherein tumor antigens recognized by antibodies produced in the living
body of a cancer patient in response to the cancer of the patient himself are identified
by application of a gene expression cloning method, was reported (Patent Document
1, Non-patent Document 2), and several cancer antigens have been isolated by this
method. Using a part of the cancer antigens as targets, clinical tests for cancer
immunotherapy have started.
[0004]
On the other hand, as in human, a number of tumors such as mammary gland
tumor and squamous cell carcinoma are known in dogs and cats, and they rank high
also in the statistics of diseases in dogs and cats. However, at present, no
therapeutic agent, prophylactic agent or diagnostic agent exists which is effective for
cancers in dogs and cats. Most of tumors in dogs and cats are realized by owners
only after they advanced to grow bigger, and in many cases, it is already too late to
visit a hospital to receive surgical excision of the tumor or administration of a human
drug (an anticancer drug or the like), so that those dogs and cats often die shortly
after the treatment. Under such circumstances, if therapeutic agents and
prophylactic agents for cancer effective for dogs and cats become available, their
uses for canine cancers are expected to be developed.
[0005]
PDS5A (PDS5, regulator of cohesion maintenance, homolog A) is a protein
also called SSC-112 which was identified as a cell cycle regulator involved in
distribution of chromosomes, and reported to show higher expression in
nasopharyngeal carcinoma, renal cancer, liver cancer and a certain type of breast
cancer cells, compared to normal tissues (Patent Document 2, Non-patent Documents
3 to 5). It has been reported that the growth of cancer cells can be suppressed by
suppressing expression of PDS5A in cancer cells using an antisense nucleic acid,
ribozyme or siRNA against the PDS5A gene or using an antibody that specifically
binds to the PDS5A protein, and that cancer cells can be induced to cause apoptosis
by administering the full-length PDS5A protein or a partial peptide of the PDS5A
protein (Patent Document 3). Further, in Patent Document 3, increase in the mRNA
level of the PDS5A protein in cancer cells was confirmed. However, there is no
report suggesting that the PDS5A protein and a partial peptide of the protein has an
action to induce immunity against cancer cells and hence the protein and a partial
peptide of the protein is useful for therapy or prophylaxis of cancer, and whether or
not the PDS5A protein has a function as a marker that can be used for diagnosis of
cancer has not been confirmed.
PRIOR ART DOCUMENTS
[0006]
Patent Documents
[Patent Document 1] US 5698396 B
[Patent Document 2] WO2006/109943
[Patent Document 3] WO2002/081641
[0007]
Non-patent Documents
[Non-patent Document 1] Science, 254: 1643-1647 (1991)
[Non-patent Document 2] Proc. Natl. Acad. Sci. USA, 92: 11810-11813
(1995)
[Non-patent Document 3] Gene. 17; 328: 187-96 (2004)
[Non-patent Document 4] J. Cell. Sci. 15; 118 (Pt 10): 2133-41 (2005)
[Non-patent Document 5] J. Cancer Res. Clin. Oncol.: 134(4):453-62 (2008)
SUMMARY OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0008]
The present invention aims to discover a novel polypeptide useful for a
therapeutic and/or prophylactic agent for cancer or useful for detection of cancer, to
provide the polypeptide for use in an immunity-inducing agent or in detection of
cancer
MEANS FOR SOLVING THE PROBLEMS
[0009]
By the SEREX method using a canine breast cancer-derived cDNA library
and serum obtained from a tumor-bearing dog, the present inventors intensively
studied to obtain a cDNA encoding a protein which binds to antibodies existing in
the serum derived from a tumor-bearing living body, and, based on the cDNA, the
canine PDS5 protein, a regulator of cohesion maintenance, homolog A (hereinafter
also referred to as PDS5A), having the amino acid sequence shown in SEQ ID NO:2
was prepared. Further, based on human and murine homologous genes of the
obtained gene, human PDS5A having the amino acid represented by SEQ ID NO:4 or
44 (SEQ ID NO:4 corresponds to a partial sequence of SEQ ID NO:44) and murine
PDS5A having the amino acid sequence shown in SEQ ID NO:6 were prepared.
The present inventors then discovered that that these PDS5A are specifically
expressed in tissues or cells of breast cancer, brain tumor, esophagus cancer, lung
cancer, renal cancer, colon cancer, perianal adenocarcinoma, neuroblastoma and
leukemia. Further, the present inventors discovered that, by administration of these
PDS5A to a living body, immunocytes against PDS5A can be induced in the living
body, and a tumor in the living body expressing PDS5A can be regressed. Further,
the present inventors discovered that a recombinant vector which can express a
polynucleotide encoding the full-length PDS5A protein or a fragment thereof can
induce an anti-tumor effect against cancer expressing PDS5A in the living body.
[0010]
Further, the present inventors discovered that a partial peptide of PDS5A has
a capacity to be presented by antigen-presenting cells, thereby allowing activation
and growth of cytotoxic T cells specific to the peptide (immunity-inducing activity),
and therefore that the peptide is useful for therapy and/or prophylaxis of cancer, and,
further, that antigen-presenting cells which have contacted with the peptide and T
cells which have contacted with the antigen-presenting cells are useful for therapy
and/or prophylaxis of cancer, thereby completing the present invention.
[0011]
Thus, the present invention has the following characteristics.
[0012]
(1) An immunity-inducing agent comprising as an effective ingredient(s) at least
one polypeptide selected from the polypeptides (a) to (c) below, the polypeptide(s)
having an immunity-inducing activity/activities, or as an effective ingredient(s) a
recombinant vector(s) which comprise(s) a polynucleotide(s) encoding the
polypeptide(s) and is/are capable of expressing the polypeptide(s) in vivo:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12
and 44 in SEQUENCE LISTING;
(b) a polypeptide having a sequence identity of not less than 90% with the
polypeptide (a) and consisting essentially of not less than 7 amino acids; and
(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence
thereof.
[0013]
(2) The immunity-inducing agent according to (1), wherein the polypeptide (b)
has a sequence identity of not less than 95% with the polypeptide (a).
[0014]
(3) The immunity-inducing agent according to (1), wherein each of the
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
consisting essentially of not less than 7 consecutive amino acids in any one of the
amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44, or a
polypeptide comprising the polypeptide as a partial sequence thereof; or a
polypeptide having the same amino acid sequence as a polypeptide consisting
essentially of not less than 7 consecutive amino acids in any one of the amino acid
sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 except that one or several
amino acids are deleted, substituted and/or added, or a polypeptide comprising the
polypeptide as a partial sequence thereof.
[0015]
(4) The immunity-inducing agent according to (3), wherein each of the
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
having any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8,10, 12
and 44 in SEQUENCE LISTING.
[0016]
(5) The immunity-inducing agent according to (3), wherein each of the
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
consisting essentially of not less than 7 consecutive amino acids in the region of
aal11-140, aa211-240, aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022,
aa994-1057 or aal018-1080 in any one of the amino acid sequences shown in SEQ
ID NOs:2, 6, 8, 10, 12 and 44 in SEQUENCE LISTING, or a polypeptide comprising
the polypeptide as a partial sequence thereof; or a polypeptide having the same amino
acid sequence as a polypeptide consisting essentially of not less than 7 consecutive
amino acids in the region of aal 11-140, aa211-240, aa248-278, aa327-357, aa459-
522, aa909-972, aa959-1022, aa994-1057 or aal018-1080 in any one of the amino
acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44 in SEQUENCE
LISTING except that one or several amino acids are deleted, substituted and/or added,
or a polypeptide comprising the polypeptide as a partial sequence thereof.
[0017]
(6) The immunity-inducing agent according to (5), wherein each of the
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
having any one of the amino acid sequences shown in SEQ ID NOs:27 to 35 in
SEQUENCE LISTING, or a polypeptide comprising the polypeptide as a partial
sequence thereof and having 10 to 12 amino acid residues; or a polypeptide having
the same amino acid sequence as a polypeptide having any one of the amino acid
sequences shown in SEQ ID NOs:27 to 35 in SEQUENCE LISTING except that one
or several amino acids are deleted, substituted and/or added, or a polypeptide
comprising the polypeptide as a partial sequence thereof and having 10 to 12 amino
acid residues.
[0018]
(7) The immunity-inducing agent according to any one of (1) to (6), for
prophylaxis of a cancer in an animal.
[0019]
(8) The immunity-inducing agent according to (5) or (6), for therapy of a cancer
in an animal.
[0020]
(9) The immunity-inducing agent according to (7) or (8), wherein the cancer is a
cancer expressing PDS5A.
[0021]
(10) The immunity-inducing agent according to any one of (7) to (9), wherein the
cancer is breast cancer, brain tumor, esophagus cancer, lung cancer, renal cancer,
colon cancer, perianal adenocarcinoma, neuroblastoma or leukemia.
[0022]
(11) The immunity-inducing agent according to any one of (1) to (10), further
comprising an immunoenhancer.
[0023]
(12) An isolated antigen-presenting cell comprising a complex between the
polypeptide having an immunity-inducing activity and an MHC molecule.
[0024]
(13) An isolated T cell which selectively binds to a complex between the
polypeptide having an immunity-inducing activity and an MHC molecule.
[0025]
(14) A polypeptide having any one of the amino acid sequences shown in SEQ ID
NOs:27 to 35 in SEQUENCE LISTING, or a polypeptide comprising the polypeptide
as a partial sequence thereof and having 10 to 12 amino acid residues; or a
polypeptide having the same amino acid sequence as a polypeptide having any one of
the amino acid sequences shown in SEQ ID NOs:27 to 35 in SEQUENCE LISTING
except that one or several amino acids are deleted, substituted and/or added, or a
polypeptide comprising the polypeptide as a partial sequence thereof and having 10
to 12 amino acid residues, which polypeptide has an immunity-inducing activity.
[0026]
(15) A method for detecting a cancer, the method comprising measurement of
expression of a polypeptide having any one of the amino acid sequences shown in
SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING or a polypeptide
having a sequence identity of not less than 90% with the polypeptide, in a sample
separated from a living body.
[0027]
(16) A method for inducing immunity, the method comprising administering to an
individual at least one polypeptide selected from the polypeptides (a) to (c) below,
the polypeptide(s) having an immunity-inducing activity/activities, or a recombinant
vector(s) which comprise(s) a polynucleotide(s) encoding the polypeptide(s) and
is/are capable of expressing the polypeptide(s) in vivo:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12
and 44 in SEQUENCE LISTING;
(b) a polypeptide having a sequence identity of not less than 90% with the
polypeptide (a) and consisting essentially of not less than 7 amino acids; and
(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence
thereof.
EFFECT OF THE INVENTION
[0028]
By the present invention, a novel immunity-inducing agent useful for therapy
and/or prophylaxis and/or the like of cancer is provided. As particularly described
in later-mentioned Examples, by administering the polypeptide used in the present
invention to a living body, immunocytes can be induced in the living body, and a
cancer which has already occurred can be reduced or regressed. Therefore, the
polypeptide is useful for therapy and/or prophylaxis of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029]
Fig. 1 shows the expression patterns of the identified PDS5A gene in canine
normal tissues, tumor tissues and tumor cell lines. Reference numeral 1, the
expression patterns of the canine PDS5A gene in various canine tissues and cell
lines; reference numeral 2, the expression patterns of the canine GAPDH gene in
various canine tissues and cell lines.
Fig. 2 shows the expression patterns of the identified PDS5A gene in human
normal tissues, tumor tissues and tumor cell lines. Reference numeral 3, the
expression patterns of the human PDS5A gene in various human tissues and cell
lines; reference numeral 4, the expression patterns of the human GAPDH gene in
various human tissues and cell lines.
Fig. 3 shows the expression patterns of the identified PDS5A gene in murine
normal tissues, tumor tissues and tumor cell lines. Reference numeral 5, the
expression patterns of the murine PDS5A gene in various murine tissues and cell
lines; reference numeral 6, the expression patterns of the murine GAPDH gene in
various murine tissues and cell lines.
Fig. 4 is a graph showing that an anti-tumor effect (therapeutic model:
neuroblastoma cell line) was observed by administration of PDS5A. Immunization
was carried out with a vector alone or a plasmid encoding PDS5A using a gene gun,
and the evaluation was carried out based on the area of the cancerous part and the
ratio of living mice. For each group, 10 individuals of mice were used. The mice
were observed twice a week. The data are represented by the mean value ± SD.
Reference numeral 7, the group wherein a plasmid vector was administered;
reference numeral 8, the group wherein a plasmid encoding PDS5A was administered.
Fig. 5 is a graph showing that an anti-tumor effect (prophylactic model:
neuroblastoma cell line) was observed by administration of PDS5A. Immunization
was carried out with a vector alone or a plasmid encoding PDS5A using a gene gun,
and the evaluation was carried out based on the area of the cancerous part and the
ratio of living mice. For each group, 10 individuals of mice were used. The mice
were observed twice a week. The data are represented by the mean value ± SD.
Reference numeral 9, the group wherein a plasmid vector was administered;
reference numeral 10, the group wherein a plasmid encoding PDS5A was
administered.
Fig. 6 shows the ratio of living mice in the experiment in Fig. 4. Reference
numeral 11, the group wherein a plasmid vector was administered; reference numeral
12, the group wherein a plasmid encoding PDS5A was administered.
Fig. 7 shows the ratio of living mice in the experiment in Fig. 5. Reference
numeral 13, the group wherein a plasmid vector was administered; reference numeral
14, the group wherein a plasmid encoding PDS5A was administered.
Fig. 8 is a graph showing that an anti-tumor effect (therapeutic model: colon
cancer cell line) was observed by administration of PDS5 A. Immunization was
carried out with a vector alone or a plasmid encoding PDS5A using a gene gun, and
the evaluation was carried out based on the area of the cancerous part and the ratio of
living mice. For each group, 10 individuals of mice were used. The mice were
observed twice a week. The data are represented by the mean value ± SD.
Reference numeral 15, the group wherein a plasmid vector was administered;
reference numeral 16, the group wherein a plasmid encoding PDS5A was
administered.
Fig. 9 is a graph showing that an anti-tumor effect (prophylactic model: colon
cancer cell line) was observed by administration of PDS5A. Immunization was
carried out with a vector alone or a plasmid encoding PDS5A using a gene gun, and
the evaluation was carried out based on the area of the cancerous part and the ratio of
living mice. For each group, 10 individuals of mice were used. The mice were
observed twice a week. The data are represented by the mean value ± SD.
Reference numeral 17, the group wherein a plasmid vector was administered;
reference numeral 18, the group wherein a plasmid encoding PDS5 A was
administered.
Fig. 10 shows the ratio of living mice in the experiment in Fig. 8. Reference
numeral 19, the group to which a plasmid vector was administered; reference
numeral 20, the group to which a plasmid encoding PDS5A was administered.
Fig. 11 shows the ratio of living mice in the experiment in Fig. 9. Reference
numeral 21, the group to which a plasmid vector was administered; reference
numeral 22, the group to which a plasmid encoding PDS5A was administered.
