U.S. patent application number 10/525831 was filed with the patent office on 2006-11-09 for cancer antigens and utilization thereof.
Invention is credited to Tetsuya Nakatsura, Yasuharu Nishimura.
Application Number | 20060251666 10/525831 |
Document ID | / |
Family ID | 31980553 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060251666 |
Kind Code |
A1 |
Nakatsura; Tetsuya ; et
al. |
November 9, 2006 |
Cancer antigens and utilization thereof
Abstract
An object of the present invention to provide: a human
pancreatic cancer antigen and/or a human colon cancer antigen that
can be applied to the diagnosis and/or treatment of various types
of cancers or tumors including pancreatic cancer and colon cancer
as representative examples; a gene encoding the same; an
anti-cancer vaccine using the same; or the like. The present
invention provides a cancer antigen comprising a protein having the
amino acid sequence shown in SEQ ID NO: 1; a peptide comprising a
portion of said protein and having immune-stimulating activity; an
anti-cancer vaccine comprising said peptide; a DNA having the
nucleotide sequence shown in SEQ ID NO: 2, or its complementary
sequence or a part or full length of these sequence; an anti-cancer
vaccine comprising said DNA; and use thereof.
Inventors: |
Nakatsura; Tetsuya;
(Kumamoto, JP) ; Nishimura; Yasuharu; (Kumamoto,
JP) |
Correspondence
Address: |
GREENBLUM & BERNSTEIN, P.L.C.
1950 ROLAND CLARKE PLACE
RESTON
VA
20191
US
|
Family ID: |
31980553 |
Appl. No.: |
10/525831 |
Filed: |
August 29, 2003 |
PCT Filed: |
August 29, 2003 |
PCT NO: |
PCT/JP03/11049 |
371 Date: |
October 17, 2005 |
Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/372; 435/6.14; 435/69.1; 435/7.23; 530/350;
536/23.5 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 13/12 20180101; A61P 15/00 20180101; A61P 35/04 20180101; A61P
11/00 20180101; A61P 17/00 20180101; A61P 1/16 20180101; A61P 37/04
20180101; A61P 13/08 20180101; G01N 33/57419 20130101; A61P 5/14
20180101; A61P 35/00 20180101; A61P 1/04 20180101; C07K 14/4748
20130101; A61P 35/02 20180101; A61P 43/00 20180101; A61P 25/00
20180101; A61P 13/10 20180101; A61P 1/18 20180101; G01N 33/57438
20130101 |
Class at
Publication: |
424/185.1 ;
530/350; 435/069.1; 435/320.1; 435/372; 536/023.5; 435/006;
435/007.23 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12Q 1/68 20060101 C12Q001/68; G01N 33/574 20060101
G01N033/574; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 5/08 20060101 C12N005/08; C07K 14/82 20060101
C07K014/82 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 30, 2002 |
JP |
2002-255668 |
Nov 25, 2002 |
JP |
2002-341168 |
Claims
1. A cancer antigen comprising a protein of any of the following
(A) or (B): (A) a protein having the amino acid sequence shown in
SEQ ID NO: 1; or (B) a protein having an amino acid sequence
comprising a substitution, deletion, insertion, and/or addition of
one or several amino acids with respect to the amino acid sequence
shown in SEQ ID NO: 1, and also having immune-stimulating
activity.
2. An immune-stimulating agent used for cancers, which comprises
the cancer antigen of claim 1.
3. A peptide comprising a portion of the cancer antigen of claim 1
and having immune-stimulating activity.
4. The peptide of claim 3 which can activate cytotoxic T
lymphocytes recognizing a cancer antigen protein.
5. The peptide of claim 3 which has the amino acid sequence shown
in any one of SEQ ID NOS: 3 to 22.
6. A peptide, which has an amino acid sequence comprising a
substitution, deletion, insertion, and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in any one of SEQ ID NOS: 3 to 22, and has immune-stimulating
activity.
7. The peptide of claim 6 which can activate cytotoxic T
lymphocytes which recognize a cancer antigen protein.
8. An immune-stimulating agent used for cancers, which comprises
the peptide of claim 3.
9. A DNA encoding the cancer antigen of claim 1.
10. A DNA of any one of the following (a), (b), and (c): (a) DNA
having the nucleotide sequence shown in SEQ ID NO: 2; (b) DNA
hybridizing with the DNA having the nucleotide sequence shown in
SEQ ID NO: 2 under stringent conditions, and encoding a protein
having immune-stimulating activity; and (c) DNA having a partial
sequence of the DNA of (a) or (b) above, and encoding a protein
having immune-stimulating activity.
11. An antibody against a cancer antigen comprising a protein of
any of the following (A) or (B): (A) a protein having the amino
acid sequence shown in SEQ ID NO: 1; or (B) a protein having an
amino acid sequence comprising a substitution, deletion, insertion,
and/or addition of one or several amino acids with respect to the
amino acid sequence shown in SEQ ID NO: 1, and also having
immune-stimulating activity or the peptide of claim 3.
12. A helper T cells, cytotoxic T lymphocytes, or an immunocyte
population comprising these cells, which are induced by in vitro
stimulation using a cancer antigen comprising a protein of any of
the following (A) or (B): (A) a protein having the amino acid
sequence shown in SEQ ID NO: 1; or (B) a protein having an amino
acid sequence comprising a substitution, deletion, insertion,
and/or addition of one or several amino acids with respect to the
amino acid sequence shown in SEQ ID NO: 1, and also having
immune-stimulating activity or the peptide of claim 3 or a mixture
thereof.
13. A helper T cells, cytotoxic T lymphocytes, or an immunocyte
population comprising these cells, which are induced by in vitro
stimulation using a cancer antigen comprising a protein of any of
the following (A) or (B): (A) a protein having the amino acid
sequence shown in SEQ ID NO: 1; or (B) a protein having an amino
acid sequence comprising a substitution, deletion, insertion,
and/or addition of one or several amino acids with respect to the
amino acid sequence shown in SEQ ID NO: 1, and also having
immune-stimulating activity or the peptide of claim 3 or a mixture
thereof, and an immune activator.
14. The helper T cells, cytotoxic T lymphocytes or the immunocyte
population comprising these cells according to claim 13, wherein
the immune activator is a cell growth factor or cytokine.
15. A method for suppressing a tumor, which comprises introducing
the helper T cells, cytotoxic T lymphocytes, or the immunocyte
population comprising these cells of claim 12 into a body.
16. The method of claim 15 which is used to prevent and/or treat
cancers.
17. A cell culture solution used to produce the helper T cells or
cytotoxic T lymphocytes or an immunocyte population comprising
these cells of claim 12, which comprises a cancer antigen
comprising a protein of any of the following (A) or (B): (A) a
protein having the amino acid sequence shown in SEQ ID NO: 1; or
(B) a protein having an amino acid sequence comprising a
substitution, deletion, insertion, and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in SEQ ID NO: 1, and also having immune-stimulating activity or a
peptide, or a mixture thereof.
18. A cell culture kit for producing the helper T cells or
cytotoxic T lymphocytes or an immunocyte population comprising
these cells of claim 12, which comprises a cell culture solution
and a cell culture vessel.
19. A cancer vaccine comprising a cancer antigen comprising a
protein of any of the following (A) or (B): (A) a protein having
the amino acid sequence shown in SEQ ID NO: 1; or (B) a protein
having an amino acid sequence comprising a substitution, deletion,
insertion, and/or addition of one or several amino acids with
respect to the amino acid sequence shown in SEQ ID NO: 1, and also
having immune-stimulating activity and/or at least one type of
peptide of claim 3.
20. The cancer vaccine of claim 19 which further comprises an
adjuvant.
21. A cancer vaccine, which comprises the DNA of claim 9 or
recombinant virus or recombinant bacteria comprising said DNA.
22. The cancer vaccine of claim 21 which further comprises an
adjuvant.
23. A probe for diagnosing cancers, which comprises the DNA of
claim 9.
24. An agent for diagnosing cancers, which comprises the cancer
diagnostic probe of claim 23 and/or an antibody.
