DESCRIPTION
Immunity-inducing Agent and Method for Detection of Cancer
TECHNICAL FIELD
[0001]
The present invention relates to a novel immunity-inducing agent useful as a
therapeutic and/or prophylactic agent for cancer. Further, the present invention
relates to a novel method for detection of 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 in
combination with radiotherapy and chemotherapy. In spite of the developments of
new surgical methods and discovery of new anti-cancer agents in recent years,
treatment results of cancers are not 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 (Non-patent
Literature 1).
[0003]
In immunotherapy, to reduce side effects, it is necessary that the peptide,
polypeptide or protein 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 Literature 2). 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 (Non-patent Literature
3; Patent Literature 1), and several cancer antigens have been isolated by this method
(Non-patent Literatures 4 to 9). Using a part thereof 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
cancer, leukemia and lymphoma 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 and prophylactic agent exist which are 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
preparation 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]
Since early detection of cancer leads to good treatment results, a method for
detecting cancer which can be easily carried out by testing serum, urine or the like
without physical and economical burden to cancer patients is demanded. Recently,
methods wherein tumor products such as tumor markers are measured have been
widely used as diagnostic methods using blood or urine. Examples of the tumor
products include tumor-related antigens, enzymes, specific proteins, metabolites,
tumor genes, products of tumor genes, and tumor-suppressor genes, and, in some
cancers, a carcinoembryonic antigen CEA, glycoproteins CA19-9 and CA125, a
prostate-specific antigen PSA, calcitonin which is a peptide hormone produced in
thyroid, and the like are utilized as tumor markers in cancer diagnosis (Non-patent
Literature 10). However, in most types of cancers, there are no tumor markers
useful for cancer diagnosis. Further, since most of the tumor markers currently
known exist only in very small amounts (e.g., in the order of pg/mL) in body fluid,
their detection requires a highly sensitive measurement method or a special technique.
Under such circumstances, if a novel cancer detection method by which various
cancers can be detected by simple operations is provided, its use for diagnosis of
various cancers are expected to be developed.
[0006]
CD179b is known to be a part of the surrogate light chain of immunoglobulin
and to be expressed on the membrane surfaces of precursor cells of B cells (pre-B
cells and pro-B cells). It disappears upon differentiation of B cells and is not
expressed in mature B cells. However, CD 179b is known to be expressed in
leukemia (pre-B cell leukemia) cells produced by cancerization of pre-B cells (Non-
patent Literatures 10 and 11). Further, CD 179b is known to be expressed also in
lymphoma (pre-B cell lymphoma) cells produced by cancerization of pre-B cells, and
able to be used as a diagnostic marker for pre-B cell lymphoma (Non-patent
Literature 12). However, its specific expression has not been reported for leukemia
cells other than pre-B cell leukemia cells, lymphomas other than pre-B cell
lymphoma, breast cancer cells and the like. Further, there has been no report
suggesting that enhancement of immunity against CD 179b is useful for therapy
and/or prophylaxis of cancer.
PRIOR ART LITERATURES
Patent Literature
[0007]
Patent Literature 1: US 5698396 B
Non-patent Literatures
[0008]
Non-patent Literature 1: Tsuyoshi Akiyoshi, "Cancer and Chemotherapy",
1997, Vol. 24, pp. 551-519
Non-patent Literature 2: Bruggen P. et ah, Science, 254:1643-1647 (1991)
Non-patent Literature 3: Proc. Natl. Acad. Sci. USA, 92:11810-11813 (1995)
Non-patent Literature 4: Int. J. Cancer, 72:965-971 (1997)
Non-patent Literature 5: Cancer Res., 58:1034-1041 (1998)
Non-patent Literature 6: Int. J. Cancer, 29:652-658 (1998)
Non-patent Literature 7: Int. J. Oncol., 14:703-708 (1999)
Non-patent Literature 8: Cancer Res., 56:4766-4772 (1996)
Non-patent Literature 9: Hum. Mol. Genet 6:33-39 (1997)
Non-patent Literature 10: Adv. Immunol., 63:1-41 (1996)
Non-patent Literature 11: Blood, 92:4317-4324 (1998)
Non-patent Literature 12: Modern Pathology, 17:423-429 (2004)
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0009]
The present invention aims to discover a novel polypeptide useful as an agent
for therapy and/or prophylaxis and/or the like of cancer, thereby providing use of the
polypeptide for an immunity-inducing agent. The present invention also aims to
provide a method for detection of cancer, which is useful for diagnosis of cancer.
MEANS FOR SOLVING THE PROBLEMS
[0010]
The present inventors intensively studied to obtain, by the SEREX method
using serum from a canine patient from which a canine breast cancer tissue-derived
cDNA library was prepared, cDNA encoding a protein which binds to antibodies
existing in the serum derived from the tumor-bearing living body, and, based on a the
cDNA, canine CD 179b polypeptides having the amino acid sequences shown in the
odd number IDs of SEQ ID NOs:5 to 95 (that is, SEQ ID NOs:5, 7, 9, 11, 13. 15, ...,
91 and 93) in SEQUENCE LISTING were prepared. Further, based on a human
homologous gene of the obtained genes, a human CD 179b polypeptide having the
amino acid sequence shown in SEQ ID NO:3 was prepared, and, similarly, based on
a bovine homologous gene, a bovine CD 179b polypeptide having the amino acid
sequence shown in SEQ ID NO:95 was prepared. The present inventors then
discovered that that these CD 179b polypeptides are specifically expressed in breast
cancer, leukemia and lymphoma cells. Further, the present inventors discovered
that, by administration of these CD 179b to a living body, immunocytes against
CD 179b can be induced in the living body, and a tumor in the living body expressing
CD 179b can be regressed. Further, the present inventors discovered that a
recombinant vector comprising a polynucleotide encoding a CD 179b polypeptide or
a fragment thereof such that it can be expressed induces an anti-tumor effect against
cancer expressing CD 179b in the living body.
[0011]
Further, the present inventors discovered that a partial polypeptide in a
CD 179b protein 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 contacted with the
peptide and T cells contacted with the antigen-presenting cells are useful for the
therapy and/or prophylaxis of cancer. Further, the present inventors discovered that,
since a recombinant polypeptide prepared based on the amino acid sequence of the
above CD 179b protein specifically reacts only with serum of a tumor-bearing living
body, cancer can be detected therewith. Based on the above discoveries, the present
inventors completed the present invention.
[0012]
Thus, the present invention has the following characteristics.
[0013]
(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 the odd number IDs of SEQ
ID NOs:3 to 95 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.
[0014]
(2) The immunity-inducing agent according to (1) above, wherein the
polypeptide (b) has a sequence identity of not less than 95% with the polypeptide (a).
[0015]
(3) The immunity-inducing agent according to (1) above, 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 the odd number IDs of SEQ ID NOs:3 to 95 in
SEQUENCE LISTING, or a polypeptide comprising the polypeptide as a partial
sequence thereof.
[0016]
(4) The immunity-inducing agent according to (3) above, 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 the odd number IDs of SEQ ID
NOs:3 to 95 in SEQUENCE LISTING.
[0017]
(5) The immunity-inducing agent according to (3) above, 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 aal-
34 or aa52-75 in any one of the amino acid sequences shown in the odd number IDs
of SEQ ID NOs:3 to 95 in SEQUENCE LISTING, or a polypeptide comprising the
polypeptide as a partial sequence thereof.
[0018]
(6) The immunity-inducing agent according to (5) above, wherein each of the
polypeptide(s) having an immunity-inducing activity/activities is a polypeptide
consisting essentially of the amino acid sequence shown in SEQ ID NO: 108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ
IDNO:114, SEQ ID NO:115, SEQ ID NO:116 or SEQ ID NO:117 in SEQUENCE
LISTING, or a polypeptide comprising as a partial sequence thereof the amino acid
sequence shown in SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID NO: 112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ
ID NO: 116 or SEQ ID NO: 117 in SEQUENCE LISTING, the polypeptide having 8
to 12 amino acid residues.
[0019]
(7) The immunity-inducing agent according to any one of (1) to (6) above,
comprising one or more of the polypeptides as an effective ingredient(s).
[0020]
(8) The immunity-inducing agent according to (7) above, wherein the
polypeptide(s) is/are an agent(s) for treating antigen-presenting cells.
[0021]
(9) The immunity-inducing agent according to any one of (1) to (8) above,
which is for therapy and/or prophylaxis of an animal cancer(s).
[0022]
(10) The immunity-inducing agent according to (9) above, wherein the
cancer(s) is/are a cancer(s) expressing the CD179b gene.
[0023]
(11) The immunity-inducing agent according to (10), wherein the cancer(s)
is/are breast cancer, leukemia and/or lymphoma.
[0024]
(12) The immunity-inducing agent according to any one of (1) to (11) above,
further comprising an immunoenhancer.
[0025]
(13) An isolated antigen-presenting cell comprising a complex between the
polypeptide having an immunity-inducing activity and an HLA molecule.
[0026]
(14) An isolated T cell which selectively binds to a complex between the
polypeptide having an immunity-inducing activity and an HLA molecule.
[0027]
(15) 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 the odd number IDs of SEQ
ID NOs:3 to 95 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.
[0028]
(16) A method for detecting a cancer(s), which method is applied to a sample
separated from a living body and comprises measuring expression of at least one of
the polypeptides (a) to (c) below:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in the odd number IDs of SEQ
ID NOs:3 to 95 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.
(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence
thereof.
[0029]
(17) The method according to (16) above, wherein the measurement of
expression of the polypeptide(s) is carried out by measuring an antibody/antibodies
which may be contained in the sample by immunoassay, which antibody/antibodies
was/were induced in the living body against the polypeptide(s) to be measured.
[0030]
(18) A method for detecting a cancer(s), which is applied to a sample
separated from a living body and comprises investigation of expression of the
CD 179b gene having a coding region having any one of the base sequences shown in
SEQ ID N0:1 and the even number IDs of SEQ ID NOs:4 to 94 in SEQUENCE
LISTING in a sample derived from a cancer patient, and comparison thereof with the
expression level of the gene in a sample derived from a healthy individual.
[0031]
(19) A reagent for detecting a cancer(s), the reagent comprising a polypeptide
which undergoes antigen-antibody reaction with an antibody induced in a living body
against the polypeptide of any one of (a) to (c) below:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in the odd number IDs of SEQ
ID NOs:3 to 95 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
[0032]
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 tumor-bearing animal, immunocytes can be induced in the body of the
tumor-bearing animal, and a cancer which has already occurred can be reduced or
regressed.
[0033]
Further, by the present invention, a novel method for detection of cancer is
provided. Since measurement of expression of the polypeptide in a sample by the
method of the present invention enables detection of invisible small cancers and
cancers which exist in deep parts of a body, the method is also useful for early
detection of cancers in medical examinations and the like. If the method of the
present invention is used in following-up of patients after cancer therapy, recurrence
of the cancer can be detected in its early stage. Moreover, the method of the present
invention makes it possible to assess the stage of cancer progression such as growth
of the tumor, invasion of the tumor to the surrounding tissues, and metastasis of the
cancer to lymph nodes and distant organs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034]
Fig. 1 is a diagram showing the expression patterns of the gene encoding the
CD 179b protein in normal tissues and tumor cell lines. Reference numeral 1
represents the expression pattern of the gene encoding the CD 179b protein; and
reference numeral 2 represents the expression pattern of the GAPDH gene. In Fig.