Fig. 12 is a diagram showing that CD8-positive T cells specific to each of the
polypeptides having the amino acid sequences shown in SEQ ID NOs:27 to 35 in
SEQUENCE LISTING recognize the complex between the polypeptide and HLA-
A0201, and produce IFN-y. In Fig. 12, the reference numerals 25 to 33 along the
abscissa indicate the abilities of HLA-A0201-positive CD8-positive T cells to
produce IFN-y in response to stimulation by T2 cells pulsed with the respective
peptides of SEQ ID NOs:27 to 35. The reference numeral 23 shows a result
obtained when the above treatment was carried out without addition of a polypeptide,
and the reference numeral 24 shows a result obtained when the above treatment was
carried out with addition of the polypeptide shown in SEQ ID NO:36, which is
outside the scope of the present invention.
Fig. 13 is a diagram showing the cytotoxic activities, against cancer cells, of
CD8-positive T cells specific to each of the polypeptides having the amino acid
sequences shown in SEQ ID NOs:27 to 35 in SEQUENCE LISTING. In Fig. 13,
the reference numerals 36 to 44 along the abscissa indicate the cytotoxic activities,
against T98G cells, of HLA-A0201-positive CD8-positive T cells stimulated with the
respective peptides of SEQ ID NOs:27 to 35. The reference numeral 34 shows the
cytotoxic activity of CD8-positive T cells induced without addition of a polypeptide,
and the reference numeral 35 shows the cytotoxic activity of CD8-positive T cells
induced using a negative control peptide (SEQ ID NO:36).
BEST MODE FOR CARRYING OUT THE INVENTION
[0030]
Examples of the polypeptide contained in the immunity-inducing agent of the
present invention as an effective ingredient include the followings. In the present
invention, the term "polypeptide" means a molecule formed by a plurality of amino
acids linked together by peptide bonds, and includes not only polypeptide molecules
having large numbers of amino acids constituting them, but also low-molecular-
weight molecules having small numbers of amino acids (oligopeptides), and full-
length proteins. In the present invention, the full-length PDS5A proteins having the
amino acid sequences shown in SEQ ID NO:2, 4, 6, 8, 10, 12 and 44 are also
included therein.
[0031]
(a) A polypeptide which consists essentially of not less than 7 consecutive
amino acids in a polypeptide having the amino acid sequence shown in SEQ ID NO:2,
4, 6, 8, 10, 12 or 44 in SEQUENCE LISTING, and has an immunity-inducing
activity.
(b) a polypeptide which has a sequence identity of not less than 90% with the
polypeptide (a), consists essentially of not less than 7 amino acids, and has an
immunity-inducing activity.
(c) a polypeptide which comprises the polypeptide (a) or (b) as a partial
sequence thereof, and has an immunity-inducing activity.
[0032]
In the present invention, the term "having an amino acid sequence" means
that amino acid residues are arrayed in such an order. Therefore, for example,
"polypeptide having the amino acid sequence shown in SEQ ID NO:2" means the
polypeptide having the amino acid sequence of Met Asp Phe Thr ... (snip) ... Asp
Leu Gin Arg shown in SEQ ID NO:2, which polypeptide has a size of 1337 amino
acid residues. Further, for example, "polypeptide having the amino acid sequence
shown in SEQ ID NO:2" may be abbreviated as "polypeptide of SEQ ID NO:2".
This also applies to the term "having a base sequence". In this case, the term
"having" may be replaced with the expression "essentially consisting of.
[0033]
As used herein, the term "immunity-inducing activity" means an ability to
induce immunocytes which secrete cytokines such as interferon in a living body.
[0034]
Whether or not the polypeptide has an immunity-inducing activity can be
confirmed using, for example, the known ELISPOT assay. More particularly, for
example, as described in the Examples below, cells such as peripheral blood
mononuclear cells are obtained from a living body to which a polypeptide whose
immunity-inducing activity is to be evaluated was administered, which cells are then
cocultured with the polypeptide, followed by measuring the amount(s) of a
cytokine(s) produced by the cells using a specific antibody/antibodies, thereby
measuring the number of immunocytes in the cells, which enables evaluation of the
immunity-inducing activity.
[0035]
Alternatively, as described in the later-mentioned Examples, when a
recombinant polypeptide in any of (a) to (c) described above is administered to a
tumor-bearing animal, the tumor can be regressed by its immunity-inducing activity.
Thus, the above immunity-inducing activity can be evaluated also as an ability to
suppress the growth of cancer cells or to cause reduction or disappearance of a cancer
tissue (tumor) (hereinafter referred to as "anti-tumor activity"). The anti-tumor
activity of a polypeptide can be confirmed by, for example, as more particularly
described in the Examples below, observation of whether or not a tumor is reduced
when the polypeptide was actually administered to a tumor-bearing living body.
[0036]
Alternatively, the anti-tumor activity of a polypeptide can be evaluated also
by observation of whether or not T cells stimulated with the polypeptide (that is, T
cells brought into contact with antigen-presenting cells presenting the polypeptide)
show a cytotoxic activity against tumor cells in vitro. The contact between the T
cells and the antigen-presenting cells can be carried out by coculture of the both in a
liquid medium, as mentioned below. Measurement of the cytotoxic activity can be
carried out by, for example, the known method called 5lCr release assay described in
Int. J. Cancer, 58: p 317, 1994. In cases where the polypeptide is to be used for
therapy and/or prophylaxis of cancer, the evaluation of the immunity-inducing
activity is preferably carried out using the anti-tumor activity as an index, although
the index is not restricted.
[0037]
The amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in
SEQUENCE LISTING are the amino acid sequences of the PDS5A proteins which
were isolated, by the SEREX method using a canine testis-derived cDNA library and
serum of a tumor-bearing dog, as a polypeptide that specifically binds to an antibody
existing in the serum of the tumor-bearing dog and homologous factors of the
polypeptide in human (SEQ ID NOs:4 and 44), mouse (SEQ ID NO:6), cow (SEQ ID
NO:8), horse (SEQ ID NO:10) and chicken (SEQ ID NO:12) (see Example 1).
Human PDS5A, which is a human homologous factor of canine PDS5A, has a
sequence identity of 94% in terms of the base sequence and 99% in terms of the
amino acid sequence; murine PDS5A, which is a murine homologous factor, has a
sequence identity of 91% in terms of the base sequence and 99% in terms of the
amino acid sequence; bovine PDS5A, which is a bovine homologous factor, has a
sequence identity of 95% in terms of the base sequence and 99% in terms of the
amino acid sequence; equine PDS5A, which is an equine homologous factor, has a
sequence identity of 96% in terms of the base sequence and 99% in terms of the
amino acid sequence; and chicken PDS5A, which is a chicken homologous factor,
has a sequence identity of 83% in terms of the base sequence and 98% in terms of the
amino acid sequence.
[0038]
The polypeptide (a) is a polypeptide which consists essentially of not less than
7 consecutive, preferably 8, 9 or not less than 10 consecutive amino acids in the
polypeptide having the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12
or 44, and has an immunity-inducing activity. The polypeptide especially preferably
has the amino acid sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44. As is
known in the art, a polypeptide having not less than about 7 amino acid residues can
exert its antigenicity and immunogenicity. Thus, a polypeptide having not less than
7 consecutive amino acid residues in the amino acid sequence shown in SEQ ID
NO:2, 4, 6, 8, 10, 12 or 44 can have an immunity-inducing activity, so that it can be
used for preparation of the immunity-inducing agent of the present invention.
[0039]
As a principle of immune induction by administration of a cancer antigenic
polypeptide, the following process is known: a polypeptide is incorporated into an
antigen-presenting cell and then degraded into smaller fragments by peptidases in the
cell, followed by presentation of the fragments on the surface of the cell. The
fragments are then recognized by a cytotoxic T cell or the like, which selectively kills
cells presenting the antigen. The size of the polypeptide presented on the surface of
the antigen-presenting cell is relatively small and about 7 to 30 amino acids.
Therefore, from the viewpoint of presenting thereof on the surface of the antigen-
presenting cell, one preferred mode of the above-described polypeptide (a) is a
polypeptide composed of about 7 to 30 consecutive amino acids in the amino acid
sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44, and more preferably, a
polypeptide composed of about 8 to 30 or about 9 to 30 amino acids is sufficient as
the polypeptide (a). In some cases, these relatively small polypeptides are presented
directly on the surface of the antigen-presenting cell without being incorporated into
the antigen-presenting cells.
[0040]
Further, since a polypeptide incorporated into an antigen-presenting cell is
cleaved at random sites by peptidases in the cell to yield various polypeptide
fragments, which are then presented on the surface of the antigen-presenting cell,
administration of a large polypeptide such as the full-length region of SEQ ID NO:2,
4, 6, 8, 10, 12 or 44 inevitably causes production of polypeptide fragments by
degradation thereof in the antigen-presenting cell, which fragments are effective for
immune induction via the antigen-presenting cell. Therefore, also for immune
induction via antigen-presenting cells, a large polypeptide can be preferably used, and
the polypeptide may be composed of not less than 30, preferably not less than 100,
more preferably not less than 200, still more preferably not less than 250 amino acids.
The polypeptide may be still more preferably composed of the full-length region of
SEQ ID NO:2, 4, 6, 8, 10, 12 or 44.
[0041]
Further, the polypeptides of the present invention can be checked with a
checking medium by which epitope peptides having binding motifs of various types
of HLA and consisting essentially of 8 to 12, preferably 9 to 10 amino acids can be
searched, for example, HLA Peptide Binding Predictions
(http://bimas.dcrt.nih.gov/molbio/hla_bind/index.html) in Bioinformatics &
Molecular Analysis Selection (BIMAS), to screen peptides which may be epitope
peptides. More particularly, a polypeptide consisting essentially of not less than 7
consecutive amino acids in the region of amino acid residue positions aal 11-140,
aa211-240, aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022,aa994-1057
or aal 018-1080 in the amino acid sequence shown in SEQ ID NO:2, 6, 8, 10, 12 or
44 is preferred, and, in the polypeptide of SEQ ID NO:4 or 44, the polypeptide
shown in any of SEQ ID NOs:27 to 35, or a polypeptide which comprises a
polypeptide having the amino acid sequence shown in any of SEQ ID NOs:27 to 35
as a partial sequence and has 10 to 12 amino acid residues is more preferred.
[0042]
The polypeptide (b) is the same polypeptide as the polypeptide (a) except that
a small number of (preferably, one or several) amino acid residues are substituted,
deleted and/or inserted, which has a sequence identity of not less than 90%,
preferably not less than 95%, more preferably not less than 98%, still more preferably
not less than 99% or not less than 99.5% to the original sequence and has an
immunity-inducing activity. It is well known in the art that, in general, there are
cases where a protein antigen retains almost the same antigenicity as the original
protein even if the amino acid sequence of the protein is modified such that a small
number of amino acids are substituted, deleted and/or inserted. Therefore, since the
polypeptide (b) may also exert an immunity-inducing activity, it can be used for
preparation of the immunity-inducing agent of the present invention. Further, the
polypeptide (b) is also preferably the same polypeptide as one having the amino acid
sequence shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 except that one or several
amino acid residues are substituted, deleted and/or inserted. As used herein, the
term "several" means an integer of 2 to 10, preferably an integer of 2 to 6, more
preferably an integer of 2 to 4.
[0043]
As used herein, the term "sequence identity" of amino acid sequences or base
sequences means the value calculated by aligning two amino acid sequences (or base
sequences) to be compared such that the number of matched amino acid residues (or
bases) is maximum between the amino acid sequences (or base sequences), and
dividing the number of matched amino acid residues (or the number of matched
bases) by the total number of amino acid residues (or the total number of bases),
which value is represented as a percentage. When the alignment is carried out, a
gap(s) is/are inserted into one or both of the two sequences to be compared as
required. Such alignment of sequences can be carried out using a well-known
program such as BLAST, FASTA or CLUSTAL W. When a gap(s) is/are inserted,
the above-described total number of amino acid residues is the number of residues
calculated by counting one gap as one amino acid residue. When the thus counted
total number of amino acid residues is different between the two sequences to be
compared, the sequence identity (%) is calculated by dividing the number of matched
amino acid residues by the total number of amino acid residues in the longer
sequence.
[0044]
The 20 types of amino acids constituting naturally occurring proteins may be
classified into groups in each of which similar properties are shared, for example,
into neutral amino acids with side chains having low polarity (Gly, He, Val, Leu, Ala,
Met, Pro), neutral amino acids having hydrophilic side chains (Asn, Gin, Thr, Ser,
Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His) and
aromatic amino acids (Phe, Tyr, Trp). It is known that, in most cases, substitutions
of amino acids within the same group do not change the properties of the polypeptide.
Therefore, in cases where an amino acid residue(s) in the polypeptide (a) of the
present invention is/are substituted, the probability that the immunity-inducing
activity can be maintained may be increased by introducing the substitution(s) within
the same group, which is preferred.
[0045]
As the polypeptide (b), which corresponds to the above-described epitope
peptide, a polypeptide which is the same as the polypeptide consisting essentially of
not less than 7 consecutive amino acids in the region of aal 11-140, aa211-240,
aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022, aa994-1057 or aal018-
1080 in any one of the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12
and 44 except that one or several amino acids are deleted, substituted and/or added,
or a polypeptide comprising the polypeptide as a partial sequence thereof and having
an immunity-inducing activity is preferred, and, in the polypeptide of SEQ ID NO:4
or 44, a polypeptide which is the same as the polypeptide having the amino acid
sequence shown in any of SEQ ID NOs:27 to 35 except that one or several amino
acids are deleted, substituted and/or added, or a polypeptide comprising the
polypeptide as a partial sequence and having 10 to 12 amino acid residues is more
preferred.
[0046]
The polypeptide (c) comprises the polypeptide (a) or (b) as a partial sequence
and has an immunity-inducing activity. That is, the polypeptide (c) has
another/other amino acid(s) or polypeptide(s) added at one or both ends of the
polypeptide (a) or (b), and has an immunity-inducing activity. Such a polypeptide
can also be used for preparation of the immunity-inducing agent of the present
invention.
[0047]
As the polypeptide (c), which corresponds to the above-described epitope, a
polypeptide comprising as a partial sequence the polypeptide consisting essentially of
not less than 7 consecutive amino acids in the region of aal 11-140, aa211-240,
aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022, aa994-1057 or aal018-
1080 in any one of the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12
and 44 is preferred, and, in the polypeptide of SEQ ID NO:4 or 44, a polypeptide
comprising as a partial sequence: a polypeptide which is the same as the polypeptide
having the amino acid sequence shown in any of SEQ ID NOs:27 to 35 except that
one or several amino acids are deleted, substituted and/or added; or a polypeptide
comprising the polypeptide as a partial sequence and having 10 to 12 amino acid
residues; is more preferred.
[0048]
The above-described polypeptides can be synthesized by, for example, a
chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl
method) or the tBoc method (t-butyloxycarbonyl method). Further, they can be
synthesized by conventional methods using various types of commercially available
peptide synthesizers. Further, the polypeptide of interest can be obtained using
known genetic engineering techniques, by preparing a polynucleotide encoding the
above polypeptide and incorporating the polynucleotide into an expression vector,
which is then introduced into a host cell, followed by allowing the polypeptide to be
produced in the host cell.
[0049]
The polynucleotide encoding the above polypeptide can be easily prepared by
a known genetic engineering technique or a conventional method using a
commercially available nucleic acid synthesizer. For example, DNA having the
base sequence shown in SEQ ID NO: 1 can be prepared by carrying out PCR using a
canine chromosomal DNA or cDNA library as a template, and a pair of primers
designed such that the base sequence shown in SEQ ID NO:l can be amplified
therewith. DNA having the base sequence of SEQ ID NO:3 or 43 can be similarly
prepared by using a human chromosomal DNA or cDNA library as the template.