25. An agent for preventing and/or treating cancers, which
comprises a cancer antigen comprising a protein of any of the
following (A) or (B): (A) a protein having the amino acid sequence
shown in SEQ ID NO: 1; or (B) a protein having an amino acid
sequence comprising a substitution, deletion, insertion, and/or
addition of one or several amino acids with respect to the amino
acid sequence shown in SEQ ID NO: 1, and also having
immune-stimulating activity, the peptide of claim 3, an antibody,
and/or helper T cells, cytotoxic T lymphocytes or immunocyte
population comprising these cells.
26. The probe of claim 23 wherein the cancer is pancreatic cancer,
colon cancer, brain tumor, malignant melanoma, chronic myelocytic
leukemia, acute myelocytic leukemia, lymphoma, esophageal cancer,
kidney cancer, prostatic cancer, lung cancer, breast cancer,
stomach cancer, hepatic cancer, gallbladder cancer, testicular
cancer, uterine cancer, ovarian cancer, thyroid cancer, bladder
cancer, or sarcoma.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel human cancer
antigen that is useful for diagnosis of various types of cancers
such as pancreatic cancer or colon cancer and for immunotherapy,
and use thereof.
BACKGROUND ART
[0002] At present, cancer is the number one cause of death.
Occurrence mechanisms, diagnostic methods, and therapeutic methods
for cancer have been developed. However, a large number of advanced
cancers have not yet been treated under the present circumstances.
In order to improve the current situation, it is considered to be
necessary to develop a novel early diagnostic method and
therapeutic method.
[0003] Immunotherapy has long been anticipated as a method for
treating cancers, and various attempts have been made regarding
such therapy. However, sufficient antitumor effects have not yet
been exhibited. Conventionally, immunotherapy for cancers had
previously been centered on nonspecific immunotherapy. In recent
years, however, it has been clarified that T cells play an
important role in tumor rejection in living bodies. As a result,
efforts are now focused on the isolation of a T cell-recognizing
cancer antigen that is capable of inducing cytotoxic T lymphocytes
(CTL) and the determination of an MHC class I-binding epitope.
[0004] To date, many cancer antigens have been isolated by the
conventional cDNA expression cloning method, using CTL. This method
requires the establishment of a cell line from tumor and the
establishment of CTL. Thus, it is difficult to isolate a tumor
antigen from carcinomas other than melanomas. In addition, in order
to enhance the effects of immunotherapy, it is considered that a
treatment method involving mixing many peptides is effective. In
order to establish such a treatment method, it is necessary to
isolate a large number of antigens. Thus, the conventional cDNA
expression cloning method is problematic in that it takes enormous
manpower and time to isolate even a single antigen.
[0005] In 1995, Pfreundschuh et al. in Germany and Old et al. in
U.S.A. have reported the SEREX method, which detects a cancer
antigen protein recognized by an antibody in the serum of a cancer
patient (Serological Identification of Recombinant cDNA Expression
Cloning; Proc. Natl. Acad. Sci. USA 92, 11810-11813, 1995). Many
tumor antigens have been isolated by this method. Among antigens
isolated by this method, antigens such as MAGE-1 or tyrosinase that
induce CTL have also been included. Accordingly, it is pointed out
that this method is also useful as a method for detecting an
antigen recognized by cell-mediated immunity. Moreover, it has been
reported that a cancer antigen recognized by the IgG antibody of a
patient was isolated by the above-described method (Int. J. Cancer
72, 965-971, 1997; Cancer Res. 58, 1034-1041, 1998; Int. J. Cancer
29, 652-658, 1998; Int. J. Oncol. 14, 703-708, 1999; Cancer Res.
56, 4766-4772, 1996; and Hum. Mol. Genet 6, 33-39, 1997).
DISCLOSURE OF THE INVENTION
[0006] It is an object of the present invention to provide: a human
pancreatic cancer antigen and/or a human colon cancer antigen that
can be applied to the diagnosis and/or treatment of various types
of cancers or tumors including pancreatic cancer and colon cancer
as representative examples; a gene encoding the same; and an
anti-cancer vaccine using the same.
[0007] That is to say, the present invention provides a cancer
antigen comprising a protein of any of the following (A) or
(B):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1;
or
(B) a protein having an amino acid sequence comprising a
substitution, deletion, insertion, and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in SEQ ID NO: 1, and also having immune-stimulating activity.
[0008] In another aspect, the present invention provides an
immune-stimulating agent used for cancers, which comprises the
aforementioned cancer antigen of the present invention.
[0009] In another aspect, the present invention provides a peptide
comprising a portion of the aforementioned cancer antigen of the
present invention and having immune-stimulating activity. The
peptide of the present invention can preferably activate cytotoxic
T lymphocytes recognizing a cancer antigen protein. The peptide of
the present invention preferably has the amino acid sequence shown
in any one of SEQ ID NOS: 3 to 22.
[0010] In another aspect, the present invention provides a peptide,
which has an amino acid sequence comprising a substitution,
deletion, insertion, and/or addition of one or several amino acids
with respect to the amino acid sequence shown in any one of SEQ ID
NOS: 3 to 22, and also has immune-stimulating activity. The
above-described peptide can preferably activate cytotoxic T
lymphocytes which recognize a cancer antigen protein.
[0011] In another aspect, the present invention provides an
immune-stimulating agent used for cancers, which comprises any one
of the above-described peptides.
[0012] In another aspect, the present invention provides DNA
encoding the aforementioned cancer antigen of the present
invention.
[0013] In another aspect, the present invention provides DNA of any
one of the following (a), (b), and (c):
(a) DNA having the nucleotide sequence shown in SEQ ID NO: 2;
(b) DNA hybridizing with the DNA having the nucleotide sequence
shown in SEQ ID NO: 2 under stringent conditions, and encoding a
protein having immune-stimulating activity; and
(c) DNA having a partial sequence of the DNA of (a) or (b) above,
and encoding a protein having immune-stimulating activity.
[0014] In another aspect, the present invention provides an
antibody against the aforementioned cancer antigen or peptide of
the present invention.
[0015] In another aspect, the present invention provides helper T
cells, cytotoxic T lymphocytes, or an immunocyte population
comprising these cells, which are induced by in vitro stimulation
using the aforementioned cancer antigen or peptide of the present
invention, or a mixture thereof.
[0016] In another aspect, the present invention provides helper T
cells, cytotoxic T lymphocytes, or an immunocyte population
comprising these cells, which are induced by in vitro stimulation
using the aforementioned cancer antigen or peptide of the present
invention, or a mixture thereof, and an immune activator. The
immune activator is preferably a cell growth factor or
cytokine.
[0017] In another aspect, the present invention provides a method
for suppressing a tumor, which comprises introducing the
above-described helper T cells, cytotoxic T lymphocytes, or an
immunocyte population comprising these cells into a body. The
above-described method is preferably used to prevent and/or treat
cancers.
[0018] In another aspect, the present invention provides a cell
culture solution used to produce the helper T cells or cytotoxic T
lymphocytes of the present invention or an immunocyte population
comprising these cells, which comprises the aforementioned cancer
antigen or peptide of the present invention, or a mixture
thereof.
[0019] In another aspect, the present invention provides a cell
culture kit for producing the helper T cells or cytotoxic T
lymphocytes of the present invention or an immunocyte population
comprising these cells, which comprises the above-described cell
culture solution and a cell culture vessel.
[0020] In another aspect, the present invention provides a cancer
vaccine comprising the aforementioned cancer antigen and/or at
least one type of peptide of the present invention. The
above-described cancer vaccine preferably further comprises an
adjuvant.
[0021] In another aspect, the present invention provides a cancer
vaccine, which comprises the aforementioned DNA of the present
invention, or recombinant virus or recombinant bacteria comprising
the above-described DNA. The above-described cancer vaccine
preferably further comprises an adjuvant.
[0022] In another aspect, the present invention provides a probe
for diagnosing cancers, which comprises the aforementioned DNA of
the present invention.
[0023] In another aspect, the present invention provides an agent
for diagnosing cancers, which comprises the aforementioned cancer
diagnostic probe and/or antibody of the present invention.
[0024] In another aspect, the present invention provides an agent
for preventing and/or treating cancers, which comprises the
aforementioned cancer antigen, peptide, antibody, and/or helper T
cells, cytotoxic T lymphocytes, or an immunocyte population
comprising these cells of the present invention.