1, reference numeral 1 in the ordinate represents the expression pattern of the gene
identified as described above, and reference numeral 2 represents the expression
pattern of the GAPDH gene as the control for comparison.
In Fig. 2, reference numerals 3,4, 5, 6, 7, 8, 9 and 10 in the abscissa indicate
the IFN-?-producing abilities of HLA-A0201-positive CD8-positive T cells due to
stimulation from T2 cells pulsed with the peptides of SEQ ID NOs:108, 109, 110,
113, 114, 115, 116 and 117, respectively. Reference numeral 11 indicates the result
for the peptide of SEQ ID NO: 118 used as the negative control (peptide having a
sequence outside the scope of the present invention).
In Fig. 3, reference numerals 12,13,14,15,16,17,18 and 19 in the abscissa
indicate the cytotoxic activities of HLA-A0201 -positive CD8-positive T cells against
Namalwa cells, which cells were stimulated using SEQ ID NOs:108, 109, 110, 113,
114,115, 116 and 117, respectively. Reference numeral 20 indicates the cytotoxic
activity of CD8-positive T cells induced using the peptide of the negative control
(SEQ ID NO: 118).
In Fig. 4, reference numerals 21, 22, 23, 24 and 25 in the abscissa indicate the IFN-?-producing abilities of HLA-A24-positive CD8-positive T cells due to
stimulation from JTK-LCL cells pulsed with the peptides of SEQ ID NOs: 110, 111,
112,115 and 116, respectively. Reference numeral 26 indicates the result for the
peptide of SEQ ID NO: 118 used as the negative control.
In Fig. 5, reference numerals 27,28,29,30 and 31 indicate the cytotoxic
activities of HLA-A24-positive CD8-positive T cells stimulated with the peptides of
SEQ ID NO: 110, 111, 112, 115 and 116, respectively, against JTK-LCL cells.
Reference numeral 32 indicates the cytotoxic activity of CD8-positive T cells
induced using the peptide of the negative control (SEQ ID NO:l 18).
BEST MODE FOR CARRYING OUT THE INVENTION
[0035]

Examples of the polypeptide contained in the immunity-inducing agent of the
present invention as an effective ingredient include one or more polypeptide(s)
selected from the polypeptides of (a), (b) and (c) below:
(a) a polypeptide which consists essentially of not less than 7 consecutive
amino acids in a polypeptide having any one of the amino acid sequences shown in
the odd number IDs of SEQ ID NOs:3 to 95 in SEQUENCE LISTING (that is, SEQ
ID NO:3, 5, 7, 9, 11, 13, 15,..., 93 and 95) and has an immunity-inducing activity;
(b) a polypeptide having a sequence identity of not less than 90% with the
polypeptide (a), consisting essentially of not less than 7 amino acids, and having an
immunity-inducing activity; and
(c) a polypeptide comprising the polypeptide (a) or (b) as a partial sequence
thereof and having an immunity-inducing activity.
[0036]
As used herein, 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 molecules. In the present invention, proteins
constituted by the total lengths of SEQ ID NO:3, 5, 7, 9,11,13,15,..., 93 and 95 are
also included therein.
[0037]
As used herein, the term "having an amino acid sequence" means that amino
acid residues are arrayed in a specific order. Therefore, for example, "a polypeptide
having the amino acid sequence shown in SEQ ID NO:3" means a polypeptide
having the amino acid sequence of Leu Leu Arg Pro ... (snip) ... Ala Glu Cys Ser
shown in SEQ ID NO:3, which polypeptide has a size of 176 amino acid residues.
Further, for example, "polypeptide having the amino acid sequence shown in SEQ ID
NO:3" may also be abbreviated as "polypeptide of SEQ ID NO:3". This also
applies to the term "having a base sequence".
[0038]
As used herein, the term "immunity-inducing activity" means an ability to
induce immunocytes which secrete cytokines such as interferon in a living body.
[0039]
Whether or not a 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
cocultivated with the polypeptide, followed by measuring the amount(s) of a
cytokine(s) and/or a chemokine(s) such as IFN-? and/or interleukin (IL) 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.
[0040]
Alternatively, as described in the later-mentioned Examples, when a
recombinant polypeptide prepared based on the amino acid sequence of SEQ ID
NO:3, 5, 7, 9, 11, 13, 15, ..., 93 or 95 is administered to a tumor-bearing animal, the
tumor can be reduced or 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 expressing the polypeptide of SEQ ID NO:3, 5, 7, 9, 11,13, 15,
..., 93 or 95, 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, observation of whether or not the
tumor is reduced or regressed when the polypeptide was administered to a tumor-
bearing living body, as more particularly described in the Examples below.
[0041]
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 cocultivation of the both
in a liquid medium, as mentioned below. Measurement of the cytotoxic activity can
be carried out by, for example, a known method called 51Cr release assay described in
Int. J. Cancer, 58: p317, 1994.
[0042]
In cases where the polypeptide is 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.
[0043]
The amino acid sequence shown in each of SEQ ID NOs:3, 5, 7, 9, 11, 13, 15,
... 93 and 95 in SEQUENCE LISTING is the amino acid sequence of a polypeptide
which binds to an antibody specifically existing in serum derived from a tumor-
bearing dog in the SEREX method using serum of the canine patient from which a
canine mammary gland cancer-derived cDNA library was prepared, or the amino acid
sequence of CD 179b isolated as a human homologous factor (homologue) of the
polypeptide (see Example 1 below). The polypeptide (a) is a polypeptide which
consists essentially of not less than 7 consecutive amino acids, preferably 8,9 or not
less than 10 consecutive amino acids in a polypeptide having any one of the amino
acid sequences shown in SEQ ID NO:3, 5, 7, 9, 11, 13, 15, ...,93 and 95 in
SEQUENCE LISTING and has an immunity-inducing activity. As 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:3,
5, 7, 9, 11, 13, 15,..., 93 or 95 can have an immunity-inducing activity, so that it can
be used for preparation of the immunity-inducing agent of the present invention.
[0044]
As a principle of immune induction by administration of a cancer antigenic
polypeptide, the following process is known: the 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 view point of presenting thereof on the surface of the antigen-
presenting cell, one preferred mode of the polypeptide (a) is a polypeptide composed
of about 7 to 30 consecutive amino acids in the amino acid sequence shown in SEQ
ID N0:3, 5, 7, 9, 11, 13, 15, ..., 93 or 95, and more preferably, a polypeptide
composed of 8 to 30 or 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 cells without incorporation thereof into the antigen-
presenting cells.
[0045]
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 entire region of SEQ ID NO:3, 5,7,
9, 11, 13, 15,..., 93 or 95 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 used, and the
polypeptide may be composed of not less than 30, preferably not less than 100, more
preferably not less than 200 amino acids, which polypeptide may be still more
preferably composed of the entire region of SEQ ID NO:3, 5, 7, 9, 11, 13, 15,..., 93
or 95.
[0046]
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 composed 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 composed of not less than 7 consecutive
amino acids in the region of aal-34 or aa52-75 in the amino acid sequence shown in
SEQ ID N0:3, 5, 7, 9, 11, 13, 15, ...,93 or 95 is preferred, and, in the polypeptide of
SEQ ID NO:3, the polypeptides shown in SEQ ID NOs:108 to 117 are more
preferred.
[0047]
The polypeptide (b) is the same polypeptide as the polypeptide (a) except that
a small number of amino acid residues are substituted, deleted, added and/or inserted,
which has a sequence identity of not less than 80%, preferably not less than 90%,
more preferably not less than 95%, still more preferably not less than 98%, 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 substantially the same antigenicity or immunogenicity as the
original even if the amino acid sequence of the protein is modified such that a small
number of amino acids are substituted, deleted, added 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:3, 5, 7, 9, 11, 13, 15, ...,93 or 95 except that
one or several amino acid residues are substituted, deleted, added and/or inserted.
[0048]
As used herein, the term "sequence identity" in relation to 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 (Karlin and Altschul,
Proc. Natl. Acad. Sci. U.S.A., 87:2264-2268, 1993; Altschul et al., Nucleic Acids
Res., 25:3389-3402, 1997). When a gap(s) is/are inserted, the above-described
number of the total amino acid residues (or the total bases) is the number of residues
(or bases) calculated by counting one gap as one amino acid residue (or base).
When the thus counted numbers of the total amino acid residues (or bases) are
different between the two sequences to be compared, the identity (%) is calculated by
dividing the number of matched amino acid residues (or bases) by the number of the
total amino acid residues (or the total bases) in the longer sequence.
[0049]
Among substitutions of amino acid residues, conservative amino acid
substitutions are preferred. The 20 types of amino acids constituting the naturally
occurring proteins may be classified into groups each of which has similar properties,
for example, into neutral amino acids with side chains having low polarity (Gly, IIe,
Val, Leu, Ala, Met, Pro), neutral amino acids having hydrophilic side chains (Asn,
Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys,
His) and aromatic amino acids (Phe, Tyr, Trp, His). It is known that, in most cases,
substitutions of amino acids within the same group, that is, conservative substitutions,
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 made high
by conducting the substitution(s) within the same group.
[0050]
The polypeptide (c) comprises the polypeptide (a) or (b) as a partial sequence
thereof and has an immunity-inducing activity. That is, the polypeptide (c) has
another/other amino acid(s) or polypeptide(s) added at one end or the 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.
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 transfectedintroduced into a host cell, followed by allowing the
polypeptide to be produced in the host cell.
[0051]
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:4 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:4 can be amplified
therewith. In the case of DNA having the base sequence of SEQ ID NO: 1, this 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. Methods, conditions and the like of PCR are described in,
for example, Ausubel et al., Short Protocols in Molecular Biology, 3rd ed.. A
compendium of Methods from Current Protocols in Molecular Biology (1995), John
Wiley & Sons (in particular, Chapter 15). 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: 1 to 95 in
SEQUENCE LISTING in the present specification, and screening a cDNA library of
human, dog, bovine 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:3, 5, 7, 9, 11, 13, 15, ...,93 or 95. The above-described operations such as
preparation of the probe(s) or primer(s), construction of a cDNA library, screening of
the cDNA library and cloning of the gene of interest are known to those skilled in the
art, and can be carried out according to the methods described in, for example,
Sambrook et al., Molecular Cloning, Second Edition, Current Protocols in Molecular
Biology (1989); and Ausubel et al. (described above). From the thus obtained DNA,
DNA encoding the polypeptide (a) can be obtained. Further, since codons encoding
each amino acid are known, a base sequence of a polynucleotide encoding a specific
amino acid sequence can be easily specified. Therefore, the base sequences of
polynucleotides encoding the polypeptide (b) and polypeptide (c) can also be easily
specified, so that such polynucleotides can also be easily synthesized using a
commercially available nucleic acid synthesizer according to a conventional method.