The reaction conditions for the PCR can be set appropriately, and examples thereof
include, but are not limited to, repeating the reaction process of 94°C for 30 seconds
(denaturation), 55°C for 30 seconds to 1 minute (annealing) and 72°C for 2 minutes
(extension) for, for example, 30 cycles, followed by the reaction at 72°C for 7
minutes. Further, the desired DNA can be isolated by preparing an appropriate
probe(s) or primer(s) based on the information of the base sequences and the amino
acid sequences shown in SEQ ID NO:l, 3, 5, 7, 9, 11 and 43 in SEQUENCE
LISTING in the present specification, and screening a cDN A library of dog, human
or the like using the probe(s) or primer(s). The cDNA library is preferably prepared
from a cell, organ or tissue expressing the protein of SEQ ID NO:2, 4, 6, 8, 10, 12 or
44. The above-described operations such as preparation of a probe(s) or primer(s),
construction of a cDNA library, screening of a cDNA library and cloning of a gene of
interest are known to those skilled in the art, and can be carried out according to the
methods described in Molecular Cloning, Second Edition; Current Protocols in
Molecular Biology; and/or the like. From the thus obtained DNA, DNA encoding
the polypeptide (a) can be obtained. Further, since the codons encoding each amino
acid are known, the base sequence of a polynucleotide encoding a specific amino
acid sequence can be easily specified. Therefore, since the base sequence of a
polynucleotide encoding the polypeptide (b) or polypeptide (c) can also be easily
specified, such a polynucleotide can also be easily synthesized using a commercially
available nucleic acid synthesizer according to a conventional method.
[0050]
The host cells are not restricted as long as those can express the above-
described polypeptide, and examples thereof include, but are not limited to,
prokaryotic cells such as E. coli; and eukaryotic cells such as mammalian cultured
cells including monkey kidney cells COS1 and Chinese hamster ovary cells CHO;
budding yeast; fission yeast; silkworm cells; and Xenopus laevis egg cells.
[0051]
In cases where prokaryotic cells are used as the host cells, an expression
vector in which an origin that enables replication of the vector in a prokaryotic cell,
promoter, ribosome binding site, DNA cloning site, terminator and/or the like is/are
contained is used. Examples of the expression vector for E. coli include the pUC
system, pBluescriptll, pET expression system and pGEX expression system. By
incorporating a DNA encoding the above polypeptide into such an expression vector
and transforming prokaryotic host cells with the vector, followed by culturing the
resulting transformants, the polypeptide encoded by the DNA can be expressed in the
prokaryotic host cells. In this process, the polypeptide can also be expressed as a
fusion protein with another protein.
[0052]
In cases where eukaryotic cells are used as the host cells, an expression vector
for eukaryotic cells having a promoter, splicing site, poly(A) addition site and/or the
like is used as the expression vector. Examples of such an expression vector
include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector,
pRS, pcDNA3, pMSG and pYES2. In the same manner as described above, by
incorporating a DNA encoding the above polypeptide into such an expression vector
and transforming eukaryotic host cells with the vector, followed by culturing the
resulting transformants, the polypeptide encoded by the DNA can be expressed in the
eukaryotic host cells. In cases where pIND/V5-His, pFLAG-CMV-2, pEGFP-Nl,
pEGFP-Cl or the like is used as the expression vector, the above polypeptide can be
expressed as a fusion protein wherein a tag such as a His tag, FLAG tag, myc tag, HA
tag or GFP was added.
[0053]
For the introduction of the expression vector into the host cells, a well-known
method such as electroporation, the calcium phosphate method, the liposome method
or the DEAE dextran method may be used.
[0054]
Isolation and purification of the polypeptide of interest from the host cells can
be carried out by a combination of known separation operations. Examples of the
known separation operations include, but are not limited to, treatment with a
denaturant such as urea or with a surfactant; ultrasonication treatment; enzyme
digestion; salting-out or solvent fractional precipitation; dialysis; centrifugation;
ultrafiltration; gel filtration; SDS-PAGE; isoelectric focusing; ion-exchange
chromatography; hydrophobic chromatography; affinity chromatography; and
reversed-phase chromatography.
[0055]
The polypeptides obtained by the above method also include, as mentioned
above, those in the form of a fusion protein with another arbitrary protein.
Examples thereof include fusion proteins with glutathion S-transferase (GST) and
with a His tag. Such a polypeptide in the form of a fusion protein is also included
within the scope of the present invention as the above-described polypeptide (c).
Further, in some cases, a polypeptide expressed in a transformed cell is modified in
various ways in the cell after translation. Such a post-translationally modified
polypeptide is also included within the scope of the present invention as long as it has
an immunity-inducing activity. Examples of such a post-translational modification
include: elimination of N-terminal methionine; N-terminal acetylation; glycosylation;
limited degradation by an intracellular protease; myristoylation; isoprenylation; and
phosphorylation.
[0056]
As described more particularly in the later-mentioned Examples, by
administration of the polypeptide having an immunity-inducing activity or an
expression vector comprising the gene encoding the polypeptide to a tumor-bearing
living body, an already existing tumor can be regressed. Further, by administration
of the polypeptide having an immunity-inducing activity or the gene encoding the
polypeptide to a living body before occurrence of cancer, development of a tumor can
be prevented. Therefore, the immunity-inducing agent of the present invention can
be used as a therapeutic and/or prophylactic agent for cancer. Further, the
polypeptide having an immunity-inducing activity can be used for a method of
therapy and/or prophylaxis of cancer by immune induction.
[0057]
As used herein, the terms "tumor" and "cancer" mean a malignant neoplasm,
and are used interchangeably
[0058]
In this case, the cancer to be treated is not restricted as long as PDS5A is
expressed in the cancer, and the cancer is preferably breast cancer, brain tumor,
esophagus cancer, lung cancer, renal cancer, colon cancer, perianal adenocarcinoma,
neuroblastoma or leukemia.
[0059]
The subject animal is preferably a mammal, more preferably a mammal such
as a primate, pet animal, domestic animal or sport animal, especially preferably
human, dog or cat.
[0060]
The administration route of the immunity-inducing agent of the present
invention to a living body may be either oral administration or parenteral
administration, and is preferably parenteral administration such as intramuscular
administration, subcutaneous administration, intravenous administration or
intraarterial administration. In cases where the immunity-inducing agent is used for
therapy of cancer, it may be administered to a regional lymph node in the vicinity of
the tumor to be treated, as described in the Examples below, in order to enhance its
anticancer activity. The dose may be any dose as long as the dose is effective for
immune induction, and, for example, in cases where the agent is used for therapy
and/or prophylaxis of cancer, the dose may be one effective for therapy and/or
prophylaxis of the cancer. The dose effective for therapy and/or prophylaxis of
cancer is appropriately selected depending on the size and symptoms of the tumor
and the like, and the effective dose is usually, 0.0001 ug to 1000 jag, preferably 0.001
ug to 1000 ug per subject animal per day, which may be administered once or in
several times. The agent is preferably administered in several times, every several
days to several months. As concretely shown in the Examples below, the immunity-
inducing agent of the present invention can cause regression of an already occurred
tumor. Therefore, since the agent can exert its anticancer activity also against a
small number of cancer cells at an early stage, development or recurrence of cancer
can be prevented by using the agent before development of the cancer or after therapy
for the cancer. That is, the immunity-inducing agent of the present invention is
effective for both therapy and prophylaxis of cancer.
[0061]
The immunity-inducing agent of the present invention may contain only a
polypeptide or may be formulated by being mixed as appropriate with an additive
such as a pharmaceutically acceptable carrier, diluent or vehicle suitable for each
administration mode. Formulation methods and additives which may be used are
well-known in the field of formulation of pharmaceuticals, and any of the methods
and additives may be used. Specific examples of the additives include, but are not
limited to, diluents such as physiological buffer solutions; vehicles such as sugar,
lactose, corn starch, calcium phosphate, sorbitol and glycine; binders such as syrup,
gelatin, gum arabic, sorbitol, polyvinyl chloride and tragacanth; and lubricants such
as magnesium stearate, polyethylene glycol, talc and silica. Examples of the
formulation include oral preparations such as tablets, capsules, granules, powders and
syrups; and parenteral preparations such as inhalants, injection solutions,
suppositories and solutions. These formulations may be prepared by commonly
known production methods.
[0062]
The immunity-inducing agent of the present invention may be used in
combination with an immunoenhancer capable of enhancing the immune response in
a living body. The immunoenhancer may be contained in the immunity-inducing
agent of the present invention or administered as a separate composition to a patient
in combination with the immunity-inducing agent of the present invention.
[0063]
Examples of the immunoenhancer include adjuvants. Adjuvants can
enhance the immune response by providing a reservoir of antigen (extracellularly or
within macrophages), activating macrophages and stimulating specific sets of
lymphocytes, thereby enhancing the immune response and hence the anticancer
action. Therefore, especially in cases where the immunity-inducing agent of the
present invention is used for therapy and/or prophylaxis of cancer, the immunity-
inducing agent preferably comprises an adjuvant, in addition to the above-described
polypeptide as an effective ingredient. Many types of adjuvants are well-known in
the art, and any of these adjuvants may be used. Specific examples of the adjuvants
include MPL (SmithKline Beecham), homologues of Salmonella minnesota Re 595
lipopolysaccharide obtained after purification and acid hydrolysis of the
lipopolysaccharide; QS21 (SmithKline Beecham), pure QA-21 saponin purified from
an extract of Quillja saponaria; DQS21 described in PCT application W096/33739
(SmithKline Beecham); QS-7, QS-17, QS-18 and QS-L1 (So and 10 colleagues,
"Molecules and cells", 1997, Vol. 7, p. 178-186); Freund's incomplete adjuvant;
Freund's complete adjuvant; vitamin E; Montanide; alum; CpG oligonucleotides (see,
for example, Kreig and 7 colleagues, Nature, Vol. 374, p. 546-549); poly-I:C and
derivatives thereof (e.g., poly ICLC); and various water-in-oil emulsions prepared
from biodegradable oils such as squalene and/or tocopherol. Among these, Freund's
incomplete adjuvant; Montanide; poly-I:C and derivatives thereof; and CpG
oligonucleotides are preferred. The mixing ratio between the above-described
adjuvant and the polypeptide is typically about 1:10 to 10:1, preferably about 1:5 to
5:1, more preferably about 1:1. However, the adjuvant is not limited to the above-
described examples, and adjuvants known in the art other than those described above
may also be used when the immunity-inducing agent of the present invention is
administered (see, for example, Goding, "Monoclonal Antibodies: Principles and
Practice, 2nd edition", 1986). Preparation methods for mixtures or emulsions of a
polypeptide and an adjuvant are well-known to those skilled in the art of vaccination.
[0064]
Further, in addition to the above-described adjuvants, factors that stimulate
the immune response of the subject may be used as the above-described
immunoenhancer. For example, various cytokines having a property to stimulate
lymphocytes and/or antigen-presenting cells may be used as the immunoenhancer in
combination with the immunity-inducing agent of the present invention. A number
of such cytokines capable of enhancing the immune response are known to those
skilled in the art, and examples thereof include, but are not limited to, interleukin-12
(IL-12), GM-CSF, IL-18, interferon-a, interferon-P, interferon-©, interferon-y, and
Flt3 ligand, which have been shown to enhance the prophylactic action of vaccines.
Such factors may also be used as the above-described immunoenhancer, and may be
contained in the immunity-inducing agent of the present invention, or may be
prepared as a separate composition to be used in combination with the immunity-
inducing agent of the present invention, to be administered to a patient.
[0065]
By bringing the above-described polypeptide into contact with antigen-
presenting cells in vitro, the antigen-presenting cells can be made to present the
polypeptide. That is, the polypeptides (a) to (c) described above can be used as
agents for treating antigen-presenting cells. Examples of the antigen-presenting
cells which may be preferably used include dendritic cells and B cells having MHC
class I molecules. Various MHC class I molecules have been identified and are
well-known. MHC molecules in human are called HLA. Examples of HLA class
I molecules include HLA-A, HLA-B and HLA-C, more specifically, HLA-A1, HLA-
A0201, HLA-A0204, HLA-A0205, HLA-A0206, HLA-A0207, HLA-A11, HLA-A24,
HLA-A31, HLA-A6801, HLA-B7, HLA-B8, HLA-B2705, HLA-B37, HLA-Cw0401
and HLA-Cw0602.
[0066]
The dendritic cells or B cells having MHC class I molecules can be prepared
from peripheral blood by a well-known method. For example, tumor-specific
dendritic cells can be induced by inducing dendritic cells from bone marrow,
umbilical cord blood or patient's peripheral blood using granulocyte-macrophage
colony-stimulating factor (GM-CSF) and IL-3 (or IL-4), and then adding a tumor-
related peptide to the culture system.
[0067]
By administering an effective amount of such dendritic cells, a response
desired for therapy of a cancer can be induced. As the cells to be used, bone
marrow or umbilical cord blood donated by a healthy individual, or bone marrow,
peripheral blood or the like from the patient himself may be used. When autologous
cells of the patient are used, high safety can be attained and serious side effects are
expected to be avoided. The peripheral blood or bone marrow may be any of a fresh
sample, cold-stored sample and frozen sample. As for the peripheral blood, whole
blood may be cultured or the leukocyte components alone may be separated and
cultured, and the latter is more efficient and thus preferred. Further, among the
leukocyte components, mononuclear cells may be separated. In cases where the
cells are originated from bone marrow or umbilical cord blood, the whole cells
constituting the bone marrow may be cultured, or mononuclear cells may be
separated therefrom and cultured. Peripheral blood, the leukocyte components
thereof and bone marrow cells contain mononuclear cells, hematopoietic stem cells
and immature dendritic cells, from which dendritic cells are originated, and also
CD4-positive cells and the like. As for the cytokine to be used, the production
method thereof is not restricted, and a naturally-occurring or recombinant cytokine or
the like may be employed as long as its safety and physiological activity have been
confirmed. Preferably, a preparation with assured quality for medical use is used in
a minimum necessary amount. The concentration of the cytokine(s) to be added is
not restricted as long as the dendritic cells are induced at the concentration, and
usually, the total concentration of the cytokine(s) is preferably about 10 to 1000
ng/mL, more preferably about 20 to 500 ng/mL. The culture may be carried out
using a well-known medium usually used for culture of leukocytes. The culturing
temperature is not restricted as long as proliferation of the leukocytes is attained at
the temperature, and a temperature of about 37°C, which is the body temperature of
human, is most preferred. The atmospheric environment during the culturing is not
restricted as long as proliferation of the leukocytes is attained under the environment,
and 5% CO2 is preferably allowed to flow. The culturing period is not restricted as
long as a necessary number of the cells are induced therewith, and usually 3 days to 2
weeks. As for the apparatuses used for separation and culturing of the cells,
appropriate apparatuses, preferably those whose safety upon application to medical
uses have been confirmed and whose operations are stable and simple, may be
employed. In particular, as for the cell-culturing apparatus, not only a general
vessel such as a Petri dish, flask or bottle, but also a layer type vessel, multistage
vessel, roller bottle, spinner type bottle, bag type culturing vessel, hollow fiber
column or the like may be used.
[0068]
The method per se to be used for bringing the above-described polypeptide
into contact with the antigen presenting cells in vitro may be carried out by a well-
known method. For example, it may be carried out by culturing the antigen-
presenting cells in a culture medium containing the above-described polypeptide.