[0025] In the present invention, cancer is preferably pancreatic
cancer, colon cancer, brain tumor, malignant melanoma, chronic
myelocytic leukemia, acute myelocytic leukemia, lymphoma,
esophageal cancer, kidney cancer, prostatic cancer, lung cancer,
breast cancer, stomach cancer, hepatic cancer, gallbladder cancer,
testicular cancer, uterine cancer, ovarian cancer, thyroid cancer,
bladder cancer, or sarcoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a microphotograph showing the results of an
immunohistochemical analysis on hsp105 in pancreatic cancer. In the
figure, the symbols have the following meanings:
A: a pancreatic cancer portion and a peripheral non-cancerous
portion stained with hematoxylin and eosin, CA: a cancerous
portion, NP: a non-cancerous portion
B: An hsp105 protein is highly expressed in cancer cells. It is
weakly expressed also in a non-cancerous portion.
C: A non-cancerous portion is significantly expanded. An hsp105
protein is weakly expressed in the cytoplasm.
D: A cancerous portion is significantly expanded. An hsp105 protein
is highly expressed mainly in the cytoplasm of cancer cells.
[0027] FIG. 2 is a microphotograph showing the results of an
immunohistochemical analysis on hsp105 in colon cancer. In the
figure, the symbols have the following meanings:
A: a colon cancer portion and a peripheral non-cancerous portion
stained with hematoxylin and eosin, CA: a cancerous portion, NP: a
non-cancerous portion
B: An hsp105 protein is highly expressed in cancer cells. It is
weakly expressed also in a non-cancerous portion.
C: A non-cancerous portion is significantly expanded. An hsp105
protein is weakly expressed in the cytoplasm.
D: A cancerous portion is significantly expanded. An hsp105 protein
is highly expressed mainly in the cytoplasm of cancer cells. Also,
the hsp105 protein is weakly expressed in the nucleus thereof.
[0028] FIG. 3 is a graph showing the anticancer effects of an
hsp105 DNA vaccine, an hsp105 peptide vaccine, and a control on
mouse colon cancer cells Colon-26. A represents the area of a
cancerous portion, B represents the ratio of mice wherein the
cancer has developed, and C represents the ratio of surviving
mice.
[0029] FIG. 4 is a graph showing the results obtained by measuring
by .sup.51Cr release assay, the cytotoxic activities on Colon-26 of
various types of peptide vaccines derived from hsp105 proteins, or
the cytotoxic activities of DNA vaccines encoding such hsp105
proteins.
[0030] FIG. 5 shows the results of an immunohistochemical analysis
on hsp105 in tissues.
[0031] FIG. 6 shows the results obtained by analyzing the in vivo
antitumor activity of a mouse CD4 positive helper T cell line
induced by hsp105.
[0032] FIG. 7 shows the results obtained by inducing by hsp105, the
CD4 positive helper T cell line of a patient with colon cancer.
[0033] FIG. 8 shows the results obtained by examining whether or
not BALB/c mouse cytotoxic T lymphocytes (CTL) induced by an
hsp105-derived peptide can reduce a tumor mass of the colon cancer
cell line Colon-26 which highly expresses hsp105.
[0034] FIG. 9 shows the results obtained by examining whether or
not the cytotoxic T lymphocytes (CTL) of a colon cancer patient
induced by an hsp105-derived peptide can reduce a tumor mass of the
colon cancer cell line sw620, which highly expresses hsp105.
BEST MODE FOR CARRYING OUT THE INVENTION
[0035] The embodiments of the present invention will be described
in detail below.
(1) The Cancer Antigen, Peptide, and Immune-Stimulating Agent
Against Cancers According to the Present Invention
[0036] The cancer antigen of the present invention collected from
pancreatic cancer or colon cancer is a protein of any of the
following (A) or (B):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1
(hereinafter referred to as "hsp105"); or
(B) a protein having an amino acid sequence comprising a
substitution, deletion, insertion, and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in SEQ ID NO: 1, and also having immune-stimulating activity.
[0037] The term "protein having immune-stimulating activity" is
used in the present specification to mean a protein having activity
of inducing an immune response such as generation of an antibody or
cell-mediated immunity. Among them, a protein having T
cell-stimulating activity of stimulating cytotoxic T lymphocytes
(killer T cells/CTL) is particularly preferable.
[0038] hsp105 is a heat shock protein with a high molecular weight,
which belongs to the hsp110/105 family, and is composed of
hsp105.alpha. and 105.beta.. 105.alpha. is a heat shock protein of
105 kDa, and is induced by various stresses. 105.beta. is a protein
generated by splicing of mRNA of 105.alpha., and has a molecular
weight smaller than that of 105.alpha.. hsp105 that is an antigen
of pancreatic cancer or colon cancer of the present invention can
be detected, for example, by the SEREX method as described later in
examples in the present specification.
[0039] In the present invention, the scope of "one or several" in
"an amino acid sequence comprising a substitution, deletion,
insertion, and/or addition of one or several amino acids with
respect to the amino acid sequence shown in SEQ ID NO: 1" is not
particularly limited. For example, it means 1 to 20, preferably 1
to 10, more preferably 1 to 7, further preferably 1 to 5, and
particularly preferably 1 to 3 amino acids.
[0040] A method of obtaining or producing the cancer antigen
protein of the present invention is not particularly limited. A
naturally occurring protein, a chemically synthesized protein, or a
recombinant protein produced by genetic engineering may be used.
From the viewpoint that it can be produced in large volume by
relatively easy operations, a recombinant protein is
preferable.
[0041] When a naturally occurring protein is obtained, it can be
isolated from cells or tissues expressing the protein by
appropriate combined use of protein isolation and purification
methods. When a chemically synthesized protein is obtained, the
protein of the present invention can be synthesized by chemical
synthesis methods such as the Fmoc method
(fluorenylmethyloxycarbonyl method) or the tBoc method
(t-butyloxycarbonyl method). Moreover, the protein of the present
invention can also be synthesized by using various types of
commercially available peptide synthesizers.
[0042] When the cancer antigen protein of the present invention is
produced in the form of a recombinant protein, it can be produced
by obtaining DNA having a nucleotide sequence encoding the protein
(e.g. the nucleotide sequence shown in SEQ ID NO: 2), a mutant
thereof, or a homologue thereof, and introducing it into a
preferred expression system.
[0043] Any expression vector can be used, as long as it can
autonomously replicate in host cells or it can be incorporated into
the chromosomes of host cells. An expression vector containing a
promoter at a site that is capable of expressing the gene of the
present invention, is used. A transformant having a gene encoding
the protein of the present invention can be produced by introducing
the aforementioned expression vector into a host. Such a host used
herein may include bacteria, yeast, animal cells, and insect cells.
In addition, an expression vector may be introduced into a host by
known methods, depending on the type of the host.
[0044] In the present invention, the transformant having the gene
of the present invention produced as described above is cultured,
and the protein of the present invention is generated and
accumulated in a culture product. Thereafter, the protein of the
present invention is collected from the culture product, thereby
isolating a recombinant protein.
[0045] When the transformant of the present invention is prokaryote
such as Escherichia coli or eukaryote such as yeast, either a
natural medium or a synthetic medium may be used as a medium in
which these microorganisms are cultured, as long as it contains a
carbon source, a nitrogen source, and inorganic salts that can be
assimilated by the microorganisms, and the culture of the
transformant can efficiently be carried out therein. In addition,
culture may be carried out under conditions that are commonly used
for culturing microorganisms. After completion of the culture, the
protein of the present invention may be isolated and purified from
the culture product of the transformant by common protein isolation
and purification methods.
[0046] A protein having an amino acid sequence comprising a
substitution, deletion, insertion, and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in SEQ ID NO: 1, can appropriately be produced or obtained by
persons skilled in the art on the basis of the information
regarding the nucleotide sequence shown in SEQ ID NO: 2, which is
an example of the DNA sequence encoding the amino acid sequence
shown in SEQ ID NO: 1.
[0047] That is to say, a gene (mutant gene) having a nucleotide
sequence encoding a protein having an amino acid sequence
comprising a substitution, deletion, insertion, and/or addition of
one or several amino acids with respect to the amino acid sequence
shown in SEQ ID NO: 1, can be produced by any given methods that
are known to persons skilled in the art, such as chemical
synthesis, genetic engineering methods, or mutagenesis.