[0052]
The host cells are not restricted as long as they 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 COS 1, Chinese hamster ovary cells CHO, a
human embryonic kidney cell line HEK293 and a mouse embryonic skin cell line
NIH3T3; budding yeast; fission yeast; silkworm cells; and Xenopus laevis egg cells.
[0053]
In cases where prokaryotic cells are used as the host cells, an expression
vector having the origin that enables its replication in a prokaryotic cell, promoter,
ribosome binding site, multicloning site, terminator, drug resistant gene, nutrient
complementary gene and/or the like 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 (e.g., green fluorescent
protein (GFP) or glutathione S-transferase (GST)).
[0054]
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-N1,
pEGFP-Cl or the like is used as the expression vector, the above polypeptide can be
expressed as a fusion protein to which a tag such as His tag (e.g., (His)6 to (His)10),
FLAG tag, myc tag, HA tag or GFP was added.
[0055]
For the introduction of the expression vector into the host cells, well-known
methods such as electroporation, the calcium phosphate method, the liposome
method, the DEAE dextran method and microinjection can be used.
[0056]
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 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.
[0057]
The polypeptides obtained by the above method 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 polypeptide (c). Further, in some cases, a
polypeptide expressed in a transformed cell is modified in various ways in the cell
after translation thereof. Such a polypeptide modified after translation thereof 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-terminus methionine, N-terminus acetylation, glycosylation, limited
degradation by an intracellular protease, myristoylation, isoprenylation and
phosphorylation.
[0058]

As described concretely in the following Examples, the above-described
polypeptide having an immunity-inducing activity can cause regression of an already
occurred tumor when administered to a tumor-bearing animal. Therefore, the
immunity-inducing agent of the present invention can be used as a therapeutic and/or
prophylactic agent for cancer.
[0059]
The terms "cancer" and "tumor" used in the present specification mean a
malignant neoplasm, and are used interchangeably.
[0060]
In this case, cancers to be treated are those expressing the CD 179b gene, such
as cancers expressing the gene encoding the polypeptide of SEQ ID NO: 3, 5, 7, 9, 11,
13, 15,..., 93 or 95, preferably breast cancer, leukemia and lymphoma. Examples
of these particular cancers include, but are not limited to, breast cancers (mammary
gland cancer, combined mammary gland cancer, mammary gland malignant mixed
tumor, intraductal papillary adenocarcinoma and the like), leukemias (chronic
lymphocytic leukemia and the like), lymphomas (gastrointestinal lymphoma,
digestive organ lymphoma, small/medium cell lymphoma and the like).
[0061]
The above-described polypeptide, or a recombinant vector comprising a
polynucleotide encoding the polypeptide and capable of expressing the polypeptide in
vivo can be used as a therapeutic method for immune induction. Further, it can be
used as a therapeutic method for the purpose(s) of therapy and/or prophylaxis of
animal cancer, and can also be used as a therapeutic method further comprising an
immunoenhancer.
[0062]
The subject animal is a mammal such as a primate, pet animal, domestic
animal or sport animal, preferably human, dog or cat.
[0063]
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. Further, the dose may vary depending on the body
weight, sex (male or female), symptoms and the like. The dose effective for therapy
and/or prophylaxis of cancer is appropriately selected depending on the size of the
tumor, the symptom and the like, and usually, 0.0001 µg to 1000 µg, preferably 0.001
µg to 1000 µg 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.
[0064]
As concretely shown in the Examples below, the immunity-inducing agent of
the present invention can cause reduction or regression of an already occurred tumor.
Therefore, since the agent can exert its anticancer activity also against a small
number of cancer cells in the 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.
[0065]
The immunity-inducing agent of the present invention may contain only a
polypeptide or may be formulated by mixing 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 sucrose,
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.
[0066]
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.
[0067]
Here, the patient is an animal, especially a mammal, preferably human, dog or
cat.
[0068]
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) and homologues of Salmonella minnesola 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 W096/33739 (SmithKline
Beecham); QS-7, QS-17, QS-18 and QS-L1 (So et al., "Molecules and cells", 1997,
Vol. 7, p. 178-186); Freund's incomplete adjuvant; Freund's complete adjuvant;
vitamin E; Montanide; alum; CpG oligonucleotides (for example, Kreig et al.. 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 (for example, Goding, "Monoclonal Antibodies:
Principles and Practice, 2nd edition", 1986) may be used when the immunity-
inducing agent of the present invention is administered. Preparation methods for
mixtures or emulsions of a polypeptide and an adjuvant are well-known to those
skilled in the art of vaccination.
[0069]
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-ß interferon-?, interferon-?, 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 can be
contained in the immunity-inducing agent of the present invention, or can 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
[0070]

As concretely described in the Examples below, by bringing the above-
described polypeptide used in the present invention 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 include dendritic cells and B cells, and dendritic cells and B cells having MHC
class I molecules are preferably employed. The agents for treating antigen-
presenting cells mean agents for pulsing antigen-presenting cells, and, since pulsed
antigen-presenting cells can have an ability to stimulate peripheral blood
lymphocytes, the cells can be used as a vaccine.
[0071]
Various MHC class I molecules have been identified and 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.
[0072]
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.
[0073]
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 a fresh
sample, cold-stored sample or 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 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 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, 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 the proliferation
of the leukocytes is attained, and 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 the proliferation of the leukocytes is attained, and 5% CO2 is
preferably allowed to flow. The culturing period is not restricted as long as a
necessary number of the cells is induced therewith, and is usually 3 days to 2 weeks.
As for the apparatuses used for separation and culturing of the cells, appropriate
apparatuses, preferably those whose safety when applied 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 the general vessels such as a
Petri dish, flask and bottle, but also a layer type vessel, multistage vessel, roller bottle,
spinner type bottle, bag type culturing vessel, hollow fiber column and the like may
be used.
[0074]
Bringing the above-described peptide 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 µg/ml to 100 µg/ml, preferably about 5 µg/ml to 20
µg/ml. The cell density during the culturing is not restricted and usually about 103
cells/ml to 107 cells/ml, preferably about 5x 104 cells/ml to 5 x 106 cells/ml. The
culturing may be carried out according to a conventional method, and is preferably
carried out at 37°C under 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.
[0075]
By culturing the antigen-presenting cells in the coexistence of the above-
described polypeptide, the polypeptide is incorporated into MHC molecules 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 molecules 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.
[0076]
By bringing the antigen-presenting cells prepared as described above having
the complex between the above-described polypeptide and the MHC molecules 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 cocultivating 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
thereto T cells and then culturing the cells. The mixing ratio of the antigen-
presenting cells to the T cells in the cocultivation 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 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 cocultivation is preferably
carried out at 37°C under atmosphere of 5% CO2 in accordance with a conventional
method. The culturing time is not restricted, and is usually 2 days to 3 weeks,
preferably about 4 days to 2 weeks. The cocultivation 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
this case, the concentration of IL-2 and IL-7 is usually about 5 U/ml to 20 U/ml, the
concentration of IL-6 is usually about 500 U/ml to 2000 U/ml, and the concentration
of IL-12 is usually about 5 ng/ml to 20 ng/ml, but the concentrations of the
interleukins are not restricted thereto. Here, "U" indicates the unit of activity. The
above cocultivation may be repeated once to several times adding fresh antigen-
presenting cells. For example, the operation of discarding the culture supernatant
after the cocultivation and adding a fresh suspension of antigen-presenting cells to
further conduct the cocultivation may be repeated once to several times. The
conditions of the each cocultivation may be the same as described above.
[0077]
By the above-described cocultivation, 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 the complex
between the polypeptide and the MHC molecule.
[0078]
As described in the Examples below, the genes encoding the polypeptides of
SEQ ID NOs:3, 5, 7, 9, 11, 13, 15,..., 93 and 95 are expressed specifically in breast
cancer cells, leukemia cells and lymphoma cells. Therefore, it is thought that, in
these cancer species, significantly higher numbers of the polypeptides of SEQ ID
NOs:3, 5, 7, 9, 11, 13, 15,..., 93 and 95 exist than in normal cells. When cytotoxic
T cells prepared as described above are administered to a living body while a part of
the polypeptides existing in cancer cells are presented by MHC molecules on the
surfaces of the cancer cells, the cytotoxic T cells can damage the cancer cells using
the presented polypeptides as markers. Since antigen-presenting cells presenting the
above-described polypeptides can induce, and allow proliferation of, cytotoxic T
cells specific to the polypeptides 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.
[0079]
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.
[0080]
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 such as intravenous or intraarterial
administration. The dose is appropriately selected depending on the symptom, 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 per several days to once per 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.
[0081]

Also by expression of the polynucleotide encoding the polypeptide (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 a 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 the polynucleotide (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 is also
called gene vaccine.
[0082]
The vector used for production of a gene vaccine is not restricted as long as it
is a vector capable of expressing a 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.
[0083]
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 usually about 0.1 µg to 100 mg, preferably about 1 µg to 10 mg in terms of
the weight of the gene vaccine per 1 kg of body weight.
[0084]
Methods 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 the 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.
[0085]
Examples of other methods include a method wherein an expression plasmid
is directly intramuscularly administered (DNA vaccine method), the liposome
method, lipofectin method, microinjection method, calcium phosphate method and
electroporation method, and the DNA vaccine method and liposome method are
especially preferred.
[0086]
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 transfected into the body, and the ex
vivo method wherein a kind of cells are collected from the subject animal and the
gene is transfected 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 papers and the like). The in vivo method is more
preferred.
[0087]
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, symptom and so on. It may be administered by, for example,
intravenous, intraarterial, subcutaneous, intramuscular administration or the like, or
may be directly administered to the affected area in which a tumor exists. 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
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.
[0088]
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: 1, but also the
sequence complementary thereto.. Thus, "a 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 the polynucleotide
encoding the polypeptide used in the present invention is prepared, any one of these
base sequences should be appropriately selected, and those skilled in the art can
easily carry out the selection.
[0089]

In a method of the present invention for detection of cancer, expression of the
polypeptide used in the present invention is measured using a sample separated from
a living body. The method for measuring the expression of a 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
which encodes 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
three methods. In the present invention, the term "measurement" includes detection,
quantification and semi-quantification.
[0090]
Here, CD 179b was identified as a polypeptide which binds to an antibody
(cancer-specific antibody) specifically existing in serum derived from a tumor-
bearing dog, by the SEREX method using serum from a canine patient from which a
canine breast cancer-derived cDNA library was prepared (see Example 1). That is,
in the living body of a tumor-bearing dog, an antibody against CD 179b is specifically
induced. Thus, also by measuring an antibody against CD 179b in a tumor-bearing
living body, a cancer expressing CD179b can be detected (see Example 7). Further,
a canine cancer can be detected also by measuring CD 179b as an antigen by the
above Method 2. Further, since, as described in the Examples below, mRNA
encoding the antigen polypeptide is significantly more highly expressed in cancer,
especially in breast cancer and leukemia cells, than in normal tissues (see Example 1),
a canine cancer can be detected also by measuring the mRNA. As mentioned above,
CD 179b is known to be expressed on the membrane surfaces of precursor cells of B
cells (pre-B cells), and therefore it is reported that CD 179b is expressed in leukemia
(pre-B cell leukemia) cells derived by cancerization of pre-B cells, but the fact that
leukemia cells other than pre-B cell leukemia cells and breast cancer cells show
expression of CD 179b was first discovered in the present invention. Accordingly,
detection of leukemia other than pre-B cell leukemia cells, lymphoma and breast
cancer became possible by investigating expression of CD 179b.