The concentration of the peptide in the medium is not restricted, and usually about 1
to 100 ng/ml, preferably about 5 to 20 jag/ml. The cell density during the culture is
not restricted and usually about 103 to 107 cells/ml, preferably about 5*104 to 5><106
cells/ml. The culture may be carried out according to a conventional method at
37°C under the atmosphere of 5% CO2. The maximum length of the peptide which
can be presented on the surface of the antigen-presenting cells is usually about 30
amino acid residues. Therefore, in cases where the antigen-presenting cells are
brought into contact with the polypeptide in vitro, the polypeptide may be prepared
such that its length is not more than about 30 amino acid residues, although the
length is not restricted.
[0069]
By culturing the antigen-presenting cells in the coexistence of the above-
described polypeptide, the polypeptide is incorporated into an MHC molecule of the
antigen-presenting cells and presented on the surface of the antigen-presenting cells.
Therefore, using the above-described polypeptide, isolated antigen-presenting cells
containing the complex between the polypeptide and the MHC molecule can be
prepared. Such antigen-presenting cells can present the polypeptide against T cells
in vivo or in vitro, and thereby induce, and allow proliferation of, cytotoxic T cells
specific to the polypeptide.
[0070]
By bringing the thus prepared antigen-presenting cells having the complex
between the above-described polypeptide and the MHC molecule into contact with T
cells in vitro, cytotoxic T cells specific to the polypeptide can be induced and
allowed to proliferate. This may be carried out by coculturing the above-described
antigen-presenting cells and T cells in a liquid medium. For example, it may be
attained by suspending the antigen-presenting cells in a liquid medium, placing the
suspension in vessels such as wells of a microplate, adding T cells thereto and then
culturing the cells. The mixing ratio of the antigen-presenting cells with respect to
the T cells in the coculture is not restricted, and is usually about 1:1 to 1:100,
preferably about 1:5 to 1:20 in terms of the ratio between the numbers of the cells.
The density of the antigen-presenting cells to be suspended in the liquid medium is
not restricted, and is usually about 100 to 10,000,000 cells/ml, preferably about
10,000 to 1,000,000 cells/ml. The coculture is preferably carried out in accordance
with a conventional method at 37°C under the atmosphere of 5% CO2. The
culturing period is not restricted, and is usually 2 days to 3 weeks, preferably about 4
days to 2 weeks. The coculture is preferably carried out in the presence of one or
more interleukins such as IL-2, IL-6, IL-7 and/or IL-12. In such cases, the
concentration of IL-2 or IL-7 is usually about 5 to 20 U/ml, the concentration of IL-6
is usually about 500 to 2000 U/ml, and the concentration of IL-12 is usually about 5
to 20 ng/ml, but the concentrations of the interleukins are not restricted thereto. The
above coculture may be repeated once to several times with addition of fresh antigen-
presenting cells. For example, the operation of discarding the culture supernatant
after the coculture and adding a fresh suspension of antigen-presenting cells to
further conduct the coculture may be repeated once to several times. The conditions
of each coculture may be the same as described above.
[0071]
By the above-described coculture, cytotoxic T cells specific to the polypeptide
are induced and allowed to proliferate. Thus, using the above-described polypeptide,
isolated T cells can be prepared which selectively bind to the complex between the
polypeptide and the MHC molecule.
[0072]
As described in the Examples below, the gene encoding PDS5A is expressed
specifically in breast cancer cells, breast cancer tissues, brain tumor cells, brain tumor
tissues, esophagus cancer cells, esophagus cancer tissues, lung cancer cells, lung
cancer tissues, renal cancer cells, renal cancer tissues, colon cancer cells, colon
cancer tissues, perianal adenocarcinoma tissues, perianal adenocarcinoma cells,
neuroblastoma cells and leukemia cells. Therefore, it is thought that, in these
cancer species, a significantly larger amount of PDS5A exists than in normal cells.
When cytotoxic T cells prepared as described above are administered to a living body
while a part of PDS5A existing in cancer cells is presented by MHC molecules on the
surface of the cancer cells, the cytotoxic T cells can damage the cancer cells using the
presented polypeptide as a marker. Since antigen-presenting cells presenting the
above-described polypeptide can induce, and allow proliferation of, cytotoxic T cells
specific to the polypeptide also in vivo, cancer cells can be damaged also by
administering the antigen-presenting cells to a living body. That is, the cytotoxic T
cells and the antigen-presenting cells prepared using the polypeptide are also
effective as therapeutic and/or prophylactic agents for cancer, similarly to the
immunity-inducing agent of the present invention.
[0073]
In cases where the above-described isolated antigen-presenting cells or
isolated T cells are administered to a living body, these are preferably prepared by
treating antigen presenting cells or T cells collected from the patient to be treated
with the polypeptide (a) to (c) as described above in order to avoid the immune
response in the living body that attacks these cells as foreign bodies.
[0074]
The therapeutic and/or prophylactic agent for cancer comprising as an
effective ingredient the antigen-presenting cells or T cells is preferably administered
via a parenteral administration route, for example, by intravenous or intraarterial
administration. The dose is appropriately selected depending on the symptoms, the
purpose of administration and the like, and is usually 1 cell to 10,000,000,000,000
cells, preferably 1,000,000 cells to 1,000,000,000 cells, which dose is preferably
administered once every several days to once every several months. The
formulation may be, for example, the cells suspended in physiological buffered saline,
and the formulation may be used in combination with another/other anticancer
preparation(s) and/or cytokine(s). Further, one or more additives well-known in the
field of formulation of pharmaceuticals may also be added.
[0075]
Also by expressing a polynucleotide encoding any of the polypeptides (a) to
(c) in the body of the subject animal, antibody production and cytotoxic T cells can
be induced in the living body, and an effect comparable to that obtained in the case of
administration of the polypeptide can be obtained. That is, the immunity-inducing
agent of the present invention may be one comprising as an effective ingredient a
recombinant vector having a polynucleotide encoding any of the polynucleotides (a)
to (c), which recombinant vector is capable of expressing the polypeptide in a living
body. Such a recombinant vector capable of expressing an antigenic polypeptide as
shown in the later-mentioned Examples is also called a gene vaccine.
[0076]
The vector used for production of the gene vaccine is not restricted as long as
it is a vector capable of expressing the polypeptide in a cell of the subject animal
(preferably in a mammalian cell), and may be either a plasmid vector or a virus
vector, and any known vector in the field of gene vaccines may be used. The
polynucleotide such as DNA or RNA encoding the above-described polypeptide can
be easily prepared as mentioned above by a conventional method. Incorporation of
the polynucleotide into the vector can be carried out using a method well-known to
those skilled in the art.
[0077]
The administration route of the gene vaccine is preferably a parenteral route
such as intramuscular, subcutaneous, intravenous or intraarterial administration, and
the dose may be appropriately selected depending on the type of the antigen and the
like, and is usually about 0.1 jig to 100 mg, preferably about 1 ug to 10 mg in terms
of the weight of the gene vaccine per 1 kg of body weight.
[0078]
Examples of the method using a virus vector include those wherein a
polynucleotide encoding the above-described polypeptide is incorporated into an
RNA virus or DNA virus, such as a retrovirus, adenovirus, adeno-associated virus,
herpes virus, vaccinia virus, pox virus, poliovirus or Sindbis virus, and then a subject
animal is infected with the resulting virus. Among these methods, those using a
retrovirus, adenovirus, adeno-associated virus, vaccinia virus or the like are
especially preferred.
[0079]
Examples of other methods include a method wherein an expression plasmid
is directly intramuscularly administered (DNA vaccine method), liposome method,
lipofectin method, microinjection method, calcium phosphate method and
electroporation method, and the DNA vaccine method and liposome method are
especially preferred.
[0080]
Methods for actually making the gene encoding the above-described
polypeptide used in the present invention act as a pharmaceutical include the in vivo
method wherein the gene is directly introduced into the body, and the ex vivo method
wherein a certain kind of cells are collected from a subject animal and the gene is
introduced into the cells ex vivo, followed by returning the cells to the body (Nikkei
Science, 1994, April, p. 20-45; The Pharmaceutical Monthly, 1994, Vol. 36, No. 1, p.
23-48; Experimental Medicine, Extra Edition, 1994, Vol.12, No. 15; and references
cited in these literatures, and the like). The in vivo method is more preferred.
[0081]
In cases where the gene is administered by the in vivo method, the gene may
be administered through an appropriate administration route depending on the
disease to be treated, symptoms and so on. It may be administered by, for example,
intravenous, intraarterial, subcutaneous or intramuscular administration. In cases
where the gene is administered by the in vivo method, the gene may be formulated
into a preparation such as a solution, and in general, it is formulated into an injection
solution or the like containing DNA encoding the above-described peptide of the
present invention as an effective ingredient. A commonly used carrier(s) may be
also added thereto as required. In the case of a liposome or membrane fusion
liposome (Sendai virus (HVJ)-liposome or the like) containing the DNA, the
liposome may be formulated into a liposome preparation such as a suspension, frozen
preparation or centrifugally concentrated frozen preparation.
[0082]
In the present invention, "the base sequence shown in SEQ ID NO:l"
includes not only the base sequence expressly written in SEQ ID NO:l, but also the
sequence complementary thereto. Thus, "the polynucleotide having the base
sequence shown in SEQ ID NO:l" includes a single-stranded polynucleotide having
the base sequence expressly written in SEQ ID NO:l, a single-stranded
polynucleotide having the base sequence complementary thereto, and a double-
stranded polynucleotide composed of these single-stranded polynucleotides. When
a polynucleotide encoding the polypeptide used in the present invention is prepared,
any one of these base sequences is appropriately selected, and those skilled in the art
can easily carry out the selection.
[0083]
Further, since the polypeptide used in the present invention is expressed
specifically in cancer, the polypeptide specifically reacts only with the serum in a
cancer-bearing living body, so that the polypeptide of the present invention is used
also for detection of cancer.
[0084]
In the above-described method for detecting cancer, a sample separated from
a living body is used to measure expression of a polypeptide having any one of the
amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44, or a
polypeptide as a homologous factor thereof, having a sequence identity of not less
than 90%, preferably not less than 95%, more preferably not less than 98%, still more
preferably not less than 99% or not less than 99.5% to the polypeptide. Examples
of the method for measuring the expression of the polypeptide using the sample
includes a method in which an antibody against the polypeptide, which antibody is
contained in the sample, is measured by immunoassay (Method 1); a method in
which the polypeptide per se contained in the sample is measured by immunoassay
(Method 2); and a method in which mRNA contained in the sample and encoding the
polypeptide is measured (Method 3). In the method of the present invention, the
expression of the polypeptide may be measured by any of these methods. In the
present invention, the term "measurement" includes detection, quantification and
semi-quantification.
[0085]
Here, PDS5A is a polypeptide identified, by the SEREX method using a
canine breast cancer-derived cDNA library and serum obtained from the same patient
dog, as a polypeptide that binds to an antibody specifically existing in the serum
derived from the tumor-bearing dog (cancer-specific antibody) (see Example 1).
That is, in the living body of the tumor-bearing dog, an antibody against PDS5A is
specifically induced. Therefore, by measuring the antibody against PDS5A in the
living body of the tumor-bearing dog, a cancer expressing PDS5A can also be
detected. Further, also by measuring PDS5A as an antigen by Method 2, the canine
cancer can be detected. Further, since, as described in the later-mentioned
Examples, mRNA encoding the antigen polypeptide is expressed at significantly
higher levels in cancer cells and cancer tissues, especially in breast cancer cells,
breast cancer tissues, brain tumor cells, brain tumor tissues, esophagus cancer cells,
esophagus cancer tissues, lung cancer cells, lung cancer tissues, renal cancer cells,
renal cancer tissues, colon cancer cells, colon cancer tissues, perianal
adenocarcinoma cells, perianal adenocarcinoma tissues, neuroblastoma cells and
leukemia cells, compared to the normal tissues (see Example 1), the canine cancer
can be detected also by measuring the mRNA.
[0086]
In Method 1 above, measurement of the cancer-specific antibody which may
exist in the sample can be easily carried out by immunoassay using an antigenic
substance which undergoes antigen-antibody reaction with the antibody. The
immunoassay per se is a conventional well-known method as explained in detail
below. Examples of the antigenic substance which may be used in the
immunoassay include the polypeptides (a) to (c). Since antibodies have cross-
reactivity, even a molecule other than the antigenic substance corresponding to the
original immunogen may be bound to an antibody induced against the immunogen by
antigen-antibody reaction, as long as the molecule has a structure thereon similar to
an epitope of the immunogen. For example, polypeptides having a high sequence
identity therebetween often have similar epitope structures, and, in this case, the both
polypeptides may have the same antigenicity. As concretely described in the
Examples below, the human-derived polypeptide of SEQ ID NO:4 or 44 undergoes
antigen-antibody reaction with the above-described antibody induced in the body of a
cancer-bearing dog. Therefore, in Method 1 of the present invention, any
mammalian homologous factor may be used as the antigen in the immunoassay.
[0087]
An antigenic substance having a complex structure and a large molecular
weight, such as a protein, usually has a plurality of sites having different structures
on the molecule. Therefore, against such an antigenic substance, a plurality of kinds
of antibodies which recognize the respective plurality of sites are produced in a living
body. That is, an antibody induced in a living body against an antigenic substance
such as a protein is a polyclonal antibody, which is a mixture of a plurality of kinds
of antibodies. It should be noted that, in the present invention, the term "polyclonal
antibody" means antibodies which exist in serum from a living body having an
antigenic substance therein and were induced in the living body against the antigenic
substance.
[0088]
Measurement of the antibody in a sample may easily be carried out by
immunoassay using the above-described polypeptide as an antigen. Immunoassays
per se are well-known in the art, and include, when classified based on the reaction
mode, the sandwich method, competition method, agglutination method, Western
blotting and the like. When classified based on the label, immunoassays include
radioimmunoassay, fluorescence immunoassay, enzyme immunoassay, biotin
immunoassay and the like, and the immunoassay of the above-described antibody
may be carried out by any of these immunoassays. Although not restricted, the
sandwich ELISA and the agglutination method may be preferably applied as the
method of immunoassay of the above antibody in the present invention, since the
operations are simple and a large-scale apparatus is not necessary in these methods.
In cases where an enzyme is used as the label of the antibody, the enzyme is not
particularly restricted as long as it satisfies conditions such as a large turnover
number, stability upon binding with the antibody, and specific coloring of the
substrate, and examples of the enzyme which may be used include enzymes used in
an ordinary enzyme immunoassay, such as peroxidase, (3-galactosidase, alkaline
phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate
dehydrogenase, and malate dehydrogenase. An enzyme inhibitor, coenzyme and/or
the like may also be used. Binding of the enzyme with the antibody may be carried
out by a known method using a cross-linking agent such as a maleimide compound.
As a substrate, a known substance may be used depending on the type of the enzyme
to be used. For example, in cases where peroxidase is used as the enzyme,
3,3',5,5'-tetramethylbenzidine may be used; and in cases where alkaline phosphatase
is used as the enzyme, para-nitrophenol or the like may be used. As a radioisotope,
one used in an ordinary radioimmunoassay, such as 125I or 3H may be used. As a
fluorescent dye, one used in an ordinary fluorescent antibody technique, such as
fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC) or
the like may be used.