Specifically, a mutation is introduced into DNA having the
nucleotide sequence shown in SEQ ID NO: 2, so as to obtain mutant
DNA.
[0048] For example, a method of allowing DNA to come into contact
with an agent acting as a mutagen, a method of irradiating with
ultraviolet rays, a genetic engineering method, and the like, can
be applied to the DNA having the nucleotide sequence shown in SEQ
ID NO: 2. Site-directed mutagenesis, one of the genetic engineering
methods, is useful because it is capable of introducing a specific
mutation into a specific site. Site-directed mutagenesis can be
carried out according to the methods described in publications such
as Molecular Cloning: A laboratory Manual, 2.sup.nd Ed., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989
(hereinafter abbreviated as Molecular Cloning 2.sup.nd Ed.); and
Current Protocols in Molecular Biology, Supplements 1 to 38, John
Wiley & Sons (1987-1997) (hereinafter abbreviated as Current
Protocols in Molecular Biology).
[0049] The present invention also relates to a peptide that is a
portion of the aforementioned protein of the present invention and
has immune-stimulating activity. The peptide of the present
invention preferably can activate cytotoxic T lymphocytes which
recognize a cancer antigen protein. Specific examples of such a
peptide may include those having any one of the following amino
acid sequences: TABLE-US-00001 (SEQ ID NO:3)
Asn-Tyr-Gly-Ile-Tyr-Lys-Gln-Asp-Leu (SEQ ID NO:4)
Ala-Phe-Asn-Lys-Gly-Lys-Leu-Lys-Val-Leu (SEQ ID NO:5)
Lys-Tyr-Lys-Leu-Asp-Ala-Lys-Ser-Lys-Ile (SEQ ID NO:6)
Gln-Phe-Glu-Glu-Leu-Cys-Ala-Glu-Leu (SEQ ID NO:7)
Met-Tyr-Ile-Glu-Thr-Glu-Gly-Lys-Met-Ile (SEQ ID NO:8)
Thr-Phe-Leu-Arg-Arg-Gly-Pro-Phe-Glu-Leu (SEQ ID NO:9)
Glu-Tyr-Val-Tyr-Glu-Phe-Arg-Asp-Lys-Leu (SEQ ID NO:10)
His-Tyr-Ala-Lys-Ile-Ala-Ala-Asp-Phe (SEQ ID NO:11)
Lys-Tyr-Asn-His-Ile-Asp-Glu-Ser-Glu-Met (SEQ ID NO:12)
Ser-Leu-Asp-Glu-Lys-Pro-Arg-Ile-Val-Val (SEQ ID NO:13)
Arg-Leu-Tyr-Gln-Glu-Cys-Glu-Lys-Leu (SEQ ID NO:14)
Lys-Leu-Met-Ser-Ser-Asn-Ser-Thr-Asp-Leu (SEQ ID NO:15)
Leu-Met-Ser-Ser-Asn-Ser-Thr-Asp-Leu (SEQ ID NO:16)
Ser-Gln-Phe-Glu-Glu-Leu-Cys-Ala-Glu-Leu (SEQ ID NO:17)
Lys-Ile-Gly-Arg-Phe-Val-Val-Gln-Asn-Val (SEQ ID NO:18)
Tyr-Val-Tyr-Glu-Phe-Arg-Asp-Lys-Leu (SEQ ID NO:19)
Leu-Leu-Thr-Glu-Thr-Glu-Asp-Trp-Leu (SEQ ID NO:20)
Trp-Leu-Tyr-Glu-Glu-Gly-Glu-Asp-Gln-Ala (SEQ ID NO:21)
Glu-Leu-Met-Lys-Ile-Gly-Thr-Pro-Val (SEQ ID NO:22)
Val-Met-Asn-Ala-Gln-Ala-Lys-Lys-Ser-Leu
[0050] Moreover, peptides having an amino acid sequence comprising
a substitution, deletion, insertion and/or addition of one or
several amino acids with respect to the amino acid sequence shown
in any one of the above SEQ ID NOS: 3 to 22, and having
immune-stimulating activity, are also included in the scope of the
present invention. A preferred example of such a peptide may be a
peptide capable of activating cytotoxic T lymphocytes which
recognize a cancer antigen protein.
[0051] In the present invention, the scope of "one or several" in
"an amino acid sequence comprising a substitution, deletion,
insertion and/or addition of one or several amino acids with
respect to the amino acid sequence shown in any one of the above
SEQ ID NOS: 3 to 22" is not particularly limited. The number of
amino acids is, for example 1 to 5, preferably 1 to 4, more
preferably 1 to 3, further preferably 1 or 2, and particularly
preferably 1.
[0052] The peptide of the present invention can be synthesized by
chemical synthesis methods such as the Fmoc method
(fluorenylmethyloxycarbonyl method) or the tBoc method
(t-butyloxycarbonyl method). Moreover, the peptide of the present
invention can also be synthesized using various types of
commercially available peptide synthesizers.
[0053] The aforementioned cancer antigen protein and peptide of the
present invention can induce immunity against cancers, as described
later in examples. Accordingly, the present invention provides an
immune-stimulating agent against cancers, which comprises the
cancer antigen protein or peptide of the present invention.
[0054] The immune-stimulating agent against cancers of the present
invention is used in vitro or in vivo, and preferably in vitro, so
as to induce helper T cells, cytotoxic T lymphocytes, or an
immunocyte population containing these cells, thereby providing
immunity against cancers.
(2) DNA of the Present Invention
[0055] The DNA of the present invention encodes the cancer antigen
protein of the present invention described in (1) above. It is
preferably DNA of any one of the following (a), (b), and (c):
(a) DNA having the nucleotide sequence shown in SEQ ID NO: 2;
(b) DNA hybridizing with the DNA having the nucleotide sequence
shown in SEQ ID NO: 2 under stringent conditions, and encoding a
protein having immune-stimulating activity; and
(c) DNA having a partial sequence of the DNA according to (a) or
(b) above, and encoding a protein having immune-stimulating
activity.
[0056] The above term "DNA hybridizing with . . . under stringent
conditions" is used to mean the nucleotide sequence of DNA obtained
by the colony hybridization method, the plaque hybridization
method, or the Southern hybridization method, using DNA as a probe.
For example, such DNA can be identified by hybridizing a filter, on
which colony- or plaque-derived DNA or a DNA fragment thereof has
been immobilized, at 65.degree. C. in the presence of 0.7 to 1.0 M
NaCl, and then washing the filter at 65.degree. C. with a 0.1 to
2.times.SSC solution (wherein 1.times.SSC solution consists of 150
mM sodium chloride and 15 mM sodium citrate). Hybridization can be
carried out by the method described in Molecular Cloning 2.sup.nd
Ed.
[0057] DNA having a certain level of homology with the nucleotide
sequence of DNA used as a probe is an example of the above DNA
hybridizing under stringent conditions. Such DNA has homology of,
for example 70% or more, preferably 80% or more, more preferably
90% or more, further preferably 93% or more, particularly
preferably 95% or more, and most preferably 98% or more, with the
DNA used as a probe.
[0058] A method of obtaining the DNA of the present invention is
not particularly limited. Suitable probes or primers are prepared
based on the information regarding the amino acid sequence and the
nucleotide sequence shown in SEQ ID NOS: 1 and 2 in the sequence
listing in the present specification, and the cDNA library of a
human and the like is screened using such probes or primers, so as
to isolate the DNA of the present invention. Such a cDNA library is
preferably produced from a cell, organ, or tissue, which expresses
the DNA of the present invention.
[0059] It is also possible to produce the DNA having the nucleotide
sequence shown in SEQ ID NO: 2 by the PCR method. Using human
chromosomal DNA or cDNA library as a template, PCR is carried out
with a pair of primers that have been designed to amplify the
nucleotide sequence shown in SEQ ID NO: 2. PCR reaction conditions
can be determined as appropriate. For example, a reaction process
consisting of 94.degree. C. and 30 seconds (denaturation),
55.degree. C. and 30 seconds to 1 minute (annealing), and
72.degree. C. and 2 minutes (elongation) is defined as 1 cycle.