[0091]
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 immunologically reacts with the antibody. The immunoassay per
se is a conventional well-known method as explained in detail below. As the
antigenic substance which may be used in the immunoassay, the polypeptide (a) to
(c) may be used. As antibodies have cross-reactivity, a molecule may be bound to
an antibody which is induced against another immunogen, as long as the molecule
has any structure thereon which is similar to the epitope of the immunogen. For
example, polypeptides having high amino acid sequence homology to each other
often have epitopes with similar structures, and in such cases the both polypeptides
may have the same antigenicity. As concretely described in the Examples below,
the human-derived polypeptide of SEQ ID NO:3 immunologically reacts with the
antibody induced in the body of a tumor-bearing dog. Therefore, in Method 1 of the
present invention, any mammalian homologous factor may be used as an antigen in
the immunoassay.
[0092]
Antigenic substances having a large molecular weight and a complex
structure, such as proteins, usually have a plurality of sites with different structures
on their surface. Therefore, such an antigenic substance induces a plurality of kinds
of antibodies which respectively recognize each of the sites 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
an antibody which exists in serum derived from a living body having an antigenic
substance therein and is induced in the living body against the antigenic substance.
[0093]
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 includes, 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 agglutination method may be preferably used as an
immunoassay of the above antibody in the present invention, as these methods are
simple and do not require a large-scale apparatus. In cases where an enzyme is used
as a label of an antibody, the used enzyme is not particularly restricted as long as it
satisfies such conditions that the turnover number is large, that the enzyme is stable
even when it is bound to an antibody, that it specifically colors its substrate and the
like. For example, enzymes used in an ordinary enzyme immunoassay such as
peroxidase, (3-galactosidase, alkaline phosphatase, glucose oxidase,
acetylcholinesterase, glucose-6-phosphate dehydrogenase, and malate dehydrogenase
may be used. Enzyme inhibitors, coenzymes and the like may also be used.
Binding of these enzymes with an antibody may be carried out by a known method
using a cross-linking agent such as a maleimide compound. As a substrate, known
substances may be used depending on the kind of the used enzyme. 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 the radioisotope, those used in an ordinary
radioimmunoassay such as l25I or H may be used. As the fluorescent dye, one used
in an ordinary fluorescent antibody technique, such as fluorescein isothiocyanate
(FITC), tetramethylrhodamine isothiocyanate (TRJTC) or the like may be used.
[0094]
These immunoassays per se are well-known in the art, and so it is not
necessary to explain these immunoassays in the present specification. Briefly, in
sandwich immunoassays, 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. In the method for detecting cancer according to the
present invention, it is preferred to immobilize an antigen polypeptide on a solid
phase, because immobilization on a solid phase makes it possible to easily remove
the unbound secondary antibody. As the secondary antibody, for example, anti-dog
IgG antibody may be used in cases where the sample is obtained from dogs. The
secondary antibody bound to the solid phase may be measured by labeling the
secondary antibody with a labeling substance exemplified above. The thus
measured amount of the secondary antibody corresponds to the amount of the above-
mentioned antibody in a 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 is decomposed by the enzymatic activity to develop a color, and then
optically measuring the amount of decomposed substrate. In cases where a
radioisotope is used as the labeling substance, the amount of radiation from the
radioisotope may be measured with a scintillation counter or the like.
[0095]
In Method 2 of the present invention, the polypeptide shown in SEQ ID NO:3,
5, 7, 9, 11, 13, 15, ..., 93 or 95 is measured, which polypeptide may be contained in
the sample obtained from a living body. As explained above, the abundance of the
cancer-specific antibody which immunologically reacts with the polypeptide shown
in SEQ ID NO:3, 5, 7, 9,11, 13,15,..., 93 or 95 or a homologous factor thereof is
significantly high in cancer patients, which indicates that the production of the
polypeptide or a homologous factor thereof, which is the antigen of the cancer-
specific antibody, is significantly high in the cancer patients. Therefore, similarly to
Method 1 above, cancers in a living body can be detected by measuring the
polypeptide shown in SEQ ID NO:3, 5, 7, 9,11,13,15, ...,93 or 95 or a
homologous factor thereof.
[0096]
Measurement of the polypeptide in a sample may easily be carried out by a
well-known immunoassay. Specifically, for example, the polypeptide having the
amino acid sequence shown in the odd number ID of SEQ ID NOs:3 to 95 or a
homologous factor thereof which may exist in a sample may be measured by
preparing an antibody or antigen-binding fragment thereof which immunologically
reacts with the polypeptide having the amino acid sequence shown in the odd number
ID of SEQ ID NOs:3 to 95 or a homologous factor thereof, and then carrying out an
immunoassay using the prepared antibody or fragment thereof. The immunoassay
per se is a well-known conventional method as described above.
[0097]
The term "antigen-binding fragment" herein means an antibody fragment such
as the Fab fragment or the F(ab')2 fragment contained in an antibody molecule, which
has a binding capacity to an antigen. Although the antibody may be either a
polyclonal antibody or monoclonal antibody, a monoclonal antibody is preferred for
immunoassays and the like, because a high reproducibility is attained therewith.
Methods for preparing a polyclonal or monoclonal antibody using a polypeptide as an
immunogen are well-known, and the preparation may be easily carried out by a
conventional method. For example, antibodies against the polypeptide may be
induced by immunizing an animal with an immunogen, the polypeptide conjugated to
a carrier protein such as keyhole limpet hemocyanin (KLH) or casein, together with
an adjuvant. Then antibody-producing cells such as spleen cells or lymphocytes are
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:3, 5, 7, 9, 11, 13, 15, ..., 93 or 95 or a
homologous factor thereof is selected and proliferated, and then the monoclonal
antibody whose corresponding antigen is the above-mentioned protein may be
collected from the culture supernatant. The above-described method is a
conventional well-known method.
[0098]
In Method 3 of the present invention, mRNA encoding CD 179b, which may
be contained in a sample obtained from a living body, is measured. As concretely
described in the Examples below, the expression level of mRNA encoding CD 179b
is significantly high in cancer, especially, breast cancer and leukemia cells.
Therefore, cancers in a living body can be detected by measuring the mRNA in a
sample.
[0099]
In the detection method of the present invention, whether the subject living
body suffers from cancer or not or the like is determined based on the expression
level of the polypeptide measured as described above. Although the cancer
detection may be attained simply by measuring the expression of the polypeptide in
the subject living body, it is preferred to obtain the normal reference value by
determining the expression level of the polypeptide (the amount of the antibody,
polypeptide or mRNA) in one or more samples from healthy individuals to compare
the measured value in the subject living body with the normal reference value, in
view of increasing the detection accuracy. In order to further increase the detection
accuracy, the cancer reference value may be obtained by determining the expression
level of the polypeptide in samples obtained from many patients who have been
revealed to suffer from cancer to compare the measured value of the subject living
body with the both of the normal and cancer reference values. The above
mentioned reference values may be determined by expressing the expression level of
the polypeptide in each sample in values and calculating the average value thereof.
The normal and cancer reference values may be determined beforehand by measuring
the expression level of the polypeptide in many healthy and cancer subjects. Thus,
when the measured value is compared with the reference values in the method of the
present invention, the reference values may be those predetermined.
[0100]
The detection method of the present invention may be carried out in
combination with detection using other cancer antigens and/or cancer markers so that
the detection accuracy of cancers can be more improved.
[0101]
By the detection method of the present invention, cancers in a living body can
be detected. The method of the present invention can detect even an invisible small
tumor or a tumor which exists in a deep part of a body, and thus the method is useful
for early detection of cancer. Further, by applying the detection method of the
present invention to patients in the follow-up period after cancer therapy, the
recurrent cancer, if any, can be detect in its early stage.
[0102]
If the more cancer cells expressing the prescribed polypeptide to be measured
in the present invention proliferate in a tumor-bearing living body, the more the
polypeptides and mRNAs encoding them accumulate in the body, which causes the
increased amount of the antibodies against the above-mentioned polypeptides in the
serum. On the other hand, the more cancer cells decrease, the more the
accumulated polypeptides and mRNAs encoding them decrease in the living body,
which causes the decreased amount of the antibodies against the above-mentioned
polypeptides in the serum. Thus, if the expression level of the prescribed
polypeptide is high, it can be determined that tumor growth and/or metastasis of
cancer occurred, i.e., the stage of progression of cancer is advanced.
[0103]
Further, as shown in the Example below, when compared between the same
kinds of tumors, a malignant one produces significantly higher amount of the
antibodies than a benign one. Therefore, if the expression level of the prescribed
polypeptides is high, it can be determined that the grade of cancer malignancy is
higher. That is, the grade of cancer malignancy can also be detected by the method
of the present invention.
[0104]
Furthermore, the effect of the cancer therapy can be monitored based on the
increase or decrease in the expression level of the prescribed polypeptides.
Therefore, by observing the expression level of the above-mentioned polypeptides on
an individual during or after cancer therapy, a clue to assess how much the
administered anti-cancer agent was effective, or whether a portion of the tumor is left
in the patient after extirpation of the tumor can be obtained, as well as a clue to find
metastasis and/or recurrence as early as possible can be obtained during the follow-
up. Appropriate treatment of cancer results in decrease in the expression level of
the polypeptides compared to that in the tumor-bearing state before the therapy. In
such a case, it can be judged that the effect of the therapy which was (is being)
performed on the living body is/was good. In cases where the expression level of
the polypeptides increases or is sustained, or once decreases and then increases, it can
be judged that the effect of the therapy is not good enough. This may be a useful
basis for selection of a therapeutic method, such as decision to change the therapeutic
method or to change the dose of an anti-cancer agent.
[0105]
Cancers to be detected by the method of the present invention are those
expressing CD 179b (excluding pre-B cell tumors), and examples thereof include, but
are not limited to, mammary gland cancer, combined mammary gland cancer,
mammary gland malignant mixed tumor, intraductal papillary adenocarcinoma,
leukemias (preferably, chronic lymphocytic leukemia excluding those of the pre-B
cell type) and lymphomas (preferably, gastrointestinal lymphoma, digestive organ
lymphoma, small/medium cell lymphoma, medium cell lymphoma and multicentric
lymphoma, excluding those of the pre-B cell type). The living bodies to which the
method of the present invention applies are mammals, preferably humans, dogs and
cats.
[0106]
The sample to be subjected to the method of the present invention includes
body fluids such as blood, serum, plasma, ascites and pleural effusion; tissues; and
cells. In particular, 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.