[0089]
These immunoassays per se are well-known in the art and do not need to be
explained in the present specification. Briefly, in a sandwich immunoassay, for
example, the above-mentioned polypeptide used as an antigen is immobilized on a
solid phase and then reacted with a sample such as a serum. After washing the solid
phase, the resultant is reacted with an appropriate secondary antibody. After
washing the solid phase, the secondary antibody bound to the solid phase is measured.
This method is preferred as an embodiment of the method of the present invention
for detecting cancer since, in this method, immobilization of the antigen polypeptide
to the solid phase enables simple removal of unbound secondary antibodies. As the
secondary antibody, an anti-dog IgG antibody may be used in cases where, for
example, the sample is derived from a dog. By preliminarily labeling the secondary
antibody with a labeling substance exemplified above, the secondary antibody bound
to the solid phase can be measured. The thus measured amount of the secondary
antibody corresponds to the amount of the above-mentioned antibody in the serum
sample. In cases where an enzyme is used as the labeling substance, the amount of
the antibody may be measured by adding a substrate which develops a color upon
decomposition by an enzymatic activity, and then optically measuring the amount of
decomposition of the substrate. In cases where a radioisotope is used as the labeling
substance, the amount of radiation emitted from the radioisotope may be measured
with a scintillation counter or the like.
[0090]
In Method 2 of the present invention, the polypeptide of SEQ ID NO:2, 4, 6, 8,
10, 12 or 44 or a homologous factor thereof, which may be contained in a sample
obtained from a living body is measured. As mentioned above, the amount of a
cancer-specific antibody which undergoes antigen-antibody reaction with the
polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 or a homologous factor thereof is
significantly larger in cancer patients, and this indicates that the amount of
production of the polypeptide or a homologous factor thereof, which corresponds to
an antigen of the cancer-specific antibody, is significantly larger in the cancer
patients. Therefore, cancer in a living body can be detected also by measuring the
polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 or a homologous factor thereof
similarly to Method 1 described above.
[0091]
The polypeptide in a sample can be easily measured by a well-known
immunoassay. More particularly, for example, by preparing an antibody or an
antigen-binding fragment thereof which undergoes antigen-antibody reaction with the
polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 and using this in an
immunoassay, the polypeptide having the sequence shown in SEQ ID NO:2, 4, 6, 8,
10, 12 or 44 or a homologous factor thereof which may exist in the sample can be
measured. The immunoassay per se is a well-known conventional method as
described above.
[0092]
The term "antigen-binding fragment" herein means an antigen fragment such
as the Fab fragment or F(ab')2 fragment contained in the antibody molecule, which
has a binding capacity to an antigen. The antibody may be either a polyclonal
antibody or monoclonal, and a monoclonal antibody is preferred in an immunoassay
and the like because a high reproducibility can be obtained therewith. The methods
of preparation of a polyclonal antibody and a monoclonal antibody using a
polypeptide as an immunogen are well known, and can be easily carried out by
conventional methods. For example, antibodies against a polypeptide can be
induced by immunizing an animal with, as an immunogen, the polypeptide
conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) or casein,
together with an adjuvant. Antibody-producing cells such as spleen cells or
lymphocytes are then collected from the immunized animal and fused with myeloma
cells to prepare hybridomas. Among the hybridomas, one producing an antibody
which binds to the polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44, or a
homologous factor thereof is selected and proliferated, and then a monoclonal
antibody whose corresponding antigen is the above-mentioned protein can be
obtained from the culture supernatant. The above-described method is a
conventional well-known method.
[0093]
In Method 3 of the present invention, mRNA which may be contained in a
sample obtained from a living body and encodes PDS5A is measured. As
concretely shown in the Examples below, mRNA encoding PDS5A is significantly
highly expressed in tissues and cells of cancer, breast cancer, brain tumor, esophagus
cancer, lung cancer, renal cancer, colon cancer, perianal adenocarcinoma,
neuroblastoma and leukemia. Therefore, also by measuring the mRNA in the
sample, cancer in the living body can be detected.
[0094]
In the detection method of the present invention, whether or not a subject
living body is suffering from cancer is judged based on the expression level of the
polypeptide measured as described above. Although the cancer detection may be
attained simply by measuring expression of the polypeptide in the subject living body,
it is preferred, from the viewpoint of enhancement of the detection accuracy, to
obtain a normal reference value by investigating the expression level of the
polypeptide (the amount of the antibody, polypeptide or mRNA) in one or more
samples from healthy individuals, followed by comparison of the measured value in
the subject living body with the normal reference value. In cases where a higher
detection accuracy is required, a cancer reference value may be obtained by
investigating the expression level of the polypeptide in samples obtained from many
patients known to be suffering from cancer, followed by comparison of the measured
value in the subject living body both with the normal reference value and with the
cancer reference value. The reference values may be determined by, for example,
digitizing the expression level of the polypeptide in each sample and calculating the
mean value. The normal reference value and the cancer reference value may be
preliminarily determined by investigating the expression level of the polypeptide in
many healthy individuals and cancer patients. Thus, in cases where comparison
with the reference value(s) is carried out in the method of the present invention, a
preliminarily determined reference value(s) may be used.
[0095]
The detection method of the present invention may be used in combination
with detection with another cancer antigen or cancer marker. By this, the accuracy
of detection of cancer can be further increased.
[0096]
By the detection method of the present invention, cancers in a living body can
be detected. By the method of the present invention, even an invisible small cancer
or a cancer which exists in a deep part of a body can be detected, and thus the method
is useful for early detection of caners. Further, by applying the detection method of
the present invention to patients in the follow-up period after cancer therapy, a
recurrent cancer, if any, can be detected at an early stage.
[0097]
In a tumor-bearing living body, as the number of cancer cells expressing the
specific polypeptide to be measured in the present invention increases, the amounts
of accumulation of the polypeptide and the mRNA encoding it in the living body
increase, leading to increased production of antibodies against the polypeptide in the
serum. On the other hand, as the number of cancer cells decreases, the amounts of
accumulation of the polypeptide and the mRNA encoding it in the living body
decrease, leading to decrease in antibodies against the polypeptide in the serum.
Thus, in cases where the expression level of the specific polypeptide is high, it can be
determined that tumor growth and/or metastasis of cancer occurred, that is, the stage
of progression of cancer is advanced.
[0098]
Further, as shown in the Examples below, when compared between the same
kind of tumors, a malignant one produces a significantly higher amount of the
antibodies than a benign one. Therefore, in cases where the expression level of the
specific polypeptides is high, it can be determined that the grade of cancer
malignancy is high. That is, the grade of cancer malignancy can also be detected by
the method of the present invention.
[0099]
Furthermore, the effect of a cancer therapy can be monitored based on
increase or decrease of the expression level of the specific polypeptide. Therefore,
by observing the expression level of the above-mentioned polypeptide in an
individual during or after a cancer therapy, one can obtain a clue(s) to know the effect
of an anticancer drug, presence/absence of a residual tumor after extirpation of the
tumor, and/or, even during the follow-up, metastasis and/or recurrence, as early as
possible. In cases where a therapy is/was appropriate, the expression level of the
polypeptide becomes lower than that in the patient in the tumor-bearing state before
the therapy, and therefore the effect of the therapy that was (or is being) provided for
the living body can be judged to have been (or to be) excellent. In cases where the
expression level of the polypeptide increased or is maintained, or in cases where the
expression level once decreased and then increased again, the therapeutic effect can
be judged to be insufficient, and this observation can be a useful basis for selection of
a therapeutic method, such as use of another therapeutic method or alteration of the
dose of an anti-cancer agent.
[0100]
Preferred examples of the cancer as the subject of the method for detecting
cancer of the present invention include cancers expressing PDS5A, such as breast
cancer, brain tumor, esophagus cancer, lung cancer, renal cancer, colon cancer,
perianal adenocarcinoma, neuroblastoma and leukemia. The living body as the
subject of the method of the present invention is preferably a mammal, more
preferably human, dog or cat.
[0101]
The sample to be provided for the method of the present invention include
body fluids such as blood, serum, plasma, ascites and pleural effusion, and tissues
and cells. Particularly, serum, plasma, ascites and pleural effusion may be
preferably used in Method 1 and Method 2 above. A tissue sample and cell sample
are preferred in the case of Method 3 above in which mRNA is measured.
[0102]
The polypeptide used as the antigen for the immunoassay in Method 1 may be
provided as a reagent for cancer detection. The reagent may consist essentially of
the above-mentioned polypeptide, or may contain, for example, various additives
useful for stabilizing the polypeptide, and/or the like. The reagent may be provided
also in a state where it is immobilized on a solid phase such as a plate or membrane.
[0103]
When the polypeptide shown in SEQ ID NO:2, 4, 6, 8, 10, 12 or 44 or a
homologous factor thereof is to be immunoassayed in Method 2, an antibody or an
antigen-binding fragment thereof which undergoes antigen-antibody reaction with the
polypeptide or a homologous factor thereof may also be provided as a reagent for
cancer detection. Also in this case, the reagent for cancer detection may consist
essentially of the antibody or antigen-binding fragment, or may contain, for example,
various additives useful for stabilizing the antibody or antigen-binding fragment,
and/or the like. The antibody or antigen-binding fragment may also be in a state
where a metal such as manganese or iron is bound thereto. Administration of such a
metal-bound antibody or antigen-binding fragment into a living body causes higher
accumulation of the antibody or antigen-binding fragment at locations where the
antigen protein exists in a larger amount, so that measurement of the metal by MRI or
the like enables detection of existence of cancer cells that produces the antigen
protein.
[0104]
Furthermore, the above-described polynucleotide for cancer detection to be
used for measuring mRNA in Method 3 may also be provided as a reagent for cancer
detection. Also in this case, the reagent for cancer detection may consist essentially
of the polynucleotide, or may contain, for example, various additives useful for
stabilizing the polynucleotide, and/or the like. The polynucleotide for cancer
detection contained in the reagent is preferably a primer or a probe.
EXAMPLES
[0105]
The present invention will now be described more concretely by way of
Examples.
[0106]
Example 1: Obtaining Novel Cancer Antigen Protein by SEREX Method
(1) Preparation of cDNA Library
Total RNA was extracted from a breast cancer tissue of a tumor-bearing dog
by the Acid guanidium-Phenol-Chloroform method, and poly(A) NA was purified
using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co.,
Ltd.) in accordance with the protocol attached to the kit.
[0107]
Using the obtained mRNA (5 ug), a cDNA phage library was synthesized.
For the preparation of the cDNA phage library, cDNA Synthesis Kit, ZAP-cDNA
Synthesis Kit, and ZAP-cDNA Gigapack III Gold Cloning Kit (manufactured by
STRATAGENE) were used in accordance with the protocols attached to the kits.
The size of the prepared cDNA phage library was 1 x 106 pfu/ml.
[0108]
(2) Screening of cDNA Library with Serum
Using the prepared cDNA phage library, immunoscreening was carried out.
More particularly, the host E. coli (XLl-Blue MRF') was infected with the library
such that 2340 clones appear on an NZY agarose plate having a size of O90 x 15 mm,
and cultured at 42°C for 3 to 4 hours to allow the phage to form plaques. The plate
was covered with a nitrocellulose membrane (Hybond C Extra: manufactured by GE
Healthcare Bio-Science) impregnated with IPTG (isopropyl-P-D-thiogalactoside) at
37°C for 4 hours to allow induction and expression of proteins, which were thus
transferred to the membrane. Subsequently, the membrane was recovered and
soaked in TBS (10 mM Tris-HCl, 150 mM NaCl; pH 7.5) containing 0.5% non-fat
dry milk, followed by shaking it at 4°C overnight to suppress non-specific reactions.
This filter was then allowed to react with 500-fold diluted canine patient serum at
room temperature for 2 to 3 hours.
[0109]
As the above-described canine patient serum, serum collected from a canine
patient suffering from perianal tumor was used. The serum was stored at -80°C and
pretreated immediately before use. The method of the pretreatment of the serum
was as follows. That is, the host E. coli (XLl-Blue MRP') was infected with X ZAP
Express phage to which no foreign gene was inserted, and then cultured on NZY
plate medium at 37°C overnight. Subsequently, 0.2 M NaHC03 buffer (pH 8.3)
containing 0.5 M NaCl was added to the plate, and the plate was left to stand at 4°C
for 15 hours, followed by collecting the supernatant as an E. co//'/phage extract.
Thereafter, the collected E. coli/phage extract was allowed to flow through an NHS
column (manufactured by GE Healthcare Bio-Science) to immobilize proteins
derived from the E. co///phage thereon. The serum from the canine patient was
allowed to flow through and react with this protein-immobilized column to remove
antibodies adsorbed to E. coli and/or the phage. The serum fraction that passed
through the column was 500-fold diluted with TBS containing 0.5% non-fat dry milk,
and the resulting diluent was used as the material for the immunoscreening.
[0110]
The membrane on which the thus treated serum and the above-described
fusion protein were blotted was washed 4 times with TBS-T (0.05% Tween 20/TBS),
and allowed to react with goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated:
manufactured by BETHYL Laboratories) 5,000-fold diluted with TBS containing
0.5% non-fat dry milk as a secondary antibody at room temperature for 1 hour,
followed by detection by the enzyme coloring reaction using the NBT/BCIP reaction
solution (manufactured by Roche). Colonies at positions where a positive coloring
reaction was observed were recovered from the NZY agarose plate having a size of
O90 x 15 mm, and dissolved in 500 ul of SM buffer (100 mM NaCl, 10 mM
MgClSCu, 50 mM Tris-HCl, 0.01% gelatin; pH 7.5). The screening was repeated as
a second and third screening in the same manner as described above until a single
coloring reaction-positive colony was obtained, thereby isolating one positive clone
after screening of 30940 phage clones reactive with IgG in the serum.
[0111]
(3) Sequence Homology Search of Isolated Antigen Gene
To subject the single positive clone isolated by the above-described method to
a base sequence analysis, an operation of conversion of the phage vector to a plasmid
vector was carried out. More particularly, 200 ul of a solution prepared such that
the host E. coli (XLl-Blue MRF') was contained at an absorbance OD600 of 1.0 was
mixed with 100 ul of a purified phage solution and further with 1 ul of ExAssist
helper phage (manufactured by STRATAGENE), and the reaction was allowed to
proceed at 37°C for 15 minutes. To the reaction mixture, 3 ml of LB medium was
added, and the resulting mixture was cultured at 37°C for 2.5 to 3 hours, followed by
immediate incubation in a water bath at 70°C for 20 minutes. The mixture was then
centrifuged at 4°C at 1,000 xg for 15 minutes, and the supernatant was recovered as a
phagemid solution. Subsequently, 200 ul of a solution prepared such that the
phagemid host E. coli (SOLR) was contained at an absorbance OD600 of 1.0 was
mixed with 10 ul of a purified phage solution, and the reaction was allowed to
proceed at 37°C for 15 minutes. Thereafter, 50 ul of the reaction mixture was
plated on LB agar medium supplemented with ampicillin (final concentration: 50
ug/ml), and cultured at 37°C overnight. A single colony of transformed SOLR was
recovered and cultured in LB medium supplemented with ampicillin (final
concentration: 50 ug/ml) at 37°C, followed by purification of plasmid DNA having
the insert of interest using QIAGEN plasmid Miniprep Kit (manufactured by Qiagen).