Such a reaction process is carried out 30 cycles, and thereafter, a
reaction consisting of 72.degree. C. and 7 minutes is carried out.
Thereafter, the amplified DNA fragment is cloned into a suitable
vector capable of replicating in a host such as Escherichia
coli.
[0060] The aforementioned preparation of probes or primers,
construction of a cDNA library, screening of a cDNA library, and
cloning of a gene of interest are already known to persons skilled
in the art. These operations can be carried out according to the
methods described in Molecular Cloning 2.sup.nd Ed., Current
Protocols in Molecular Biology, and the like.
(3) Antibody of the Present Invention
[0061] The present invention further relates to an antibody
recognizing a portion or the entire of the aforementioned protein
or peptide of the present invention as an epitope (antigen), and
cytotoxic (killer) T lymphocytes (CTL) induced by in vitro
stimulation using the above-described protein or peptide. In
general, CTL exhibits stronger antitumor activity than an
antibody.
[0062] The antibody of the present invention may be either a
polyclonal antibody or a monoclonal antibody. It can be produced by
common methods.
[0063] For example, a polyclonal antibody can be obtained by
immunizing a mammal with the protein of the present invention as an
antigen, collecting the blood from the mammal, and then separating
and purifying an antibody from the collected blood. Examples of a
mammal to be immunized may include a mouse, a hamster, a Guinea
pig, a chicken, a rat, a rabbit, a dog, a goat, a sheep, and a
bovine. The immunization method is known to those skilled in the
art. For example, an antigen may be administered 2 or 3 times at
intervals of 7 to 30 days. The dosage may be set at approximately
0.05 to 2 mg of antigen per administration. An administration route
is not particularly limited. A suitable administration route can
appropriately be selected from subcutaneous administration,
intracutaneous administration, intraperitoneal administration,
intravenous administration, and intramuscular administration. In
addition, an antigen can be dissolved in a suitable buffer solution
containing a commonly used adjuvant such as Freund's complete
adjuvant or aluminum hydroxide, before use.
[0064] Such an immunized mammal has been bred for a certain period
of time, and when its antibody titer begins to increase, a booster
can be carried out using 100 .mu.g to 1,000 .mu.g of the antigen,
for example. 1 or 2 months after the final administration, the
blood is collected from the immunized mammal. The collected blood
is then separated and purified by common methods including
centrifugation, precipitation using ammonium sulfate or
polyethylene glycol, or chromatography such as gel filtration
chromatography, ion exchange chromatography, or affinity
chromatography, so as to obtain a polyclonal antibody recognizing
the protein of the present invention as a polyclonal antiserum.
[0065] On the other hand, a monoclonal antibody can be obtained by
preparing hybridomas. Hybridomas can be obtained by cell fusion
between antibody-generating cells and myeloma cells, for example.
Hybridomas which generate the monoclonal antibody of the present
invention can be obtained by the following cell fusion method.
[0066] Spleen cells, lymph node cells, B lymphocytes or the like
collected from the immunized animal are used as antibody-generating
cells. The protein of the present invention or a partial peptide
thereof is used as an antigen. A mouse, a rat, or the like can be
used as an animal to be immunized. The administration of an antigen
to such an animal is carried out by common methods. For example, a
suspension or emulsified liquid of an adjuvant such as Freund's
complete adjuvant or Freund's incomplete adjuvant and the protein
of the present invention used as an antigen is administered
intravenously, subcutaneously, intracutaneously, or
interperitoneally to an animal several times for immunization. For
example, spleen cells are obtained from the thus immunized animal
as antibody-generating cells, and the obtained cells are fused with
myeloma cells by a known method (G. Kohler et al., Nature, 256, 495
(1975)), so as to produce hybridomas.
[0067] Examples of a myeloma cell line used for cell fusion may
include mouse P3X63Ag8, mouse P3U1 line, and mouse Sp2/0 line. For
cell fusion, fusion promoting agents such as polyethylene glycol or
Sendai virus are used. For selection of hybridomas after completion
of the cell fusion, hypoxanthine aminopterin thymidine (HAT) medium
is used according to common methods. Hybridomas obtained by cell
fusion are cloned by limiting dilution or the like. Thereafter, as
necessary, screening is carried out by enzyme immunoassay using the
protein of the present invention, so as to obtain a cell line
generating a monoclonal antibody specifically recognizing the
protein of the present invention.
[0068] In order to produce a monoclonal antibody of interest from
the thus obtained hybridoma, the hybridoma may be cultured by the
common cell culture method or ascites formation method, and the
monoclonal antibody may be purified from the culture supernatant or
ascites. The monoclonal antibody can be purified from the culture
supernatant or ascites by common methods. For example, ammonium
sulfate fractionation, gel filtration, ion exchange chromatography,
affinity chromatography, and the like can appropriately be used in
combination.
[0069] Moreover, fragments of the aforementioned antibody are also
included in the scope of the present invention. Examples of such an
antibody fragment may include a F(ab').sub.2 fragment and a Fab'
fragment.
[0070] Furthermore, a labeled antibody obtained by labeling the
aforementioned antibody is also included in the scope of the
present invention. That is to say, the antibody of the present
invention produced as described above can be labeled before use.
The type of a substance used to label the antibody of the present
invention and a labeling method are known to persons skilled in the
art. Examples of such a labeling method may include: enzyme
labeling with horseradish peroxidase or alkaline phosphatase;
fluorescent labeling with FITC (fluorescein isothiocyanate) or
TRITC (tetramethylrhodamine B isothiocyanate); labeling with color
substances such as colloidal metal or colored latex; affinity
labeling with biotin; and isotopic labeling with .sup.125I. The
analysis or measurement of the protein of the present invention
(that is a cancer antigen) with the labeled antibody of the present
invention can be carried out according to methods widely known to
those skilled in the art, such as the enzyme antibody technique,
immunohistological staining, immunoblotting, the direct fluorescent
antibody method, or the indirect fluorescent antibody method.
(4) Helper T Cells, Cytotoxic T Lymphocytes, or Immunocyte
Population Containing these Cells
[0071] The present invention further relates to helper T cells,
cytotoxic T lymphocytes, or an immunocyte population containing
these cells, which are induced by in vitro stimulation using the
cancer antigen or peptide of the present invention or a mixture
thereof. For example, when peripheral blood lymphocytes or
tumor-infiltrating lymphocytes are stimulated in vitro with the
protein or peptide of the present invention, tumor responsive
activated T cells are induced. The activated T cells can
effectively be used for adoptive immunotherapy. Moreover, the
cancer antigen or peptide of the present invention is allowed to
express in dendritic cells that are strong antigen presenting cells
in vivo or in vitro, thereby conducting immune stimulation by
administration of the dendritic cells wherein the antigen has been
expressed.
[0072] Preferably, helper T cells, cytotoxic T lymphocytes, or an
immunocyte population containing these cells are induced by in
vitro stimulation using the cancer antigen or peptide of the
present invention or a mixture thereof and an immune activator.
Examples of an immune activator used herein may include a cell
growth factor and cytokine.
[0073] The helper T cells, cytotoxic T lymphocytes, or an
immunocyte population containing these cells obtained as described
above are transferred into a body to suppress tumor, thereby
preventing and/or treating cancers.
[0074] Moreover, helper T cells, cytotoxic T lymphocytes, or an
immunocyte population containing these cells, which can suppress
tumor as described above, can be produced using the cancer antigen
or peptide of the present invention or a mixture thereof.
Accordingly, the present invention provides a cell culture solution
containing the cancer antigen or peptide of the present invention
or a mixture thereof. Using such a cell culture solution, the
helper T cells, cytotoxic T lymphocytes, or an immunocyte
population containing these cells, which can suppress tumor, can be
produced. Further, the present invention provides a cell culture
kit for producing the helper T cells, cytotoxic T lymphocytes, or
an immunocyte population containing these cells, which comprises
the aforementioned cell culture solution and a cell culture
vessel.
(5) Cancer Vaccine of the Present Invention
[0075] Since the DNA, cancer antigen, and peptide of the present
invention can induce cancer cell-specific cytotoxic T lymphocytes,
they are anticipated as therapeutic and/or preventive agents used
for cancers. For example, BCG bacteria transformed by recombinant
DNA obtained by incorporating the DNA of the present invention into
a suitable vector, or viruses such as vaccinia virus into the
genome of which the DNA of the present invention has been
incorporated, can effectively be used as live vaccine for treating
and/or preventing human cancers. The dosage and administration
method of such a cancer vaccine are the same as those of ordinary
vaccination or BCG vaccine.