[0107]
The polypeptide used as an antigen for immunoassay in Method 1 may be
provided as a reagent for detecting cancer. The reagent may consist only of the
above-mentioned polypeptide, or may contain various additives useful for stabilizing
the polypeptide, and the like. The reagent may also be provided in the form of
being immobilized on a solid phase such as a plate or membrane.
[0108]
The antibody or an antigen-binding fragment thereof which immunologically
reacts with the polypeptide of SEQ ID NO:3, 5, 7, 9, 11, 13, 15, ..., 93 or 95 or a
homologous factor thereof, which is used for measuring the polypeptide or the
homologous factor thereof by immunoassay in Method 2, may also be provided as a
reagent for detecting cancer. The reagent may also consist only of the above-
mentioned antibody or antigen-binding fragment thereof, or may contain various
additives useful for stabilizing the antibody or antigen-binding fragment thereof, and
the like. The antibody or antigen-binding fragment thereof may also be in the form
of being conjugated with a metal such as manganese or iron. Since such a metal-
conjugated antibody or antigen-binding fragment thereof accumulates in a site in
which a large amount of antigen protein exists when administered to a body, the
existence of cancer cells which produce the antigen protein can be detected by
measuring the metal by MRI or the like.
[0109]
Furthermore, the above-described polynucleotide for cancer detection used for
measuring mRNA in Method 3 may also be provided as a reagent for detecting
cancer. The reagent for detecting cancer may also consist only of the polynucleotide,
or may contain various additives useful for stabilizing the polynucleotide and the like.
The polynucleotide for cancer detection contained in the reagent is preferably a
primer or a probe.
EXAMPLES
[0110]
The present invention will now be described more concretely by way of
Examples, but the scope of the present invention is not limited to the particular
examples below.
[0111]
Example 1: Acquisition of Novel Cancer Antigen Protein by SEREX Method
(1) Preparation of cDNA Library
From a canine mammary gland cancer tissue removed by surgery, total RNA
was extracted by the Acid guanidium-Phenol-Chloroform method, and poly(A)+
RNA was purified using the Oligotex-dT30 mRNA purification Kit (manufactured by
Takara Shuzo Co., Ltd.) according to the protocol described in the attached
instructions.
[0112]
Using the obtained mRNA (5 ug), a canine mammary gland cancer-derived
cDNA phage library was synthesized. Preparation of the cDNA phage library was
carried out using cDNA Synthesis Kit, ZAP-cDNA Synthesis Kit, and ZAP-cDNA
Gigapack III Gold Cloning Kit (manufactured by STRATAGENE) in accordance
with the protocols attached to the kits. The size of the prepared cDNA phage library
was 2.99 x 105 pfu/ml.
[0113]
(2) Screening of cDNA Library with Serum
Using the canine mammary gland cancer-derived cDNA phage library
prepared as described above, immunoscreening was carried out. More particularly,
host E. coli (XL 1-Blue MRF) was infected with the library such that 2340 clones
were included in a F90x15 mm NZY agarose plate, followed by culture at 42°C for 3
to 4 hours to allow formation of plaques. The plate was covered with a
nitrocellulose membrane (Hybond C Extra; manufactured by GE Healthcare Bio-
Science) impregnated with IPTG (isopropyl-ß-D-thiogalactoside) at 37°C for 4 hours,
to allow induction and expression of proteins, thereby transferring the proteins to the
membrane. Thereafter, the membrane was recovered and soaked in TBS (10 mM
Tris-HCl, 150 mM NaCl pH 7.5) supplemented with 0.5% non-fat dry milk, followed
by being shaken at 4°C overnight to suppress nonspecific reactions. This filter was
allowed to react with 500-fold diluted canine patient serum at room temperature for 2
to 3 hours.
[0114]
As the above-described canine patient serum, a total of 3 serum samples were
used which were collected from each of the dog from which the above mammary
gland cancer was removed and another mammary gland cancer canine patient.
These sera were stored at -80°C and pretreated immediately before use. The
pretreatment of the sera was carried out by the following method. That is, host E.
coli (XLl-BLue MRF) was infected with X ZAP Express phage into which no
exogenous gene was inserted, and cultured on an NZY plate 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 recovering
the supernatant as an E. coli/phage extract. Thereafter, the recovered E. coli/phage
extract was passed through an NHS-column (manufactured by GE Healthcare Bio-
Science) to immobilize the proteins derived from the E. coli/phage. The serum
from the canine patient was passed through this protein-immobilized column and
allowed to react with the proteins, thereby removing antibodies that adsorb to E. coli
and the phage from the serum. The serum fraction passed through the column
without being adsorbed was 500-fold diluted with TBS supplemented with 0.5% non-
fat dry milk, and the resulting dilution was used as a material for the
immunoscreening.
[0115]
The membrane to which the thus treated serum and the above-described
fusion proteins were blotted was washed with TBS-T (0.05% Tween 20/TBS) 4 times,
and goat anti-dog IgG (Goat anti Dog IgG-h+I HRP conjugated; manufactured by
BETHYL Laboratories, Inc.) which was 5000-fold diluted with TBS supplemented
with 0.5% non-fat dry milk was allowed, as a secondary antibody, to react at room
temperature for 1 hour. Detection was carried out by an enzymatic coloring reaction
using the NBT/BCIP reaction solution (manufactured by Roche), and colonies whose
positions were identical to those of positive sites of the coloring reaction were
collected from the F90x1 5mm NZY agarose plate, and dissolved into 500 µl of SM
buffer (100 mM NaCl, 10 mM MgClSO4, 50 mM Tris-HCl, 0.01% gelatin, pH7.5).
The second and third screenings were carried out by repeating the same method as
described above until the colonies positive in the coloring reaction became single
colonies, thereby isolating 45 positive clones after screening of 92820 phage clones
reactive with IgG in the serum.
[0116]
(3) Homology Search of Isolated Antigen Genes
To subject the 45 positive clones isolated by the above method to sequence
analysis, an operation to convert the phage vector to a plasmid vector was carried out.
More particularly, 200 µl of a solution prepared such that the host E. coli (XL 1-Blue
MRF') was contained to an absorbance OD600 of 1.0,250 µl of the purified phage
solution and 1 µl of ExAssist helper phage (manufactured by STRATAGENE) were
mixed together, and the resulting mixture was allowed to react at 37°C for 15
minutes, followed by adding 3 ml of LB broth thereto and culturing the resultant at
37°C for 2.5 to 3 hours. This was immediately followed by 20 minutes of
incubation in a water bath at 70°C and centrifugation at 1000xg for 15 minutes, after
which the supernatant was collected as a phagemid solution. Subsequently, 200 µl
of a solution prepared such that the phagemid host E. coli (SOLR) was contained to
an absorbance OD600 of 1.0 and 10 µl of the purified phagemid solution were mixed
together, and the resulting mixture was allowed to react at 37°C for 15 minutes,
followed by plating a 50 µl aliquot of the resultant on LB agar medium supplemented
with ampicillin (50 µg/ml final concentration) and culturing at 37°C overnight.
Single colonies of the transformed SOLR were picked up and cultured in LB medium
supplemented with ampicillin (50 µg/ml final concentration) at 37°C, followed by
purifying plasmid DNAs having inserts of interest using QIAGEN plasmid Miniprep
Kit (manufactured by QIAGEN).
[0117]
Each purified plasmid was subjected to analysis of the full-length sequence of
the insert by the primer walking method using the T3 primer shown in SEQ ID
NO:96 and the T7 primer shown in SEQ ID NO:97. By this sequence analysis, the
gene sequences shown in the even number IDs of SEQ ID NOs:4 to 92 were obtained.
Using the base sequences and the amino acid sequences (odd number IDs of SEQ ID
NOs: 5 to 93) of these genes, homology search against known genes were carried out
using a homology search program BLAST (http://www.ncbi.nlm.nih.gov/BLAST/),
and, as a result, it was revealed that all the obtained 45 genes were those encoding
CD 179b. The homologies among the 45 genes were 94 to 99% in terms of the base
sequences and 96 to 99% in terms of the amino acid sequences. The homologies
between these genes and the gene encoding a human homologous factor were 62 to
82% in terms of the base sequences and 69 to 80% in terms of the amino acid
sequences, in the region translated to a protein. The base sequence of the human
homologous factor is shown in SEQ ID NO: 1, and the amino acid sequences of the
human homologous factor are shown in SEQ ID N0s:2 and 3. Further, the
homologies between these genes and the gene encoding a bovine homologous factor
were 68 to 82% in terms of the base sequences and 56 to 77% in terms of the amino
acid sequences, in the region translated to a protein. The base sequence of the
bovine homologous factor is shown in SEQ ID NO:94, and the amino acid sequence
of the bovine homologous factor is shown in SEQ ID NO:95. The homology
between the gene encoding the human homologous factor and the gene encoding the
bovine homologous factor was 62% in terms of the base sequences and 72% in terms
of the amino acid sequences, in the region translated to a protein.
[0118]
(4) Analysis of Expression in Various Tissues
Expressions of the genes obtained by the above method in canine and human
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 5-10 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 by 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 as follows, using primers specific to
the obtained canine genes (shown in SEQ ID NOs:98 and 99) and their human
homologous gene (shown in SEQ ID NOs: 100 and 101). That is, reagents and an
attached buffer were mixed such that concentrations/amounts of 0.25 µl of a sample
prepared by the reverse transcription reaction, 2 µM each of the above primers, 0.2
mM each of dNTPs, and 0.65 U ExTaq polymerase (manufactured by Takara Shuzo
Co., Ltd.) were attained in a total volume of 25 µl, and the reaction was carried out
with 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds
using a Thermal Cycler (manufactured by BIO RAD). The above-described primers
specific to genes having the base sequences shown in SEQ ID NOs: 98 and 99 were
for amplification of the positions 32 to 341 in the base sequence shown in SEQ ID
NO:4, and for amplification of the region common to all the canine CD 179b genes
shown in the even number IDs of SEQ ID NOs: 4 to 92. Further, the primers
specific to genes having the base sequences shown in SEQ ID NOs: 100 and 101 were
for amplification of the positions 216 to 738 in the base sequence shown in SEQ ID
NO: 1. As a control for comparison, primers specific to GAPDH (shown in SEQ ID
NOs: 102 and 103) were used at the same time. As a result, as shown in Fig. 1, the
obtained canine genes did not show expression in normal canine tissues at all, but
showed strong expression in canine breast cancer tissues. In terms of expression of
the human homologous gene, bone marrow was the only human normal tissue
wherein its expression was confirmed, but, in human cancer cells, its expression was
detected in leukemia cell lines and breast cancer cell lines, so that specific expression
of CD 179b in the leukemia cell lines and the breast cancer cell lines was confirmed.
[0119]
In Fig. 1, reference numeral 1 in the ordinate represents the expression pattern
of the gene identified as described above, and reference numeral 2 represents the
expression pattern of the GAPDH gene as the control for comparison.