[0112]
The purified plasmid was subjected to analysis of the full-length sequence of
the insert by the primer walking method using the T3 primer described in SEQ ID
NO: 13 and the T7 primer described in SEQ ID NO: 14. By this sequence analysis,
the gene sequence described in SEQ ID NO: 1 was obtained. Using the base
sequence and the amino acid sequence of this gene, homology search against known
genes was carried out using a sequence homology search program BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/). As a result, it was revealed that the
obtained gene is the PDS5A gene. Human PDS5A, which is a human homologous
factor of canine PDS5A, had a sequence identity of 94% in terms of the base
sequence and 99% in terms of the amino acid sequence; murine PDS5A, which is a
murine homologous factor, had a sequence identity of 91% in terms of the base
sequence and 99% in terms of the amino acid sequence; bovine PDS5A, which is a
bovine homologous factor, had a sequence identity of 95% in terms of the base
sequence and 99% in terms of the amino acid sequence; equine PDS5A, which is a
equine homologous factor, had a sequence identity of 96% in terms of the base
sequence and 99% in terms of the amino acid sequence; and chicken PDS5A, which
is a chicken homologous factor, had a sequence identity of 83% in terms of the base
sequence and 98% in terms of the amino acid sequence. In terms of human PDS5A,
the base sequence is shown in SEQ ID NOs:3 and 43, and the amino acid sequence is
shown in SEQ ID NOs:4 and 44; in terms of murine PDS5A, the base sequence is
shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; in
terms of bovine PDS5A, the base sequence is shown in SEQ ID NO:7, and the amino
acid sequence is shown in SEQ ID NO:8; in terms of equine PDS5A, the base
sequence is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ
ID NO: 10; and in terms of chicken PDS5A, the base sequence is shown in SEQ ID
NO:l 1, and the amino acid sequence is shown in SEQ ID NO: 12.
[0113]
(4) Analysis of Expression in Various Tissues
Expression of the genes obtained by the above method in canine, human and
murine normal tissues and various cell lines were investigated by the RT-PCR
(Reverse Transcription-PCR) method. The reverse transcription reaction was
carried out as follows. That is, from 50 to 100 mg of each tissue or 5 x 106 to 10 x
106 cells of each cell line, total RNA was extracted using the TRIZOL reagent
(manufactured by INVITROGEN) according to the protocol described in the attached
instructions. Using this total RNA, cDNA was synthesized with the Superscript
First-Strand Synthesis System for RT-PCR (manufactured by INVITROGEN)
according to the protocol described in the attached instructions. As the cDNAs of
human normal tissues (brain, hippocampus, testis, colon and placenta), Gene Pool
cDNA (manufactured by INVITROGEN), QUICK-Clone cDNA (manufactured by
CLONETECH) and Large-Insert cDNA Library (manufactured by CLONETECH)
were used. The PCR reaction was carried out using gene-specific primers (the
canine primers described in SEQ ID NOs:15 and 16, the human primers described in
SEQ ID NOs:17 and 18, and the murine primers described in SEQ ID NOs:19 and
20) as described below. That is, reagents and an attached buffer were mixed such
that 0.25 \d of the sample prepared by the reverse transcription reaction, 2 uM each
of the above primers, 0.2 mM each of dNTPs, and 0.65 U ExTaq polymerase
(manufactured by Takara Shuzo Co., Ltd.) were contained in a total volume of 25 ul,
and the reaction was carried out by repeating 30 times the cycle of 94°C for 30
seconds, 55°C for 30 seconds and 72°C for 1 minute using a Thermal Cycler
(manufactured by BIO RAD). As a control for comparison, primers specific to
GAPDH (the canine and human GAPDH primers are shown in SEQ ID NOs:21 and
22; and the murine GAPDH primers are shown in SEQ ID NOs:23 and 24) were used
at the same time. As a result, as shown in Fig. 1, in terms of the canine PDS5A
gene, expression was not observed in most of the healthy canine tissues, while strong
expression was observed in the canine tumor tissues. Also in terms of the human
and murine PDS5A genes, expression was not observed in most of the healthy human
and murine tissues, while expression was detected in most of the cancer cell lines
(Figs. 2 and 3), as in the case of the canine PDS5A gene.
[0114]
Example 2: Analysis of Cancer Antigenicity and Evaluation of Pharmacological
Effect of PDS5A in Living Body
(l)Preparation of Recombinant Vector That Expresses PDS5A in Living Body
Based on the base sequence of SEQ ID NO:5, a recombinant vector that
expresses PDS5A in a living body was prepared. Reagents and an attached buffer
were mixed together such that 1 ul of the cDNA prepared from the murine cancer cell
line N2a (purchased from ATCC), which showed expression in Example 1, 0.4 uM
each of two kinds of primers having the Notl andXhol restriction sites (shown in
SEQ ID NOs:25 and 26), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase
(manufactured by Takara Shuzo Co., Ltd.) were contained in a total volume of 50 ul,
and PCR was carried out by repeating 30 times the cycle of 98°C for 10 seconds,
55°C for 15 seconds and 72°C for 4 minute using a Thermal Cycler (manufactured by
BIO RAD). The above-described two kinds of primers were those for amplification
of the region encoding the full-length of the amino acid sequence shown in SEQ ID
NO:5. After the PCR, the amplified DNA was subjected to electrophoresis using
1% agarose gel, and a DNA fragment of about 4000 bp was purified using QIAquick
Gel Extraction Kit (manufactured by QIAGEN).
[0115]
The purified DNA fragment was ligated into a cloning vector pCR-Blunt
(manufactured by Invitrogen). E. coli was transformed with the resulting ligation
product, and the plasmid was then recovered. The amplified gene fragment was
confirmed to have the same sequence as that of interest by sequencing. The plasmid
having the same sequence as that of interest was treated with restriction enzymes
Notl and Xhol, and purified using QIAquick Gel Extraction Kit, followed by inserting
the gene sequence of interest into a mammalian expression vector PCDNA3.1
(manufactured by Invitrogen) that had been treated with restriction enzymes Notl and
Xhol. Use of this vector enables production of the PDS5A protein in mammalian
cells.
[0116]
To 100 |ag of the thus prepared plasmid DNA, 50 u.g of gold particles
(manufactured by Bio Rad), 100 ul spermidine (manufactured by SIGMA) and 100 ul
of 1 M CaCb (manufactured by SIGMA) were added, and the resulting mixture was
stirred by vortexing, followed by leaving the mixture to stand for 10 minutes at room
temperature (the resulting particles are hereinafter referred to as gold-DNA particles).
The mixture was then centrifuged at 3000 rpm for 1 minute and the supernatant was
discarded, followed by rinsing the precipitate 3 times with 100% ethanol
(manufactured by WAKO). To the gold-DNA particles, 6 ml of 100% ethanol was
added, and the resulting mixture was sufficiently stirred by vortexing, followed by
pouring the gold-DNA particles into Tefzel Tubing (manufactured by Bio Rad) and
allowing the particles to precipitate on the wall surface. The ethanol in the Tefzel
Tubing to which the gold-DNA particles are attached was dried in the air, and the
tube was cut into pieces having a length appropriate for a gene gun.
[0117]
(2) Anti-tumor Effect of PDS5A by DNA Vaccine Method
Each of a murine neuroblastoma cell line N2a and a colon cancer cell line
CT26 were subcutaneously transplanted to 10 individuals of A/J mice (7 weeks old,
male, purchased from Japan SLC) and Balb/c mice (7 weeks old, male, purchased
from Japan SLC) in an amount of 1 x 106 cells. The above prepared tube was fixed
in a gene gun, and a pressure of 400 psi was applied using pure helium gas to
perform percutaneous administration of the DNA vaccine to the abdominal cavity of
each mouse whose hair had been shaved, which administration was repeated a total
of 3 times at intervals of 7 days (this corresponds to 2 ug/individual in terms of the
dose of the inoculated amount of the plasmid DNA) to evaluate the anti-tumor effect
(therapeutic model). Further, in a similar manner, the DNA vaccine was
subcutaneously administered to each of 10 individuals of A/J mice and Balb/c mice a
total of 3 times at intervals of 7 days, and N2a cells or CT26 cells were then
transplanted to each mouse to evaluate the anti-tumor effect (prophylactic model).
As a control, a plasmid DNA to which the PDS5A gene was not inserted was
administered to 10 individuals in each model.
[0118]
The anti-tumor effect was evaluated based on the size of the tumor (major
axis x minor axis / 2) and the ratio of living mice. The results are shown in Figs. 4
to 11. As a result of this study, in the therapeutic model using the neuroblastoma
cell line, the size of the tumor on Day 41 was 569 mm3 and 109 mm3 in the control
group and the PDS5 A plasmid-administered group, respectively, indicating
significant reduction of the tumor in the PDS5A plasmid-administered group (Fig. 4).
Similarly, in the prophylactic model using the neuroblastoma cell line, the size of the
tumor on Day 43 was 476 mm3 and 0 mm3 in the control group and the PDS5A
plasmid-administered group, respectively, indicating complete regression of the
tumor in the PDS5A plasmid-administered group (Fig. 5). Further, in the
therapeutic model using the colon cancer cell line, the size of the tumor on Day 41
was 589 mm and 189 mm in the control group and the PDS5A plasmid-
administered group, respectively, indicating significant reduction of the tumor in the
PDS5A plasmid-administered group (Fig. 8). Further, in the prophylactic model
using the colon cancer cell line, the size of the tumor on Day 43 was 397 mm and 43
mm in the control group and the PDS5A plasmid-administered group, respectively,
indicating significant reduction of the tumor in the PDS5A plasmid-administered
group (Fig. 9). Based on observation of the process of survival in the both models
using the neuroblastoma cell line, while all the cases in the control group died by Day
84 after the administration, 60% of the mice were alive at that time in the PDS5A
plasmid-administered group (Fig. 6). Further, in the prophylactic model, while all
the cases in the control group died by Day 90 after the administration, all the mice
were alive at that time in the PDS5A plasmid-administered group (Fig. 7). Further,
based on observation of the process of survival in the both models using the colon
cancer cell line, while all the cases in the control group died by Day 84 after the
administration, 40% of the mice were alive at that time in the PDS5A plasmid-
administered group (Fig. 10). Further, in the prophylactic model, while all the cases
in the control group died by Day 90 after the administration, 80% of the mice were
alive at that time in the PDS5A plasmid-administered group (Fig. 11).
[0119]
In the above results, a significantly higher anti-tumor effect was observed in
the PDS5A-plasmid administered group than in the control group, and, by this
observation, it was revealed that PDS5A is a cancer antigen having a strong cancer
antigenicity and effective for therapy and prophylaxis of cancer.
[0120]
Example 3: Induction of Peptide Epitope-reactive CD8-positive T Cells
For prediction of an HLA-A0201-binding motif in the amino acid sequence of
the human PDS5A protein, a computer-based prediction program using the known
BIMAS software (available at http://bimas.dcrt.nih.gov/molbio/hla_bind/) was used
to analyze the amino acid sequences shown in SEQ ID NOs:4 and 44, and thereby the
polypeptides shown in SEQ ID NOs:27 to 35, which were expected to be capable of
binding to the HLA class I molecule, were selected.
[0121]
From an HLA-A0201-positive healthy individual, peripheral blood was
separated, and the peripheral blood was overlaid on Lymphocyte separation medium
(OrganonpTeknika, Durham, NC), followed by centrifuging the resultant at 1,500
rpm at room temperature for 20 minutes. A fraction containing peripheral blood
mononuclear cells (PBMCs) was recovered and washed 3 times in a cold phosphate
buffer, to obtain PBMCs. The obtained PBMCs were suspended in 20 ml of AIM-
V medium (Life Technolo-gies, Inc., Grand Island, NY, USA), and the cells were
allowed to attach to a culture flask (Falcon) at 37°C under 5% CO2 for 2 hours.
Unattached cells were used for preparation of T cells, and attached cells were used
for preparation of dendritic cells.
[0122]
The attached cells were cultured in AIM-V medium in the presence of IL-4
(1000 U/ml) and GM-CSF (1000 U/ml). The medium was replaced 6 days later
with AIM-V medium supplemented with IL-4 (1000 U/ml), GM-CSF (1000 U/ml),
IL-6 (1000 U/ml, Genzyme, Cambridge, MA), IL-1(3 (10 ng/ml, Genzyme,
Cambridge, MA) and TNF-a (10 ng/ml, Genzyme, Cambridge, MA), and the culture
was carried out for additional 2 days to obtain a population of unattached cells, which
were employed as the dendritic cells.
[0123]
The prepared dendritic cells were suspended in AIM-V medium at a cell
density of 1 x 106 cells/ml, and each of the selected polypeptides was added at a
concentration of 10 (J-g/ml to the suspension. Using a 96-well plate, the cells were
cultured at 37°C under 5% CO2 for 4 hours. After the culture, X-ray irradiation
(3000 rad) was carried out, and the cells were washed with AIM-V medium,
followed by being suspended in AIM-V medium supplemented with 10% human AB
serum (Nabi, Miami, FL), IL-6 (1000 U/ml) and IL-12 (10 ng/ml, Genzyme,
Cambridge, MA). The cells were placed in a 24-well plate in an amount of 1 x 105
cells/well. Further, the prepared T cell population was added to each well in an
amount of 1 x 106 cells, and cultured at 37°C under 5% C02. Each culture
supernatant was discarded 7 days later, and dendritic cells obtained in the same
manner as described above by treatment with each polypeptide and the subsequent X-
ray irradiation were suspended in AIM-V medium supplemented with 10% human
AB serum (Nabi, Miami, FL), IL-7 (10 U/ml, Genzyme, Cambridge, MA) and IL-2
(10 U/ml, Genzyme, Cambridge, MA) (cell density, 1 x 105 cells/ml), which
suspension was then added to the 24-well plate in an amount of 1 x 105 cells/well,
followed by further culturing the cells. The same operation was repeated 4 to 6
times at intervals of 7 days, and stimulated T cells were then recovered, followed by
confirmation of induction of CD8-positive T cells by flow cytometry.
[0124]
Example 4: Determination of Cytotoxic T Cell Antigen Epitope in PDS5A That
Stimulates HLA-A0201-positive CD8-positive T Cells
Among the induced T cells in the respective wells, growth of T cells
stimulated by each of the polypeptides of SEQ ID NOs:27 to 35 was confirmed by
counting of the cell number under the microscope. In order to investigate the
specificity of the respective T cells, whose growth was confirmed, to each
polypeptide used for pulsing, 5 x 103 T cells were added with respect to 5 x 104 T2
cells expressing the HLA-A0201 molecule (Salter RD et al., Immunogenetics, 21:
235-246 (1985), purchased from ATCC) pulsed with the polypeptide (each
polypeptide was added to AIM-V medium at a concentration of 10 fig/ml, and the
cells were cultured therein at 37°C under 5% CO2 for 4 hours), and the cells were
cultured in AIM-V medium supplemented with 10% human AB serum in a 96-well
plate for 24 hours. After recovering the supernatant after the culture, the amount of
production of IFN-y was measured by the ELISA method. As a result, higher
production of IFN-y was confirmed in the culture supernatants in the wells containing
T2 cells pulsed with the respective polypeptides shown in SEQ ID NOs:27 to 35
compared to the culture supernatants in the wells containing T2 cells which were not
pulsed with a polypeptide (Fig. 12). Thus, it was revealed that each of the
polypeptides of SEQ ID NOs:27 to 35 is a T cell epitope peptide having a capacity to
stimulate and proliferate HLA-A0201-positive CD8-positive T cells, to induce
production of IFN-y. On the other hand, in the case where the polypeptide having
the amino acid sequence shown in SEQ ID NO:36, which is outside the scope of the
present invention, was added to perform the above-described treatment, no
production of IFN-y could be confirmed (Fig. 12).