[0076] Namely, the DNA of the present invention (as is, or in the
form of plasmid DNA that is incorporated into an expression
vector), and recombinant virus or recombinant bacteria containing
the above DNA are administered as a cancer vaccine to mammals
including a human, directly or in a state where they are dispersed
in an adjuvant. Also, the peptide of the present invention can be
administered thereto as a cancer vaccine, in a state where it is
dispersed in an adjuvant.
[0077] Examples of an adjuvant used in the present invention may
include Freund's incomplete adjuvant, BCG, trehalose dimycolate
(TDM), lipopolysaccharide (LPS), alum adjuvant, and silica
adjuvant. From the viewpoint of ability to induce antibody,
Freund's incomplete adjuvant (FIA) is preferably used.
(6) Probe for Diagnosing Cancers, Agent for Diagnosing Cancers, and
Preventive and/or Therapeutic Agent Against Cancers According to
the Present Invention
[0078] The DNA of the present invention can be used as a diagnostic
probe, in which DNAs of various types of human cancers are
extracted and the homology between them is examined. Moreover, this
probe and the above-described antibody can also be used as an agent
for diagnosing cancers.
[0079] That is to say, the present invention relates to a probe for
diagnosing cancers, which comprises an entire or a part of the
antisense strand of DNA or RNA encoding the protein of the present
invention. The present invention also relates to an agent for
diagnosing cancers, which comprises the above-described probe for
diagnosing cancers or an antibody reacting with the protein of the
present invention. The probe for diagnosing cancers of the present
invention is preferably the entire or a part of the antisense
strand of DNA (cDNA) or RNA (cRNA) encoding the protein of the
present invention, which preferably has a length that is long
enough as a probe (at least 20 bases). For example, mRNA of the
protein (cancer antigen) of the present invention obtained from an
analyte is detected using the above-described antisense strand,
thereby enabling the diagnosis of cancers. Examples of an analyte
used for detection may include genomic DNA that can be obtained by
biopsy of the cells of a subject, such as the blood, urine, saliva,
or tissues; RNA; and cDNA, but examples are not limited thereto.
When such an analyte is used, those amplified by PCR and the like
may also be used.
[0080] The type of cancer is not particularly limited in the
present specification. Specific examples of cancer may include
pancreatic cancer, colon cancer, brain tumor, malignant melanoma,
chronic myelocytic leukemia, acute myelocytic leukemia, lymphoma,
esophageal cancer, kidney cancer, prostatic cancer, lung cancer,
breast cancer, stomach cancer, hepatic cancer, gallbladder cancer,
testicular cancer, uterine cancer, ovarian cancer, thyroid cancer,
bladder cancer, and sarcoma.
[0081] The immune response of a cancer patient to cancer cells is
unexpectedly active, and it is found that IgG antibodies are
generated to various types of proteins. As described later in
examples, hsp105 that is the antigen protein of the present
invention is highly expressed specifically in pancreatic cancer,
colon cancer, breast cancer, esophageal cancer, malignant lymphoma,
pheochromocytoma, thyroid cancer, bladder cancer, and seminoma.
[0082] The protein or peptide of the present invention can induce
cancer cell-specific cytotoxic T lymphocytes as T cell epitopes.
Thus, it is useful as a preventive and/or therapeutic agent used
for human cancers. In addition, the antibody of the present
invention is also effective as a preventive and/or therapeutic
agent used for human cancers, as long as it can inhibit the
activity of the protein of the present invention that is a cancer
antigen. As a practical usage, the protein, peptide, or antibody of
the present invention may be administered directly, or together
with a pharmaceutically acceptable carrier and/or diluent, or also
together with the below-mentioned assistant agents, as necessary,
so as to create an injection. Otherwise, it may also be
administered by percutaneous absorption via the mucosa according to
a method such as spraying. The term "carrier" is used herein to
mean, for example, human serum albumin. Examples of a diluent may
include PBS and distilled water.
[0083] As a dosage, the cancer antigen, peptide, or antibody of the
present invention may be administered within a range between 0.01
mg and 100 mg per once per adult. However, a dosage is not limited
to the above range. Also, the dosage form is not particularly
limited. A freeze-dried product, or granules produced by adding an
excipient such as sugar to the cancer antigen, peptide, or antibody
of the present invention, may also be used.
[0084] Examples of an assistant agent that can be added to the
agent of the present invention to enhance the activity of inducing
cytotoxic T lymphocytes may include fungal components of BCG
bacteria or the like, ISCOM described in Nature, vol. 344, p. 873
(1990), QS-21 of saponins described in J. Immunol. vol. 148, p.
1438 (1992), liposome, and aluminum hydroxide. In addition, immune
activators such as lenthinan, schizophyllan, or Picibanil may also
be used as assistant agents. Moreover, cytokines which promote the
growth or differentiation of T cells, such as IL-2, IL-4, IL-12,
IL-1, IL-6, or TNF, may also be used as assistant agents.
[0085] Furthermore, the above-described antigen peptide is added to
cells collected from a patient or cells having the same HLA
haplotype in a test tube, so as to allow the cells to present an
antigen. Thereafter, it is administered into the blood vessel of
the patient, so that cytotoxic T lymphocytes can effectively be
induced in the patient body. Moreover, the above-described peptide
is added to the peripheral blood lymphocytes of a patient, and it
is then cultured in a test tube, so that cytotoxic T lymphocytes
can be induced in the test tube and then returned to the blood
vessel of the patient. Such a treatment involving cell transfer has
already been conducted as a cancer treatment, and thus, it has been
well known to persons skilled in the art.
[0086] A target antigen in the specific antitumor immunotherapy is
required to be an antigen recognized by cytotoxic T lymphocytes
(killer T cells/CTL). The antigen of the present invention has
increased in vitro killer T cell-stimulating activity on HLA-A24
that is widely found in Japanese, or on HLA-A2 that is widely found
over the world. Thus, the injection of the antigen of the present
invention into a body induces the activation of CTL, and as a
result, it can be anticipated that antitumor effects be obtained.
Moreover, when lymphocytes are stimulated with the antigen of the
present invention in vitro, activated T cells are induced. The thus
activated T cells are injected into an affected area. Thus, this
method can effectively be used as an adoptive immunotherapy.
EXAMPLES
[0087] The antigen of the present invention, the production method
thereof, and the effects thereof will be described in the following
examples. However, these examples are not intended to limit the
scope of the present invention.
Example 1
<Collection of Serum>
[0088] The serum was collected from a patient with pancreatic
cancer. The collected serum was conserved at -80.degree. C. From
this serum sample, an antibody reacting with Escherichia coli and
.lamda. phage was eliminated by using a column that was filled with
dissolved matter of Escherichia coli and phage and sepharose 4B.
Thereafter, the resultant serum was 100 to 800 times diluted and
then used.
<cDNA Library and Production of Protein>
[0089] A phage cDNA library produced by inserting cDNA of a
pancreatic cancer cell line CFPAC-1 into a .lamda.ZAP express
vector was purchased from Stratagene, La Jolla, Calif. Escherichia
coli was infected with this .lamda. phage cDNA library, and it was
then cultured on NZY plate medium at 42.degree. C. for 6 hours, so
as to produce plaques. Thereafter, the plate was covered with a
nitrocellulose membrane into which isopropyl
.beta.-D-thiogalactoside (IPTG) had been infiltrated at 37.degree.
C. for 3 hours, so as to produce a protein encoded by the cDNA that
had been incorporated into .lamda. phage in the plaques.