[0120]
Example 2: Preparation of Canine and Human Novel Cancer Antigen Protein
(1) Preparation of Recombinant Protein
Based on the gene of SEQ ID NO:4 obtained in Example 1, a recombinant
protein was prepared by the following method. That is, reagents and an attached
buffer were mixed such that concentrations/amounts of 1 µl of the vector prepared
from the phagemid solution obtained in Example 1 and subjected to the sequence
analysis, 0.4 µM each of two kinds of primers having NdeI and KpnI restriction sites
(described in SEQ ID NOs:104 and 105), 0.2 mM dNTP, and 1.25 U PrimeSTAR HS
polymerase (manufactured by Takara Shuzo Co., Ltd.) were attained in a total
volume of 50 µl, and PCR was carried out with 30 cycles of 98°C for 10 seconds and
68°C for 40 seconds using a Thermal Cycler (manufactured by BIO RAD). The
above-described two kinds of primers were those for amplification of the region
encoding the 5th to 120th amino acids in the amino acid sequence shown in SEQ ID
NO:5. After the PCR, the amplified DNA was subjected to electrophoresis using
2% agarose gel, and a DNA fragment of about 350 bp was purified using QIAquick
Gel Extraction Kit (manufactured by QIAGEN).
[0121]
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 plasmids were recovered thereafter, followed by confirming, by
sequencing, that the sequence of the amplified gene fragment matches the sequence
of interest. The plasmid having the sequence that matched the sequence of interest
was treated with restriction enzymes Ndel and Kpnl and purified using QIAquick Gel
Extraction Kit, followed by inserting the gene sequence of interest into an expression
vector for E. coli, pET30b (manufactured by Novagen) that had been treated with
restriction enzymes Ndel and Kpnl. Usage of this vector enables production of a
His-tag fusion recombinant protein. E. coli for expression, BL21 (DE3), was
transformed with this plasmid, and expression of the protein of interest was induced
in E. coli with 1 mM IPTG.
[0122]
On the other hand, based on the gene of SEQ ID NO: 1, a recombinant protein
of the human homologous gene was prepared by the following method. Reagents
and an attached buffer were mixed such that concentrations/amounts 1 µl of the
cDNA prepared in Example 1 whose expression could be confirmed by the RT-PCR
method in cDNAs from various tissues/cells, 0.4 uM each of two kinds of primers
having EcoKl and Sail restriction sites (described in SEQ ID NOs: 106 and 107), 0.2
mM dNTP, and 1.25 U PrimeSTAR HS polymerase (manufactured by Takara Shuzo
Co., Ltd.) were attained in a total volume of 50 µl, and PCR was carried out with 30
cycles of 98°C for 10 seconds and 68°C for 40 seconds using a Thermal Cycler
(manufactured by BIO RAD). The above-described two kinds of primers were
those for amplification of the total length the amino acid sequence shown in SEQ ID
NO:3. After the PCR, the amplified DNA was subjected to electrophoresis using
2% agarose gel, and a DNA fragment of about 540 bp was purified using QIAquick
Gel Extraction Kit (manufactured by QIAGEN).
[0123]
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 plasmids were recovered thereafter, followed by confirming, by
sequencing, that the sequence of the amplified gene fragment matches the sequence
of interest. The plasmid having the sequence that matched the sequence of interest
was treated with restriction enzymes EcoRI and SalI 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 EcoRI and SalI. Usage of this vector enables
production of a His-tag fusion recombinant protein. E. coli for expression, BL21
(DE3), was transformed with this plasmid, and expression of the protein of interest
was induced in E. coli with 1 mM IPTG.
[0124]
(2) Purification of Recombinant Protein
The above-obtained recombinant E. coli cells that expresse SEQ ID NO:1 and
SEQ ID NO:4, respectively, were cultured in LB medium supplemented with 30
µg/ml kanamycin at 37°C until the absorbance at 600 nm reached about 0.7, and
then isopropyl-P-D-1-thiogalactopyranoside was added thereto such that its final
concentration should be 1 mM, followed by culturing them at 30°C for 20 hours.
Subsequently, the 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 cells.
[0125]
The cells were suspended in phosphate-buffered saline and subjected to
sonication on ice. The sonicated solution of E. coli was centrifuged at 7,000 rpm
for 20 minutes to obtain the supernatant as the soluble fraction and the precipitate as
the insoluble fraction.
[0126]
The insoluble fraction was suspended in 4% Triton-X100 solution and
centrifuged at 7,000 rpm for 20 minutes. This operation was repeated twice and an
operation of removal of proteases was carried out.
[0127]
The residue was suspended in 20 mM phosphate buffer (pH 8.0) containing
6M guanidine hydrochloride, and the resulting suspension was left to stand at 4°C for
20 hours to denature proteins. Thereafter, the suspension was centrifuged at 7,000
rpm for 20 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: 20 mM
phosphate buffer (pH 8.0) containing 6M guanidine hydrochloride). The fraction
that was not adsorbed to the column was washed away with 10 column volumes of
20 mM phosphate buffer (pH 8.0) containing 6M guanidine hydrochloride and 20
mM phosphate buffer (pH 8.0) containing 10 mM imidazole, and elution was
immediately carried out with a four-step density gradient of 50 mM-500 mM
imidazole, to obtain a purified fraction, which was used thereafter as a material for
administration tests.
[0128]
To 1 ml of a reaction buffer (20 mM Tris-HCl, 50 mM NaCl, 2 mM CaCl2;
pH 7.4), 200 µl of the purified preparation obtained by the above-described method
was aliquoted, and 2 µl of enterokinase (manufactured by Novagen) was then added
thereto, followed by leaving it to stand at room temperature overnight to cleave His
tag. The resulting product was purified using Enterokinase Cleavage Capture Kit
(manufactured by Novagen) in accordance with the protocol attached to the kit.
Subsequently, the buffer contained in 1.2 ml of the purified preparation obtained by
the above-described method was replaced with physiological phosphate buffer
(manufactured by Nissui Pharmaceutical) by ultrafiltration using NANOSEP 1 OK
OMEGA (manufactured by PALL), and the resulting solution was filtered aseptically
using HT Tuffryn Acrodisc 0.22 µm (manufactured by PALL) and used in the
following experiments.
[0129]
Example 3: Test of Administration of Recombinant Protein to Cancer-bearing Dog
(1) Antitumor Assay
The anti-tumor effect of the recombinant protein which was purified as
described above was assessed in a tumor-bearing dog (breast cancer) having an
epidermal tumor.
[0130]
An equal amount of Freund's incomplete adjuvant (manufactured by Wako
Pure Chemicals) was mixed with 100 µg (0.5 ml) of the recombinant polypeptide
purified as described above, to prepare a therapeutic agent for cancer. This was
administered to a regional lymph node in the vicinity of the tumor a total of 3 times,
by carrying out the subsequent administrations 3 days and 7 days after the first
administration. As a result, the tumor with a size of about 55 mm3 at the time of
administration of the therapeutic agent for cancer was reduced in size to 30 mm3 10
days after the first administration; to 16 mm 20 days after the first administration;
and to 10 mm 30 days after the first administration.
[0131]
Further, to another canine patient suffering from mammary gland cancer, a
mixture of 100 µg (0.5 ml) of the above-described polypeptide derived from dog and
0.5 ml of Freund's incomplete adjuvant was administered in the same manner as
described above a total of 3 times. Further, concurrently with the respective
administrations, 100 µg of canine interleukin 12 was administered subcutaneously.
As a result, the tumor with a size of about 155 mm3 at the time of administration of
the therapeutic agent for cancer completely regressed 24 days after the first
administration.
[0132]
(2) Immune Inducibility Assay
Blood of the canine patient in which the anti-tumor effect was obtained in the
administration test in the above-described (1) was collected before the administration
of the therapeutic agent for cancer, and 10 days and 30 days after the first
administration. Peripheral blood mononuclear cells were isolated according to a
conventional method, and by the ELISPOT assay for IFN? using them, the immune
inducibility of each administered recombinant protein was assayed.
[0133]
In a 96-well plate manufactured by Millipore (MultiScreen-IP, MAIPS 4510),
100 µL/well of 70% ethanol was placed and the plate was left to stand for 5 minutes,
followed by removal of the ethanol by aspiration. The plate was washed with sterile
water and 300 uL/well of 200 raM Sodium Bicarbonate (pH8.2) was placed therein.
After leaving it to stand for 5 minutes, Sodium Bicarbonate was removed by
aspiration, and then the plate was washed. Subsequently, 0.5 µl/well of anti-canine
interferon y monoclonal antibody (manufactured by R&D, clone 142529, MAB781)
mixed with 200 mM Sodium Bicarbonate was placed in wells, and the plate was
incubated at 37°C overnight to immobilize the primary antibody. After removal of
the primary antibody by aspiration, 300 uL/well of a blocking solution (1% BSA-5%
sucrose-200 mM Sodium Bicarbonate (pH8.2)) was added to the wells, and the plate
was incubated at 4°C overnight to block the plate. After removal of the blocking
solution by aspiration, 300 uL/well of 10% fetal calf serum-containing RPMI
medium (manufactured by Invitrogen) was placed in the wells, and the plate was left
to stand for 5 minutes, followed by removal of the medium by aspiration.
Subsequently, 5 x 105 cells/well of the canine peripheral blood mononuclear cells
suspended in 10% fetal calf serum-containing RPMI medium were placed in the plate,
and 10 µL/well of the canine-derived polypeptide or human-derived polypeptide used
in each administration was added thereto, followed by culturing the cells under the
conditions of 37°C and 5% CO2 for 24 hours, to allow immunocytes that might exist
in the peripheral blood mononuclear cells to produce interferon y. After the culture,
the medium was removed, and the wells were washed 6 times with a washing
solution (0.1 % Tween 20-200mM Sodium Bicarbonate (pH8.2)). In each well, 100
µl of rabbit anti-dog polyclonal antibody 1000-fold diluted with the above-described
blocking solution was placed, and the plate was incubated at 4°C overnight. After
washing the wells 3 times with the above-described washing solution, 100 µl of
HRP-labeled anti-rabbit antibody 1000-fold diluted with the above-described
blocking solution was placed in each well, and the reaction was allowed to proceed at
37°C for 2 hours. After washing the wells 3 times with the above-described
washing solution, the resultant was colored with Konica Immunostain (manufactured
by Konica), and the wells were washed with water to stop the reaction. Thereafter,
the membrane was dried, and the number of the appeared spots was counted using
KS ELISPOT (manufactured by Carl Zeiss, Inc.). As a result, in peripheral blood
mononuclear cells sampled before the administration of the polypeptide, no spot was
detected. On the other hand, in the canine patient after the administration of the
polypeptide, 18 and 87 spots were detected in the peripheral blood mononuclear cells
sampled 10 days and 30 days, respectively, after the administration.
[0134]
From the above results, it was confirmed that immunocytes which specifically
react with the administered recombinant protein and produce interferon y were
induced in the canine patient to which the recombinant protein was administered, and
it was thought that the anti-tumor effect described in the above-described (1) was
exerted by immunoreactions in which these immunocytes were mainly involved.