[0125]
Subsequently, whether or not the respective polypeptides shown in SEQ ID
NOs:27 to 35, which are polypeptides to be used in the present invention, are
presented on HLA-A0201 molecules on HLA-A0201 -positive tumor cells expressing
PDS5A, and whether or not CD8-positive cells stimulated with the polypeptides can
damage HLA-A0201 -positive tumor cells expressing PDS5A, were studied.
[0126]
In a 50-ml centrifuge tube, 105 cells of a malignant brain tumor cell line T98G,
whose expression of PDS5A had been confirmed (Stein GH et at., J. Cell Physiol.,
99: 43-54 (1979), purchased from ATCC), were collected, and 100 uCi of chromium
51 was added to the tube, followed by incubation at 37°C for 2 hours. Subsequently,
the cells were washed 3 times with AIM-V medium supplemented with 10% human
AB serum, and placed in a 96-well V-bottomed plate in an amount of 103 cells per
well, followed by further addition, to each well, of 105, 5><104, 2.5 x 104 or 1.25 x 104
HLA-A0201-positive CD8-positive T cells suspended in AIM-V medium
supplemented with 10% human AB serum, which cells were stimulated with the
respective polypeptides shown in SEQ ID NOs:27 to 35. The cells were then
cultured at 37°C under 5% CO2 for 4 hours. Thereafter, the amount of chromium
51 released from damaged tumor cells into the culture supernatant was measured, and
thereby the cytotoxic activity of the CD8-positive T cells stimulated with each of the
polypeptides shown in SEQ ID NOs:27 to 35 was calculated.
[0127]
As a result, it was revealed that the HLA-A0201-positive CD8-positive T
cells stimulated with the respective polypeptides shown in SEQ ID NOs:27 to 35
have the cytotoxic activity against T98G (Fig. 13). Therefore, it became clear that
the polypeptides shown in SEQ ID NOs:27 to 35, which are polypeptide to be used in
the present invention, are presented on HLA-A0201 molecules on HLA-A0201-
positive tumor cells expressing PDS5A, and that these polypeptides have a capacity
to induce CD8-positive cytotoxic T cells which can damage such tumor cells. On
the other hand, in the case where the polypeptide having the amino acid sequence
shown in SEQ ID NO:36, which is outside the scope of the present invention, was
added to perform the above-described treatment, no cytotoxic activity could be
observed (Fig. 13).
[0128]
The cytotoxic activity was determined by, as described above, mixing 105
CD8-positive T cells stimulated and induced with each of the peptides of the present
invention and 103 cells of a malignant brain tumor cell line T98G to which chromium
51 was incorporated; culturing the resultant for 4 hours; measuring the amount of
chromium 51 released into the culture medium after the culturing; and calculating the
cytotoxic activity of the CD8-positive T cells against T98G according to the
following equation*.
[0129]
*Equation: cytotoxic activity (%) = (Amount of chromium 51 released from
T98G when CD8-positive T cells were added) / (Amount of chromium 51 released
from the target cells to which IN hydrochloric acid was added) x 100.
[0130]
Example 5: Preparation, and Evaluation of Pharmacological Effect, of Recombinant
PDS5A Protein; Detection of Cancer; and Cancer Diagnosis
(1) Preparation of Recombinant PDS5A Protein
Based on the gene of SEQ ID NO:l obtained in Example 1, a recombinant
protein was prepared by the following method. Regents and an attached buffer were
mixed such that 1 jlxI of the vector obtained in Example 1 which was prepared from
the phagemid solution and subjected to the sequence analysis, 0.4 jaM each of two
kinds of primers having the Notl andXhol restriction sites (shown in SEQ ID NOs:37
and 38), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase (manufactured by
Takara Shuzo Co., Ltd.) were contained in a total volume of 50 ul, and PCR was
carried out by repeating 30 times the cycle of 98°C for 10 seconds, 55°C for 15
seconds and 72°C for 4 minute using a Thermal Cycler (manufactured by BIO RAD).
The above-described two kinds of primers were those for amplification of the region
encoding the full-length of the amino acid sequence shown in SEQ ID NO:2. After
the PCR, the amplified DNA was subjected to electrophoresis using 1% agarose gel,
and a DNA fragment of about 4000 bp was purified using QIAquick Gel Extraction
Kit (manufactured by QIAGEN).
[0131]
The purified DNA fragment was ligated into a cloning vector pCR-Blunt
(manufactured by Invitrogen). E. coli was transformed with the resulting ligation
product, and the plasmid was then recovered. The amplified gene fragment was
confirmed to have the same sequence as that of interest by sequencing. The plasmid
having the same sequence as that of interest was treated with restriction enzymes
Notl and Xhol, and purified using QIAquick Gel Extraction Kit, followed by inserting
the gene sequence of interest into an expression vector for E. coli, pET30a
(manufactured by Novagen) that had been treated with restriction enzymes Notl and
Xhol. Use of this vector enables production of a His tag-fused recombinant protein.
E. coli for expression, BL21 (DE3), was transformed with this plasmid, and
expression was induced with 1 mM IPTG, to allow expression of the protein of
interest in E. coli.
[0132]
Further, based on the gene of SEQ ID NO:43, a recombinant protein of
human PDS5A was prepared by the following method. Regents and an attached
buffer were mixed such that 1 ul of the cDNA prepared in Example 1 whose
expression could be confirmed with cDNAs from various tissues and cells by the RT-
PCR method, 0.4 uM each of two kinds of primers having the Notl and Xhol
restriction sites (shown in SEQ ID NOs:39 and 40), 0.2 mM dNTP and 1.25 U
PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) were
contained in a total volume of 50 ul, and PCR was carried out by repeating 30 times
the cycle of 98°C for 10 seconds, 55°C for 15 seconds and 72CC for 4 minute using a
Thermal Cycler (manufactured by BIO RAD). The above-described two kinds of
primers were those for amplification of the region encoding the full-length of the
amino acid sequence shown in SEQ ID NO:44. After the PCR, the amplified DNA
was subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about
4000 bp was purified using QIAquick Gel Extraction Kit (manufactured by
QIAGEN).
[0133]
The purified DNA fragment was ligated into a cloning vector pCR-Blunt
(manufactured by Invitrogen). E. coli was transformed with the resulting ligation
product, and the plasmid was then recovered. The amplified gene fragment was
confirmed to have the same sequence as that of interest by sequencing. The plasmid
having the same sequence as that of interest was treated with restriction enzymes
Notl and Xhol, and purified using QIAquick Gel Extraction Kit, followed by inserting
the gene sequence of interest into an expression vector for E. coli, pET30a
(manufactured by Novagen) that had been treated with restriction enzymes Notl and
Xhol. Use of this vector enables production of a His tag-fused recombinant protein.
E. coli for expression, BL21 (DE3), was transformed with this plasmid, and
expression was induced with 1 mM IPTG, to allow expression of the protein of
interest in E. coli.
[0134]
Further, based on the gene of SEQ ID NO:5, a recombinant protein of murine
PDS5A was prepared by the following method. Regents and an attached buffer
were mixed such that 1 ul of the cDNA prepared in Example 1 whose expression
could be confirmed with cDNAs from various tissues and cells by the RT-PCR
method, 0.4 uM each of two kinds of primers having the Notl and Xhol restriction
sites (shown in SEQ ID NOs:41 and 42), 0.2 mM dNTP and 1.25 U PrimeSTAR HS
polymerase (manufactured by Takara Shuzo Co., Ltd.) were contained in a total
volume of 50 ul, and PCR was carried out by repeating 30 times the cycle of 98°C
for 10 seconds, 55°C for 15 seconds and 72°C for 4 minute using a Thermal Cycler
(manufactured by BIO RAD). The above-described two kinds of primers were
those for amplification of the region encoding the full-length of the amino acid
sequence shown in SEQ ID NO:6. After the PCR, the amplified DNA was
subjected to electrophoresis using 1% agarose gel, and a DNA fragment of about
4000 bp was purified using QIAquick Gel Extraction Kit (manufactured by
QIAGEN).
[0135]
The purified DNA fragment was ligated into a cloning vector pCR-Blunt
(manufactured by Invitrogen). E. coli was transformed with the resulting ligation
product, the plasmid was then recovered. The amplified gene fragment was
confirmed to have the same sequence as that of interest by sequencing. The plasmid
having the same sequence as that of interest was treated with restriction enzymes
Notl and Xhol, and purified using QIAquick Gel Extraction Kit, followed by inserting
the gene sequence of interest into an expression vector for E. coli, pET30a
(manufactured by Novagen) that had been treated with restriction enzymes Notl and
Xhol. Use of this vector enables production of a His tag-fused recombinant protein.
E. coli for expression, BL21 (DE3), was transformed with this plasmid, and
expression was induced with 1 mM IPTG, to allow expression of the protein of
interest in E. coli.
[0136]
(2) Purification of PDS5A Protein
Each of the above obtained recombinant E. coli that expresses SEQ ID NO.-2,
SEQ ID NO:44 or SEQ ID NO:6 was cultured in LB medium supplemented with 100
ug/ml ampicillin at 37°C until the absorbance at 600 nm reached about 0.7, and then
isopropyl-P-D-1-thiogalactopyranoside was added thereto to a final concentration of
1 mM, followed by further culturing the recombinant E. coli at 37°C for 4 hours.
Subsequently, the bacterial cells were collected by centrifugation at 4,800 rpm for 10
minutes. The pellet of the cells was suspended in phosphate-buffered saline and
further subjected to centrifugation at 4,800 rpm for 10 minutes to wash the bacterial
cells.
[0137]
The bacterial cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and
subjected to sonication on ice. The liquid obtained by the sonication of E. coli was
centrifuged at 6000 rpm for 20 minutes, to obtain the supernatant as the soluble
fraction and the precipitate as the insoluble fraction.
[0138]
The insoluble fraction was suspended in 50 mM Tris-HCl buffer (pH 8.0) and
centrifuged at 6000 rpm for 15 minutes. This operation was repeated twice to
perform an operation of removal of proteases.
[0139]
The residue was suspended in 50 mM Tris-HCl buffer (pH 8.0) supplemented
with 6 M guanidine hydrochloride and 0.15 M sodium chloride, and left to stand at
4°C for 20 hours to denature proteins. Thereafter, the suspension was centrifuged at
6000 rpm for 30 minutes, and the obtained soluble fraction was placed in a nickel
chelate column prepared by a conventional method (carrier: Chelating Sepharose
(trademark) Fast Flow (GE Health Care); column volume: 5 mL; equilibration buffer:
50 mM Tris-HCl buffer (pH 8.0) supplemented with 6M guanidine hydrochloride and
0.15 M sodium chloride), followed by leaving the resultant to stand at 4°C overnight
to allow adsorption of the proteins to the nickel-chelated carrier. This column
carrier was centrifuged at 1500 rpm for 5 minutes and the supernatant was then
recovered. The column carrier was suspended in phosphate-buffered saline and
refilled into the column.
[0140]
The fraction not adsorbed to the column was washed with 10 column volumes
of 0.1 M acetate buffer (pH 4.0) supplemented with 0.5 M sodium chloride, and
immediately thereafter, proteins were eluted with 0.1 M acetate buffer (pH 3.0)
supplemented with 0.5 M sodium chloride, to obtain a purified fraction, which was
used later as a material for an administration test. The protein of interest in each
eluted fraction was confirmed by Coomassie staining carried out according to a
conventional method.
[0141]
The buffer of the purified preparation obtained by the above method was
replaced with a reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl2
(pH8.0)), and the resulting sample was subjected to cleavage of the His tag with
factor Xa protease and purification of the protein of interest, using Factor Xa
Cleavage Capture Kit (manufactured by Novagen) in accordance with the protocol
attached to the kit. Subsequently, the buffer of 12 ml of the purified preparation
obtained by the above method was replaced with a physiological phosphate buffer
(manufactured by Nissui Pharmaceutical) using ultrafiltration NANOSEP 10K
OMEGA (manufactured by PALL), and the resulting sample was subjected to aseptic
filtration through HT Tuffryn Acrodisc 0.22 um (manufactured by PALL) and used
in the experiment.
[0142]
(3) Anti-tumor Effect of Recombinant Murine PDS5A Protein in Tumor-bearing
Mouse
The murine neuroblastoma cell line N2a was subcutaneously transplanted to
A/J mice (7 weeks old, male, purchased from Japan SLC) in an amount of 1 x 106
cells. When the tumor volume reached an average of 50 to 100 mm3 (typically 7
days after the inoculation of the tumor), the mice were randomly divided into groups
each of which contains 10 individuals, and subjected to evaluation of the anti-tumor
effect of the recombinant murine PDS5A protein (therapeutic model). With 100 ug
(0.5 ml) of the recombinant murine PDS5A protein purified as described above, 50
(j.g of poly I:C was mixed to prepare a therapeutic agent for cancer, and this
therapeutic agent was subcutaneously administered to the tumor-bearing mice a total
of 3 times at intervals of 1 week. As a result, on Day 31 after the administration of
the therapeutic agent for cancer, complete regression of the tumor was achieved.
On the other hand, in the negative control group to which PBS(-) was administered
and the group to which poly I:C alone (50 jag) was administered, the mean tumor
volumes on Day 31 after the administration were 1657 mm and 932 mm ,
respectively.
[0143]
Further, a therapeutic agent for cancer wherein 100 \xg (0.5 ml) of the
recombinant murine PDS5A protein and 50 u.g poly I:C were mixed was prepared,
and subcutaneously administered to A/J mice a total of 3 times at intervals of 1 week,
followed by transplantation of 1 x 106 N2a cells to the mice and evaluation of the
anti-tumor effect (prophylactic model). Ten individuals were included in each
group, and, as controls for comparison, a negative control group to which PBS(-) was
administered and a group to which poly I:C alone (50 ug) was administered were
provided. As a result, in the group to which the therapeutic agent for cancer was
administered, no development of a tumor was observed even on Day 40 after the
administration of the therapeutic agent for cancer. On the other hand, in the
negative control group to which PBS(-) was administered and the group to which
poly I:C alone (50 ug) was administered, the mean tumor volumes on Day 40 after
the administration were 1989 mm3 and 1843 mm3, respectively.
[0144]
The same experiment was carried out also for a colon cancer model. The
colon cancer cell line CT26 was subcutaneously transplanted to Balb/c mice (7 weeks
old, male, purchased from Japan SLC) in an amount of 1 * 106 cells. When the
tumor volume reached an average of 50 to 100 mm3 (typically 7 days after the
inoculation of the tumor), the mice were randomly divided into groups each of which
contains 10 individuals, and subjected to evaluation of the anti-tumor effect of the
recombinant murine PDS5A protein (therapeutic model). With 100 ug (0.5 ml) of
the recombinant murine PDS5A protein purified as described above, 50 ug of poly
I:C was mixed to prepare a therapeutic agent for cancer, and this therapeutic agent
was subcutaneously administered to the tumor-bearing mice a total of 3 times at
intervals of 1 week. As a result, on Day 24 after the administration of the
therapeutic agent for cancer, complete regression of the tumor was achieved. On
the other hand, in the negative control group to which PBS(-) was administered and
the group to which poly I:C alone (50 ug) was administered, the mean tumor volumes
on Day 24 after the administration were 1449 mm and 835 mm , respectively.