<Immunoscreening>
[0090] The protein produced by the aforementioned method was
transferred into a nitrocellulose membrane. After blocking, the
nitrocellulose was washed, and it was then allowed to react with
the above-described serum at 4.degree. C. for 15 hours. After
washing, a horseradish peroxidase (HRP)-labeled mouse anti-human
IgG antibody was used as a secondary antibody, and it was allowed
to react with the membrane. After washing, chemoluminescence was
detected on an X-ray film, and it was compared with the plate on a
photograph, so that positive plaques were picked up together with
the peripheral negative plaques. Plaques corresponding to positive
sites in a color reaction were collected from a 15-cm NZY agarose
plate. The collected plaques were then dissolved in an SM buffer
solution (100 mM NaCl, 10 mM MgSO.sub.4, 50 mM Tris-HCl, and 0.01%
gelatin; pH 7.5). The color reaction positive plaques were
subjected to a second screening and a third screening by the same
above method, until they became a single colony, thereby obtaining
a single phage clone with which an IgG antibody in the serum
reacts. By the above-described method, 63 positive clones were
isolated from cDNA derived from the pancreatic cancer cell
line.
<Homology Search of Isolated Antigen Gene>
[0091] Insert DNA was amplified by the PCR method, and it was used
for the subsequent analysis. The obtained PCR product was sequenced
using Big Dye DNA sequencing kit (PE Biosystems, CA), so as to
determine a nucleotide sequence thereof. Using homology search
program BLAST (Basic Local Alignment Search Tool), each of the thus
determined nucleotide sequences of 63 types of genes was compared
with the gene information registered in the NCBI databank.
<hsp105>
[0092] As a result, 18 positive clones shown in Table 1 were found.
One of the positive clones was hsp105. TABLE-US-00002 TABLE 1 Genes
Isolated by SEREX of a Pancreatic Ductal Adenocarcinoma Cell Line
SEREX Gene database designation Gene/sequence identity search.sup.a
KM-PA-1 apg-2 (heat shock protein 110 family) NGO-St-81, NY-CO-40,
NY-CO-32 KM-PA-2 EST (KIAA0124) -- KM-PA-3 .beta.-actin -- KM-PA-4
coactosin-like protein (CLP) -- KM-PA-5 HALPHA44 (alpha-tubulin) --
KM-PA-6 unknown -- KM-PA-7 CDC-like kinase (CLK3) -- KM-PA-8
cytokeratin 18 -- KM-PA-9 polyA binding protein -- KM-PA-10
very-long-chain-acyl-CoA-dehydrogenase -- (VLCAD) KM-PA-11 unknown
-- KM-PA-12 HLA-Cw heavy chain (MHC Class I) LONY-BR-26 KM-PA-13
unknown -- KM-PA-14 CGI 55 protein -- KM-PA-15
glycosylation-inhibiting factor (GIF) Mz19-16a, Hom-HD1-21 KM-PA-16
unknown NGO-St-95, NGO-St-103 KM-PA-17 DNA binding protein A (dbpA)
-- KM-PA-18 heat shock protein 105 (KIAA0201) NY-CO-25 .sup.aDash
means no strong homology.
<Confirmation of Expression of hsp105>
[0093] The presence or absence of the expression of an hsp105
protein was immunohistochemically analyzed in various types of
cancer tissues and in normal tissues. As a result, it was found
that hsp105 is expressed in pancreatic cancer tissues and in colon
cancer tissues, as shown in FIGS. 1 and 2.
Example 2
<Peptide Constituting hsp105>
[0094] A motif binding to HLA-A24, for which 60% of Japanese people
get positive, is identical to a motif, to which K.sup.d of a BALB/c
mouse binds. A peptide that is shared by human hsp105 and mouse
hsp105 and is predicted to bind to both HLA-A24 and K.sup.d is
selected from the sequence of hsp105, using HLA-peptide binding
prediction (http://bimas/dcrt.nih.gov/molbio/hla_bind/). Nine types
of peptides consisting of 9 or 10 amino acids were synthesized by
the Fmoc/PyBOP method. The sequences of the peptides and the
estimated binding values to K.sup.d are shown in Table 2.
TABLE-US-00003 TABLE 2 hsp105-derived peptides hsp105-derived
peptides No. Position Sequence Binding Score 1 hsp105 180-188
NYGIYKQDL 2400 2 hsp105 214-223 AFNKGKLKVL 960 3 hsp105 251-260
KYKLDAKSKI 2880 4 hsp105 305-313 QFEELCAEL 1382 5 hsp105 433-442
TFLRRGPFEL 1920 6 hsp105 570-579 MYIETEGKMI 4800 7 hsp105 597-606
ECVYEFRDKL 80 8 hsp105 682-690 HYAKIAADF 60 9 hsp105 696-705
KYNHIDESEM 432
Example 3
<DNA Vaccine>
[0095] Plasmid DNA produced by incorporating mouse hsp105 cDNA into
an expression vector pCAGGS was adjusted to be a suitable
concentration. It was then used as a vaccine for the following
performance evaluation test. With regard to this mouse
hsp105-pCAGGS DNA vaccine, Escherichia coli was cultured, and
thereafter, plasmid DNA was extracted from the Escherichia coli and
purified, so as to produce the vaccine in large scale.
<Anti-Cancer Effects of Peptide Vaccine and DNA Vaccine>
[0096] The following samples were injected into the muscle of each
BALB/c mouse: (1) a normal saline solution, (2) only a vector, (3)
a hsp105 cDNA vector, (4) only an adjuvant, (5) an adjuvant +a
peptide. Thereafter, a colon cancer cell line Colon-26 derived from
a syngeneic mouse, that highly expresses hsp105, was subcutaneously
transplanted into the back of the mouse. Thereafter, the
development of the cancer in the mice was evaluated in the
following points: (1) the area of a cancerous portion, (2) the
ratio of mice in which the cancer developed, and (3) the ratio of
surviving mice. The results are shown in FIGS. 3A, 3B, and 3C.
[0097] As shown in FIGS. 3A, 3B, and 3C, when 3.times.10.sup.4
cells of Colon-26 were transplanted, until 13 days after the
immunization, a tumor developed in all the 5 mice immunized with a
normal saline solution and in all the 5 mice immunized with only
pCAGGS. On the other hand, in the case of 5 mice immunized with
hsp105-DNA vaccine, a tumor developed in one mouse 20 days after
the immunization and in another mouse 24 days after the
immunization. However, the remaining 3 mice completely rejected the
development of a tumor. In the case of the adjuvant administration
group, a tumor developed in all the 5 mice until 24 days after the
immunization. There were observed significant differences between
the DNA vaccine-, peptide vaccine-, and adjuvant-administration
groups, and the normal saline solution- and vector-administration
groups (FIG. 3B). The same results were obtained regarding the mean
tumor area (FIG. 3A).
[0098] From these results, it is clear that the peptide vaccine and
the DNA vaccine have the effects as anticancer agents. Considering
a survival curve, 2 out of the 5 mice still survived 45 days after
the immunization in the normal saline solution-, vector-, and
adjuvant-administration groups. Moreover, all the 5 mice survived
in the DNA vaccine-administration group. The DNA vaccine group had
significant differences from the other 4 groups. The peptide
vaccine group had a significantly longer survival period than those
of the normal saline solution-, vector-, and
adjuvant-administration groups (FIG. 3C). Furthermore, the mice
that rejected the development of a tumor were pathologically
observed, and it was confirmed that there were no damage on normal
organs and that a large number of inflammatory cells filtrated into
sites that rejected the development of a tumor.
<Determination of CTL Epitope Peptide of hsp105>
[0099] In order to identify a CTL epitope peptide, pancreatic cells
were recovered from the mice, on which the DNA vaccine-peptide
vaccine had worked. The recovered cells were stimulated once with
the 9 types of peptides shown in Table 2, and the cytotoxic
activity on Colon-26 was analyzed by .sup.51Cr release assay. As a
result, it was found that among the above-described 9 types of
peptides, the following 5 types of peptides 1, 2, 3, 4, and 5 are
useful (FIG. 4). TABLE-US-00004 Asn-Tyr-Gly-lle-Tyr-Lys-Gln-Asp-Leu
(1) Ala-Phe-Asn-Lys-Gly-Lys-Leu-Lys-Val-Leu (2)
Lys-Tyr-Lys-Leu-Asp-Ala-Lys-Ser-Lys-Ile (3)
Gln-Phe-Glu-Glu-Leu-Cys-Ala-Glu-Leu (4)
Met-Tyr-Ile-Glu-Thr-Glu-Gly-Lys-Met-Ile (5)
<Agent for Diagnosing Cancers>
[0100] Using an hsp105 antibody, the pathological diagnosis of the
following cancers can be conducted: pancreatic cancer, colon
cancer, brain tumor, malignant melanoma, chronic myelocytic
leukemia, acute myelocytic leukemia, lymphoma, esophageal cancer,
kidney cancer, prostatic cancer, lung cancer, breast cancer,
stomach cancer, hepatic cancer, gallbladder cancer, testicular
cancer, uterine cancer, ovarian cancer, thyroid cancer, bladder
cancer, and sarcoma.