[0135]
Example 4: Induction of CD8-positive T Cells Reactive with Epitopes of CD 179b-
derived Peptide
(1) Prediction of Peptide Motifs Which Bind to HLA-A0201 and HLA-A24
Information on the amino acid sequence of the human CD 179b protein was
obtained from GenBank. For prediction of HLA-A0201 and HLA-A24 binding
motifs, the amino acid sequence of the human CD 179b protein was analyzed
employing a computer-based prediction program using a known BIMAS software
(available at http://bimas.dcrt.nih.gov/molbio/hla_bind/). As a result, 8 kinds of
peptides shown in SEQ ID NOs:108 to 110 and SEQ ID NOs:113 to 117, which were
expected to be capable of binding to the HLA-A0201 molecule; and 5 kinds of
peptides shown in SEQ ID NOs:110 to 112, SEQ ID NO:115 and SEQ ID NO:116,
which were expected to be capable of binding to the HLA-A24 molecule; were
selected.
[0136]
(2) Induction of Peptide Epitope-reactive CD8-positive T Cells
From an HLA-A0201-positive healthy individual, peripheral blood was
isolated, and the peripheral blood was overlaid on Lymphocyte separation medium
(OrganonpTeknika, Durham, NC), followed by centrifugation thereof at 1,500 rpm at
room temperature for 20 minutes. A PBMC-containing fraction was recovered and
washed 3 times (or more) with cold phosphate buffer to obtain peripheral blood
mononuclear cells (PBMCs). The obtained PBMCs were suspended in 20 ml of
AIM-V medium (manufactured by Life Technologies, Inc., Grand Island, NY), and
allowed to adhere to a culturing flask (manufactured by Falcon) at 37°C under 5%
CO2 for 2 hours. The cells which were not adhered were used for the preparation of
T cells, and the adhered cells were used for the preparation of dendritic cells.
[0137]
The adhered cells were cultured in AIM-V medium in the presence of IL-4
(1000 U/ml) and GM-CSF (1000 U/ml). Six days later, the medium was replaced
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-1ß (10 ng/ml, Genzyme,
Cambridge, MA) and TNF-a (10 ng/ml, Genzyme, Cambridge, MA), and the
culturing was continued for another 2 days. The obtained population of cells which
did not adhere was used as the dendritic cells.
[0138]
The prepared dendritic cells were suspended in AIM-V medium at a cell
density of 1 x 106 cells/ml, and the peptide shown in SEQ ID NOs: 108 to 110 or SEQ
ID NOs:l 13 to 117, which are sequences selected in the above (1) and expected to be
capable of binding to the HLA-A201 molecule, was added to the resulting suspension
at a concentration of 10 µg/ml, followed by culture using a 96-well plate under the
conditions of 37°C, 5% CO2 for 4 hours. Thereafter, the cells were irradiated with
X-ray (3000 rad), washed with AIM-V medium, suspended in AIM-V medium
containing 10% human AB serum (Nabi, Miami, FL), IL-6 (1000 U/ml) and IL-12
(10 ng/ml, Genzyme, Cambridge, MA), and placed in wells of a 24-well plate at a
population of 1 x 103 cells/well. The prepared T cell population was added to the
wells at a population of 1 x 106 cells/well, and the cells were cultured at 37°C under
5% CO2. Seven days later, each culture supernatant was discarded, and the cells
were treated with each of the peptides obtained in the same manner as described
above. After irradiation with X-ray, the dendritic cells were suspended in AIM-V
medium containing 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 105cells/ml), and the cells were placed in wells of a 24-well plate at a cell
population of 1 x 105 cells/well and further cultured. The same operations were
repeated 4 to 6 times at intervals of 7 days, and the stimulated T cells were then
recovered, after which induction of CD8-positive T cells were confirmed by flow
cytometry.
[0139]
Also for the peptides shown in SEQ ID NOs:110, 111, 112, 115 and 116,
which were expected to be capable of binding to the HLA-A24 molecule, induction
of peptide epitope-reactive CD8-positive T cells was attempted using dendritic cells
and a T cell population induced from peripheral blood of an HLA-A24-positive
healthy individual.
[0140]
As a negative control, a peptide outside the scope of the present invention
(SEQ ID NO: 118) was used.
[0141]
Example 5: Determination of CD179b-derived Cytotoxic T Cell Antigen Epitopes
Which Stimulate HLA-A0201 -positive CD8-positive T Cells
(1) IFN-?-Producing Ability
In order to examine the specificity of each of the T cells, whose growth was
confirmed among the T cells induced as described above, to peptide epitopes, 5x103
T cells were added to 5x104 T2 cells (Salter RD et al., Immunogenetics, 21:235-246
(1985), purchased from ATCC) which were pulsed with each peptide and expresses
the HLA-A0201 molecule (cultured in AIM-V medium supplemented with each
peptide at a concentration of 10 ng/ml, at 37°C under 5% CO2 for 4 hours), and the
cells were cultured in AIM-V medium containing 10% human AB serum in a 96-well
plate for 24 hours. The supernatant after the culturing was recovered and the
production amount of IFN-? was measured by ELISA. As a result, production of
IFN-? was confirmed in the culture supernatants in the wells of T2 cells pulsed with
the peptides of SEQ ID NOs:108 to 110 and SEQ ID NOs:113 to 117, when
compared with the culture supernatants in the wells of T2 cells which were not
pulsed with a peptide (Fig. 2). From these results, it was revealed that the above-
described peptides are T cell epitope peptides having a capacity to specifically
stimulate, and allow proliferation of, the HLA-A0201-positive CD8-positive T cells,
thereby inducing production of IFN-?.
[0142]
In Fig. 2, reference numerals 3, 4, 5, 6, 7, 8, 9 and 10 in the abscissa indicate
the IFN-?-producing abilities of the HLA-A0201-positive CD8-positive T cells due
to stimulation from the T2 cells pulsed with the peptides of SEQ ID NOs: 108, 109,
110, 113, 114, 115, 116 and 117, respectively. Reference numeral 11 indicates the
result for the peptide of SEQ ID NO: 118 used as the negative control.
[0143]
(2) Cytotoxicity Assay
Subsequently, whether or not the peptides of SEQ ID NOs: 108 to 110 and
SEQ ID NOs: 113 to 117 used in the present invention are presented on the HLA-
A0201 molecules on tumor cells which are HLA-A0201 -positive and express
CD 179b, and whether or not the CD8-positive T cells stimulated by these peptides
can damage the tumor cells which are HLA-A0201-positive and express CD 179b
were examined. In a 50-ml centrifugal tube, 106 cells of a B cell leukemia cell line,
Namalwa cells (purchased from ATCC), whose expression of CD 179b had been
confirmed, were collected, and 100 µCi of chromium 51 was added thereto, followed
by incubation at 37°C for 2 hours. Thereafter, the cells were washed 3 times with
RPMI medium (manufactured by Gibco) containing 10% fetal calf serum
(manufactured by Gibco), and placed in wells of a 96-well V-bottom plate in an
amount of 103 cells/well. Further, to each well, 5x104 T cells suspended in RPMI
medium containing 10% fetal bovine serum, which cells were stimulated by each
peptide, and HLA-A0201-positive, peptide epitope-reactive and CD8-positive, were
added, followed by culture at 37°C under 5% CO2 for 4 hours. Thereafter, by
measuring the amount of chromium 51 in the culture supernatant, which was released
from the damaged tumor cells, the cytotoxic activity of the CD8-positive T cells
stimulated by each peptide was calculated. As a result, it was revealed that the
HLA-A0201-positive CD8-positive T cells stimulated by the peptide have a cytotoxic
activity against Namalwa cells (Fig. 3). The CD8-positive T cells induced using the
negative control peptide (SEQ ID NO: 118) did not show a cytotoxic activity. Thus,
it was proved that each of the peptides used in the present invention (SEQ ID
NOs:108 to 110 and SEQ ID NOs:l 13 to 117) is presented on the HLA-A0201
molecules on tumor cells which are HLA-A0201-positive and express CD 179b, and
that the peptide has an ability to induce CD8-positive cytotoxic T cells which can
damage such tumor cells.
[0144]
The cytotoxic activity was determined by, as described above, mixing 105
CD8-positive T cells stimulated and induced with each of the peptides used in the
present invention and 103 cells of the B cell leukemia cell line Namalwa which were
made to incorporate chromium 51; culturing the resulting mixture for 4 hours;
measuring the amount of chromium 51 released to the culture medium after the
culturing; and calculating the cytotoxic activity of the CD8-positive T cells against
the Namalwa cells according to the following equation*.
[0145]
*Equation: Cytotoxic activity (%) = the amount of chromium 51 released from
Namalwa cells upon addition of CD8-positive T cells / the amount of chromium 51
released from the target cells upon addition of 1 N hydrochloric acid x 100.
[0146]
In Fig. 3, reference numerals 12, 13, 14, 15, 16, 17, 18 and 19 in the abscissa
indicate the cytotoxic activities of the HLA-A0201-positive CD8-positive T cells
against the Namalwa cells, which T cells were stimulated using SEQ ID NOs: 108,
109, 110, 113, 114, 115, 116 and 117, respectively. Reference numeral 20 indicates
the cytotoxic activity of CD8-positive T cells induced using the peptide of the
negative control (SEQ ID NO:118).
[0147]
Example 6: Determination of CD179b-derived Cytotoxic T Cell Antigen Epitopes
Which Stimulate HLA-A24-positive CD8-positive T Cells
(1) IFN-?-Producing Ability
In order to examine the specificity of the peptide epitope-reactive CD8-
positive T cells induced in Example 3(2) to peptide epitopes in the same manner as in
Example 5(1), 5 x 103 cells of the above-described T cells were added to 5x104 JTK-
LCL cells expressing HLA-A24 molecules (purchased from RIKEN), which JTK-
LCL cells were pulsed using the peptide of SEQ ID NOs: 110, 111, 112, 115 or 116
(cultured in AIM-V medium supplemented with each peptide at a concentration of 10
µg/ml, at 37°C under 5% CO2 for 4 hours), and the cells were cultured in AIM-V
medium containing 10% human AB serum in a 96-well plate for 24 hours. The
supernatant after the culturing was recovered and the production amount of IFN-?
was measured by ELISA. As a result, production of IFN-? was confirmed in the
culture supernatants in the wells of JTK-LCL cells pulsed with the peptides of SEQ
ID NOs: 110,111,112,115 and 116, when compared with the culture supernatants in
the wells of JTK-LCL cells which were not pulsed with a peptide (Fig. 4). From
these results, it was revealed that the above-described peptides are T cell epitope
peptides having a capacity to specifically stimulate, and allow proliferation of, the
HLA-A24-positive CD8-positive T cells, thereby inducing production of IFN-?.
[0148]
In Fig. 4, reference numerals 21, 22, 23, 24 and 25 in the abscissa indicate the
IFN-?-producing abilities of the HLA-A24-positive CD8-positive T cells due to
stimulation from the JTK-LCL cells pulsed with the peptides of SEQ ID NOs:l 10,
111, 112, 115 and 116, respectively. Reference numeral 26 indicates the result for
the peptide of SEQ ID NO: 118 used as the negative control.