[0145]
Further, a therapeutic agent for cancer wherein 100 ug (0.5 ml) of the
recombinant murine PDS5 A protein and 50 ug poly I:C were mixed was prepared,
and subcutaneously administered to Balb/c mice a total of 3 times at intervals of 1
week, followed by transplantation of 1 x 106 CT26 cells to the mice and evaluation
of the anti-tumor effect (prophylactic model). Ten individuals were included in
each group, and, as controls for comparison, a negative control group to which PBS(-
) was administered and a group to which poly I:C alone (50 ug) was administered
were provided. As a result, in the group to which the therapeutic agent for cancer
was administered, no development of a tumor was observed even on Day 31 after the
administration of the therapeutic agent for cancer. On the other hand, in the
negative control group to which PBS(-) was administered and the group to which
poly I:C alone (50 ug) was administered, the mean tumor volumes on Day 31 after
the administration were 1781 mm and 1675 mm , respectively.
[0146]
From these results, it was revealed that the recombinant PDS5A protein is
effective for therapy and prophylaxis of cancer.
[0147]
(4) Anti-tumor Effect of Recombinant PDS5A Protein in Tumor-bearing Dog
The anti-tumor effect of the recombinant protein described in Example 5
below in 3 individuals of tumor-bearing patient dogs (3 individuals having a
mammary gland tumor) having a tumor mass in the epidermis was evaluated.
Before administration, the antibody titer against the recombinant protein in the serum
of each patient dog was measured by the method described in Example 5 (3), and, as
a result, an antibody titer higher than that of a healthy dog was detected. From these
results, it was suggested that the protein having the amino acid sequence shown in
SEQ ID NO:2 was expressed as a cancer antigen in the tumor tissue in the living
body of these tumor-bearing patient dogs.
[0148]
With 500 jag (2.5 ml) each of the recombinant PDS5A proteins (dog-derived
and human-derived) purified as described above, the same amount of Freund's
incomplete adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) was
mixed to prepare 2 kinds of therapeutic agents for cancer, each of which was
administered to a regional lymph node in the vicinity of the tumor a total of 3 times
at 1-week intervals. As a result, complete regression of the tumor, which had had a
size of about 500 mm3 or 1000 mm3 at the time of administration of each therapeutic
agent for cancer, was achieved on Day 13 or Day 21, respectively. On the other
hand, in the negative control group to which PBS(-) was administered, the tumor
volume, which had been about 800 mm3 at the time of administration of PBS,
became 1625 mm3 on Day 21 after the administration.
[0149]
With 500 ug (2.5 ml) of the canine recombinant PDS5A protein purified as
described in Example 5 below, the same amount of Freund's incomplete adjuvant
(manufactured by Wako Pure Chemical Industries, Ltd.) was mixed to prepare a
therapeutic agent for cancer, and this therapeutic agent was subcutaneously
administered in the vicinity of the tumor in 1 individual each of patient dogs
suffering from perianal adenocarcinoma and epidermal squamous cell carcinoma a
total of 4 times at 1-week interval. As a result, complete regression of the tumor,
which had had a size of about 370 mm3 or 280 mm3, respectively, at the time of
administration of the therapeutic agent for cancer, was achieved on Day 35 or Day 42,
respectively.
[0150]
(5) Detection of Cancer Using Recombinant PDS5A Protein
Blood was collected from 112 patient dogs wherein malignant tumor was
found and 30 healthy dogs, and sera were separated therefrom. Using the canine
PDS5A protein (SEQ ID NO:2) prepared in the above-described (2), the titer of
antibodies specifically reactive with the protein in each serum was measured by the
ELISA method. Immobilization of the prepared protein was carried out by placing
100 uL/well of the recombinant protein solution diluted to 5 ug/mL with phosphate-
buffered saline in a 96-well Immobilizer Amino plate (manufactured by Nunc),
followed by leaving the plate to stand at 4°C overnight. Blocking was carried out
by adding 100 uL of 50 mM sodium bicarbonate buffer (pH 8.4) supplemented with
3% BSA (bovine serum albumin, manufactured by Sigma-Aldrich Co.) (hereinafter
referred to as the blocking solution) to each well and shaking the plate at room
temperature for 1 hour. The sera were 1000-fold diluted with the blocking solution
and added to the wells in an amount of 100 uL/well, and the plate was shaken at
room temperature for 3 hours to allow the reaction to proceed. The wells were
washed 3 times with phosphate-buffered saline supplemented with 0.05% Tween 20
(manufactured by Wako Pure Chemical Industries, Ltd.) (hereinafter referred to as
PBS-T), and 100 uL/well of an HRP-modified anti-dog IgG antibody (Goat anti Dog
IgG-h+I HRP conjugated: manufactured by BETHYL Laboratories) 3000-fold diluted
with the blocking solution was added thereto, followed by shaking the plate at room
temperature for 1 hour to allow the reaction to proceed. After washing the wells 3
times with PBS-T, 100 ul/well of an HRP substrate TMB (1-Step Turbo TMB
(tetramethylbenzidine), PIERCE) was added, and the enzyme-substrate reaction was
allowed to proceed at room temperature for 30 minutes. Thereafter, 100 ul/well of
0.5 M sulfuric acid solution (manufactured by Sigma-Aldrich Japan) was added to
the wells to stop the reaction, and the absorbance at 450 nm was measured using a
microplate reader. To prepare controls for comparison, experiments were carried
out in the same manner as described above except that the prepared recombinant
protein was not immobilized or except that the tumor-bearing dog serum was not
reacted.
[0151]
All the 112 samples used for the above-described cancer diagnosis were those
which had been definitely diagnosed as malignant by pathological diagnosis using
extirpated tumor tissues.
[0152]
Specifically, the samples were those diagnosed as cancers such as malignant
melanoma, malignant mixed tumor, hepatocellular carcinoma, basal cell carcinoma,
intraoral tumor, perianal adenocarcinoma, anal sac tumor, anal sac apocrine
carcinoma, Sertoli cell tumor, vulva cancer, sebaceous adenocarcinoma, sebaceous
epithelioma, sebaceous adenoma, sweat gland carcinoma, intranasal adenocarcinoma,
nasal adenocarcinoma, thyroid cancer, colon cancer, bronchial adenocarcinoma,
adenocarcinoma, ductal carcinoma, mammary adenocarcinoma, combined mammary
adenocarcinoma, mammary gland malignant mixed tumor, intraductal papillary
adenocarcinoma, fibrosarcoma, hemangiopericytoma, osteosarcoma, chondrosarcoma,
soft tissue sarcoma, histiocytic sarcoma, myxosarcoma, undifferentiated sarcoma,
lung cancer, mastocytoma, cutaneous leiomyoma, intra-abdominal leiomyoma,
leiomyoma, squamous cell carcinoma, chronic lymphocytic leukemia, lymphoma,
gastrointestinal lymphoma, digestive organ lymphoma, small cell or medium cell
lymphoma, adrenomedullary tumor, granulosa cell tumor and pheochromocytoma.
[0153]
Sera from these cancer-bearing dogs showed significantly higher antibody
titers against the recombinant protein than sera from the healthy dogs. It was
revealed that, by diagnosing a sample showing a value not less than twice as high as
the average value in healthy dogs as malignant, 94 samples, which corresponds to
83.9% of the malignant cases, could be successfully diagnosed as malignant. The
types of the cancers in these 94 samples were as described below. It should be
noted that, although a part of the samples were suffering from a plurality of types of
cancers, each value shown below is the cumulative total for each type of cancer.
[0154]
Malignant melanoma, 5 cases; lymphoma, 10 cases; granulosa cell tumor, 1
case; hepatocellular carcinoma, 3 cases; malignant testicular tumor, 3 cases; intraoral
tumor, 3 cases; perianal adenocarcinoma, 5 cases; sarcoma, 9 cases; mammary
adenocarcinoma, 35 cases; lung cancer, 1 case; ductal carcinoma, 4 cases; sebaceous
adenocarcinoma, 2 cases; mastocytoma, 5 cases; leiomyosarcoma, 1 case; squamous
cell carcinoma, 4 cases; malignant mixed tumor, 2 cases; and hemangiopericytoma, 1
case.
[0155]
When cancer diagnosis was carried out in the same manner as described
above using the human PDS5A protein (SEQ ID NO:44) prepared in the above-
described (2), a similar result was obtained.
[0156]
From the above results, it was revealed that, by using the PDS5A protein to
measure the titer of antibodies specifically reactive with the protein in the serum,
detection and diagnosis of cancer is possible.
INDUSTRIAL APPLICABILITY
[0157]
The immunity-inducing agent of the present invention comprising a
polypeptide that exerts an anti-tumor activity against various types of cancers is
useful for therapy and/or prophylaxis of cancer, and/or detection of cancer.
We Claim:
1. An immunity-inducing agent comprising as an effective ingredient(s) at least
one polypeptide selected from the polypeptides (a) to (c) below, said polypeptide(s)
having an immunity-inducing activity/activities, or as an effective ingredient(s) a
recombinant vector(s) which comprise(s) a polynucleotide(s) encoding said
polypeptide(s) and is/are capable of expressing said polypeptide(s) in vivo:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12
and 44 in SEQUENCE LISTING;
(b) a polypeptide having a sequence identity of not less than 90% with said
polypeptide (a) and consisting essentially of not less than 7 amino acids; and
(c) a polypeptide comprising said polypeptide (a) or (b) as a partial sequence
thereof.
2. The immunity-inducing agent according to claim 1, wherein said polypeptide
(b) has a sequence identity of not less than 95% with said polypeptide (a).
3. The immunity-inducing agent according to claim 1, wherein each of said
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
consisting essentially of not less than 7 consecutive amino acids in any one of the
amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44, or a
polypeptide comprising said polypeptide as a partial sequence thereof; or a
polypeptide having the same amino acid sequence as a polypeptide consisting
essentially of not less than 7 consecutive amino acids in any one of the amino acid
sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 except that one or several
amino acids are deleted, substituted and/or added, or a polypeptide comprising said
polypeptide as a partial sequence thereof.
4. The immunity-inducing agent according to claim 3, wherein each of said
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
having any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12
and 44 in SEQUENCE LISTING.
5. The immunity-inducing agent according to claim 3, wherein each of said
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
consisting essentially of not less than 7 consecutive amino acids in the region of
aal11-140, aa211-240, aa248-278, aa327-357, aa459-522, aa909-972, aa959-1022,
aa994-1057 or aal018-1080 in any one of the amino acid sequences shown in SEQ
ID NOs:2, 6, 8, 10, 12 and 44 in SEQUENCE LISTING, or a polypeptide comprising
said polypeptide as a partial sequence thereof; or a polypeptide having the same
amino acid sequence as a polypeptide consisting essentially of not less than 7
consecutive amino acids in the region of aal 11-140, aa211-240, aa248-278, aa327-
357, aa459-522, aa909-972, aa959-1022, aa994-1057 or aal018-1080 in any one of
the amino acid sequences shown in SEQ ID NOs:2, 6, 8, 10, 12 and 44 in
SEQUENCE LISTING except that one or several amino acids are deleted, substituted
and/or added, or a polypeptide comprising said polypeptide as a partial sequence
thereof.
6. The immunity-inducing agent according to claim 5, wherein each of said
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
having any one of the amino acid sequences shown in SEQ ID NOs:27 to 35 in
SEQUENCE LISTING, or a polypeptide comprising said polypeptide as a partial
sequence thereof and having 10 to 12 amino acid residues; or a polypeptide having
the same amino acid sequence as a polypeptide having any one of the amino acid
sequences shown in SEQ ID NOs:27 to 35 in SEQUENCE LISTING except that one
or several amino acids are deleted, substituted and/or added, or a polypeptide
comprising said polypeptide as a partial sequence thereof and having 10 to 12 amino
acid residues.
7. The immunity-inducing agent according to any one of claims 1 to 6, for
prophylaxis of a cancer in an animal.
8. The immunity-inducing agent according to claim 5 or 6, for therapy of a
cancer in an animal.
9. The immunity-inducing agent according to claim 7 or 8, wherein said cancer
is a cancer expressing PDS5A.
10. The immunity-inducing agent according to any one of claims 7 to 9, wherein
said cancer is breast cancer, brain tumor, esophagus cancer, lung cancer, renal cancer,
colon cancer, perianal adenocarcinoma, neuroblastoma or leukemia.
11. The immunity-inducing agent according to any one of claims 1 to 10, further
comprising an immunoenhancer.
12. An isolated antigen-presenting cell comprising a complex between said
polypeptide having an immunity-inducing activity and an MHC molecule.
13. An isolated T cell which selectively binds to a complex between said
polypeptide having an immunity-inducing activity and an MHC molecule.
14. A polypeptide having any one of the amino acid sequences shown in SEQ ID
NOs:27 to 35 in SEQUENCE LISTING, or a polypeptide comprising said
polypeptide as a partial sequence thereof and having 10 to 12 amino acid residues; or
a polypeptide having the same amino acid sequence as a polypeptide having any one
of the amino acid sequences shown in SEQ ID NOs:27 to 35 in SEQUENCE
LISTING except that one or several amino acids are deleted, substituted and/or added,
or a polypeptide comprising said polypeptide as a partial sequence thereof and having
10 to 12 amino acid residues, which polypeptide has an immunity-inducing activity.
15. A method for detecting a cancer, said method comprising measurement of
expression of a polypeptide having any one of the amino acid sequences shown in
SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING or a polypeptide
having a sequence identity of not less than 90% with said polypeptide, in a sample
separated from a living body.
16. A method for inducing immunity, said method comprising administering to an
individual at least one polypeptide selected from the polypeptides (a) to (c) below,
said polypeptide(s) having an immunity-inducing activity/activities, or a recombinant
vector(s) which comprise(s) a polynucleotide(s) encoding said polypeptide(s) and
is/are capable of expressing said polypeptide(s) in vivo:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10,12
and 44 in SEQUENCE LISTING;
(b) a polypeptide having a sequence identity of not less than 90% with said
polypeptide (a) and consisting essentially of not less than 7 amino acids; and
(c) a polypeptide comprising said polypeptide (a) or (b) as a partial sequence
thereof.

ABSTRACT
An immunity-inducing agent comprising as an effective ingredient(s) a
polypeptide(s) selected from the polypeptides: (a) a polypeptide consisting essentially
of not less than 7 consecutive amino acids in any one of the amino acid sequences
shown in SEQ ID NOs:2, 4, 6, 8, 10, 12 and 44 in SEQUENCE LISTING; (b) a
polypeptide having a sequence identity of not less than 90% with the polypeptide (a)
and consisting essentially of not less than 7 amino acids; and (c) a polypeptide
comprising the polypeptide (a) or (b) as a partial sequence thereof; which
polypeptide(s) has/have an immunity-inducing activity/activities, or as an effective
ingredient(s) a recombinant vector(s) which comprise(s) a polynucleotide(s)
encoding the polypeptide(s) and is/are capable of expressing the polypeptide(s) in
vivo, is useful as a therapeutic and/or prophylactic agent for cancer, and/or the like.