[0149]
(2) Cytotoxicity Assay
Subsequently, whether or not the peptides of SEQ ID NOs: 110, 111, 112, 115
and 116 used in the present invention are presented on the HLA-A24 molecules on
cells which are HLA-A24-positive and express CD 179b, and whether or not the
CD8-positive T cells stimulated by these peptides can damage the tumor cells which
are HLA-A24-positive and express CD 179b were examined in the same manner as in
Example 5(2). In a 50-ml centrifugal tube, 106 JTK-LCL cells, which are HLA-
A24-positive and express CD 179b, were collected, and 100 µCi of chromium 51 was
added thereto, followed by incubation at 37°C for 2 hours. Thereafter, the cells
were washed 3 times with RPMI medium containing 10% fetal calf serum, and
placed in wells of a 96-well V-bottom plate in an amount of 103 cells/well. Further,
to each well, 5x104 T cells suspended in RPMI medium containing 10% fetal calf
serum, which cells were stimulated with each peptide, and HLA-A24-positive,
peptide epitope-reactive and CD8-positive, were added, followed by culture at 37°C
under 5% CO2 for 4 hours. Thereafter, by measuring the amount of chromium 51 in
the culture supernatant, which was released from the damaged cells, the cytotoxic
activity of the CD8-positive T cells stimulated by each peptide was calculated. As a
result, it was revealed that the HLA-A24-positive CD8-positive T cells stimulated by
the peptide have a cytotoxic activity against JTK-LCL cells (Fig. 5). Thus, it was
proved that each of the peptides used in the present invention (SEQ ID NOs: 110, 111.
112, 115 and 116) is presented on the HLA-A24 molecules on cells which are HLA-
A24-positive and express CD 179b, and that the peptide has an ability to induce CD8-
positive cytotoxic T cells which can damage such cells. The CD8-positive T cells
induced using the negative control peptide (SEQ ID NO:118) did not show a
cytotoxic activity.
[0150]
In Fig. 5, reference numerals 27, 28, 29, 30 and 31 indicate the cytotoxic
activities of the HLA-A24-positive CD8-positive T cells stimulated with the peptides
of SEQ ID NO: 110, 111, 112, 115 and 116, respectively, against JTK-LCL cells.
Reference numeral 32 indicates the cytotoxic activity of CD8-positive T cells
induced using the peptide of the negative control (SEQ ID NO:118).
[0151]
Example 7: Detection of Cancer Using Recombinant Protein
(1) Detection of Canine Cancer
From 153 canine patients whose malignant tumor was confirmed and 264
healthy dogs, blood was collected, and sera were separated therefrom. Using the
dog-derived cancer antigen protein prepared in Example 2 (the 5th to 120th amino
acids in the amino acid sequence shown in SEQ ID NO:5) and anti-dog IgG antibody,
the titer of IgG antibody in the sera which specifically reacts with the polypeptide
was measured by ELISA.
[0152]
Immobilization of the prepared polypeptide on a solid phase was carried out
by placing 100 µL/well of the recombinant protein solution diluted to 100 µg/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 µL/well of a solution, which was prepared by dissolving 4
g of Block Ace powder (manufactured by DS Pharma Biomedical Co., Ltd.) into 100
ml of purified water, into the wells, and shaking the plate at room temperature for 1
hour. The serum 1000-fold diluted with the blocking solution was added to the
wells in an amount of 100 µL/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 containing 0.05% Tween 20 (manufactured by Wako Pure
Chemical Industries, Ltd.)(hereinafter referred to as PBS-T), and 100 µL/well of
HRP-modified dog IgG antibody (Goat anti Dog IgG(-H+L)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 µL/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 µL/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 ran was measured using a microplate reader. As
a control, a case where the same operation was 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, was designed for
comparison.
[0153]
As the cancer species to be used for the above detection of cancer, 112
samples of breast cancer, 31 samples of lymphoma and 10 samples of leukemia
which had been definitely diagnosed as malignant by pathological diagnosis were
used.
[0154]
These sera derived from the living bodies of the tumor-bearing dogs showed
significantly high antibody titers against the recombinant protein. It was revealed
that, by diagnosing a sample showing twice the average value of healthy canine
samples as malignant, 61 samples (54%) of breast cancer, 21 samples (71%) of
lymphoma and 7 samples (70%) of leukemia could be successfully diagnosed as
malignant. When the test was similarly carried out using sera from 30 canine
patients having a mammary gland tumor which had been definitely diagnosed as
benign, the number of samples showing twice the average value of healthy canine
samples was 0.
[0155]
In the same manner, using the human-derived cancer antigen protein prepared
in Example 2 (the amino acid sequence shown in SEQ ID NO:3) and anti-dog IgG
antibody, the titer of IgG antibody which specifically reacts with the polypeptide in
each of the above-described tumor-bearing dog serum samples was measured by
ELISA. As a result, it was revealed that 56 samples (50%) of breast cancer, 18
samples (58%) of lymphoma and 5 samples (50%) of leukemia could be judged as
malignant.
[0156]
When the detection was carried out in the same manner as described above
using pleural effusion and ascites collected from canine patients with terminal cancer,
values similar to the results obtained by the detection method using serum could be
detected, and diagnosis of the cancer was possible.
[0157]
(2) Detection of Human Cancer
In the same manner, using the human-derived cancer antigen protein (the
amino acid sequence shown in SEQ ID NO:3) used in the above detection and anti-
human IgG antibody, the titer of IgG antibody in a healthy individual which
specifically reacts with the polypeptide was measured. The secondary antibody to
be used was an HRP-modified anti-human IgG antibody (manufactured by HRP-Goat
Anti-Human IgG(H+L) Conjugate: manufactured by Zymed Laboratories) 10000-
diluted with the blocking solution. As a positive control, egg white albumin which
was prepared to 50 µg/ml with phosphate-buffered saline and immobilized on the
solid phase was used. As a result, in the case of the egg white albumin, seven
healthy individuals showed an absorbance of 0.45 at 450 nm on average, which was
high. On the other hand, in the case of the above-described polypeptide, the
absorbance was 0, which means that the reaction was not detected at all.
[0158]
Further, in the same manner as described above, using 17 samples of sera
derived from patients suffering from malignant breast cancer (purchased from
Promeddx), the titer of IgG antibody in each serum which specifically reacts with the
human-derived cancer antigen protein (amino acid sequence shown in SEQ ID NO:3)
was similarly measured. As a result, in the case of the above-described polypeptide,
the 17 breast cancer patients showed an absorbance of 0.28 at 450 nm on average,
which was high. Thus, it was revealed that cancer can be detected by the present
method also in human.
INDUSTRIAL APPLICABILITY
[0159]
The present invention is useful for therapy of cancer since it provides an
immunity-inducing agent containing a polypeptide which exerts an anti-tumor
activity against a cancer(s) (tumor(s)) such as breast cancer, leukemia and/or
lymphoma. Further, the present invention is useful for diagnosis of cancer since it
provides a novel detection method for 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 the odd number IDs of SEQ
ID NOs:3 to 95 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 the odd number IDs of SEQ ID NOs:3 to 95 in
SEQUENCE LISTING, 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 the odd number IDs of SEQ ID
NOs:3 to 95 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 aal-
34 or aa52-75 in any one of the amino acid sequences shown in the odd number IDs
of SEQ ID NOs:3 to 95 in SEQUENCE LISTING, or a polypeptide comprising the
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
consisting essentially of the amino acid sequence shown in SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO:111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ
ID NO:114, SEQ ID NO:115, SEQ ID NO:116 or SEQ ID NO:117 in SEQUENCE
LISTING, or a polypeptide comprising as a partial sequence thereof the amino acid
sequence shown in SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO:110, SEQ ID
NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ
ID NO:116 or SEQ ID NO:117 in SEQUENCE LISTING, said polypeptide having 8
to 12 amino acid residues.
7. The immunity-inducing agent according to any one of claims 1 to 6,
comprising one or more of said polypeptides as an effective ingredient(s).
8. The immunity-inducing agent according to claim 7, wherein said
polypeptide(s) is/are an agent(s) for treating antigen-presenting cells.
9. The immunity-inducing agent according to any one of claims 1 to 8, which is
for therapy and/or prophylaxis of an animal cancer(s).
10. The immunity-inducing agent according to claim 9, wherein said cancer(s)
is/are a cancer(s) expressing the CD179b gene.
11. The immunity-inducing agent according to claim 10, wherein said cancer(s)
is/are breast cancer, leukemia and/or lymphoma.
12. The immunity-inducing agent according to any one of claims 1 to 11, further
comprising an immunoenhancer.
13. An isolated antigen-presenting cell comprising a complex between said
polypeptide having an immunity-inducing activity and an HLA molecule.
14. An isolated T cell which selectively binds to a complex between said
polypeptide having an immunity-inducing activity and an HLA molecule.
15. 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 the odd number IDs of SEQ
ID NOs:3 to 95 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.
16. A method for detecting a cancer(s), which method is applied to a sample
separated from a living body and comprises measuring expression of at least one of
the polypeptides (a) to (c) below:
(a) a polypeptide produced in the living body and having a reactivity to bind,
by antigen-antibody reaction, to an antibody against a polypeptide consisting
essentially of not less than 7 consecutive amino acids in any one of the amino acid
sequences shown in the odd number IDs of SEQ ID NOs:3 to 95 in SEQUENCE
LISTING;
(b) a polypeptide produced in the living body and having a reactivity to bind,
by antigen-antibody reaction, to an antibody against 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 produced in the living body and having a reactivity to bind,
by antigen-antibody reaction, to an antibody against a polypeptide comprising said
polypeptide (a) or (b) as a partial sequence thereof.
17. The method according to claim 16, wherein the measurement of expression of
said polypeptide(s) is carried out by measuring an antibody/antibodies which may be
contained in the sample by immunoassay, which antibody/antibodies was/were
induced in the living body against said polypeptide(s) to be measured.
18. A method for detecting a cancels), which is applied to a sample separated
from a living body and comprises investigation of expression of the CD 179b gene
having a coding region having any one of the base sequences shown in SEQ ID NO:1
and the even number IDs of SEQ ID NOs:4 to 94 in SEQUENCE LISTING in a
sample derived from a cancer patient, and comparison thereof with the expression
level of the gene in a sample derived from a healthy individual.
19. A reagent for detecting a cancer(s), said reagent comprising a polypeptide
which undergoes antigen-antibody reaction with an antibody induced in a living body
against the polypeptide of any one of (a) to (c) below:
(a) a polypeptide consisting essentially of not less than 7 consecutive amino
acids in any one of the amino acid sequences shown in the odd number IDs of SEQ
ID NOs:3 to 95 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.

An immunity-inducing agent comprising as an effective ingredient(s) at least
one polypeptide selected from the following polypeptides, 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 can be used
for therapy and/or prophylaxis of cancer: (a) a polypeptide consisting essentially of
not less than 7 consecutive amino acids in any one of the amino acid sequences
shown in the odd number IDs of SEQ ID NOs:3 to 95 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. Further, since
the above polypeptide(s) react(s) with antibodies existing specifically in serum of a
cancer patient, it is possible to detect cancer in a living body by measuring the
antibodies in a sample.