<CTL Cancer Therapeutic Agent>
[0101] In the case of mice, it was clarified that killer T cells
recognizing hsp105 and/or a peptide constituting the hsp105 as an
antigen do not impair normal cells and have cytotoxic activity only
on mouse colon cancer. Thus, there is a possibility that CTL can be
used as a cancer therapeutic or preventive agent with few side
effects in human pancreatic and colon cancers wherein hsp105 is
highly expressed.
Example 4
Identification of CTL Epitope Peptide of HLA-A24 of hsp105 in
Human
[0102] In order to determine the CTL epitope peptide of HLA-A24 of
hsp105 in human, the peripheral blood lymphocytes collected from
two colon cancer patients with HLA-A24 were stimulated 4 times with
9 types of peptides shown in Table 3. Thereafter, the cytotoxic
activity on a human colon cancer cell line sw620, which highly
expresses hsp105 and also expresses HLA-A24, was analyzed by
.sup.51Cr release assay.
[0103] Specifically, monocytes were separated from the peripheral
blood. Two millions of monocytes per well on a 24-well plate were
cultured in 2 ml of a culture solution containing 10% autoserum,
IL-2 (100 IU/ml), and 10 .mu.M each peptide for 1 week. Thereafter,
every week, the above culture product was stimulated with 200,000
dendritic cells (DC), which had been induced over 1 week, pulsed
with 10 mM the above peptide and exposed to radioactive rays,
repeatedly 3 times. 6 days later, the cytotoxic activity thereof
was analyzed. Herein, two millions of monocytes were cultured in
the presence of GM-CSF (100 ng/ml) and IL-4 (100 U/ml) for 5 days,
and TNF-.alpha. (20 ng/ml) was further added thereto, followed by
culture for 2 days. The obtained culture product was used as
DC.
[0104] CTL which impairs C1RA2402 cells stimulated with the above
peptide rather than those that were not stimulated with the above
peptide was defined as peptide-specific positive. The results are
shown in Table 3. The boldface figures in Table 3 indicate that CTL
that is specific for the peptide and has cancer cell cytotoxicity
can be induced. TABLE-US-00005 TABLE 3 Peptides which can induce
peptide-specific and cancer cell cytotoxic killer T cell by
stimulating human peripheral blood lymphocytes of HLA-A24 each
peptide-induced CTLs each peptide-induced CTLs from Pt 1
(HLA-A2402/) from Pt 2 (HLA-A2402/) % Lysis to % Lysis to hsp105-
HLA-A2402- sw620 sw620 C1RA2402 derived binding (HLA-A C1RA2402
(HLA- peptide peptide Position Sequence score 0201/2402) C1RA2402
peptide 10 .mu.M A0201) C1RA2402 10 .mu.M A24-1 180-188 NYGIYKQDL
240 16 42 31 32 13 25 A24-2 214-223 AFNKGKLKVL 30 0 42 49 40 28 54
A24-3 251-260 KYKLDAKSKI 110 50 29 46 21 33 44 A24-4 305-313
QFEELCAEL 48 48 22 43 16 40 38 A24-5 433-442 TFLRRGPFEL 33 53 33 33
26 33 46 A24-6 613-622 MYIETEGKMI 90 49 22 47 29 28 52 A24-7
640-649 EYVYEFRDKL 330 40 22 45 8 26 31 A24-8 725-733 HYAKIAADF 140
41 25 37 66 28 43 A24-9 739-748 KYNHIDESEM 83 19 36 43 33 24 45
Example 5
Identification of CTL Epitope Peptide of HLA-A2 of hsp105 in
Human
[0105] In order to determine the CTL epitope peptide of HLA-A2 of
hsp105 in human, the peripheral blood lymphocytes of a colon cancer
patient with HLA-A2 and those of a healthy subject with HLA-2 were
stimulated 4 times with 30 types of peptides shown in Table 4.
Thereafter, the cytotoxic activity on a human colon cancer cell
line sw620, which highly expresses hsp105 and also expresses
HLA-A2, was analyzed. Specific experimental methods were the same
as those in Example 4.
[0106] In addition, sw620 cells, the expression level of hsp105 of
which was reduced by RNAi, was defined as sw620 hsp105-RNAi cells.
If CTL did not impair the sw620 hsp105-RNAi cells, then it was
judged that it had cytotoxic activity specific for hsp105.
Moreover, CTL impairing sw620 hsp105-RNAi cells stimulated with the
above peptide rather than those that were not stimulated with the
above peptide was defined as peptide-specific positive. The results
are shown in Table 4. The boldface figures in Table 4 indicate that
CTL that is specific for the peptide and has cancer cell
cytotoxicity can be induced. TABLE-US-00006 TABLE 4 Peptides which
can induce peptide-specific and cancer cell cytotoxic killer T cell
by stimulating human peripheral blood lymphocytes of HLA-A2 each
peptide-induced CTLs each peptide-induced CTLs from Pt 1
(HLA-A0207/3301) from HD 1 (HLA-A0201/0207) % Lysis to % Lysis to
hsp105- HLA-A0201- sw620 sw620 sw620 sw620 sw620 sw620 derived
binding (HLA- hsp105- hsp105-RNAI (HLA- hsp105- hsp105-RNAI peptide
Position Sequence score A0201) RNAI peptide 10 .mu.M A0201) RNAI
peptide 10 .mu.M A2-1 86-94 NLSYDLVPL 49 5 68 56 -- -- -- A2-2
103-111 VMYMGEEHL 41 20 41 36 -- -- -- A2-3 105-114 YMGEEHLFSV
12637 5 0 0 -- -- -- A2-4 120-128 MLLTKLKET 107 0 0 1 6 35 3 A2-5
141-149 VISVPSFFT 55 4 0 5 -- -- -- A2-6 155-163 SVLDAAQIV 37 5 7
18 4 0 13 A2-7 169-177 RLMNDMTAV 591 4 0 8 2 29 32 A2-8 190-199
SLDEKPRIVV 46 30 18 0 26 9 40 A2-9 202-210 DMGHSAFQV 21 26 0 3 --
-- -- A2-10 222-231 VLGTAFDPFL 759 0 29 20 2 0 0 A2-11 265-273
RLYQECEKL 33 18 0 28 15 0 17 A2-12 275-284 KLMSSNSTDL 276 10 1 13
10 28 58 A2-13 276-284 LMSSNSTDL 26 11 0 21 11 0 14 A2-14 300-309
KMNRSQFEEL 50 11 0 0 44 61 9 A2-15 304-313 SQFEELCAEL 32 12 0 4 21
0 9 A2-16 313-321 LLQKIEVPL 36 10 21 8 -- -- -- A2-17 323-332
SLLEQTHLKV 1055 1 76 34 32 0 0 A2-18 381-389 AILSPAFKV 205 50 0 0
22 28 9 A2-19 434-422 FLRRGPFEL 43 8 39 3 -- -- -- A2-20 458-467
KIGRFVVQNV 76 24 0 9 32 9 4 A2-21 601-610 NLVWQLGKDL 21 7 0 4 5 0 4
A2-22 602-610 LVWQLGKDL 26 19 0 3 -- -- -- A2-23 641-649 YVYEFRDKL
210 26 2 13 0 9 23 A2-24 648-657 KLCGPYEKFI 200 9 0 0 42 0 9 A2-25
668-676 LLTETEDWL 401 32 0 27 23 42 27 A2-26 675-684 WLYEEGEDQA 146
18 0 41 11 21 3 A2-27 694-702 ELMKIGTPV 19 14 0 13 22 0 0 A2-28
714-723 KMFEELGQRL 819 11 2 0 5 0 0 A2-29 757-765 EVMEWMNNV 15 1 0
0 -- -- -- A2-30 765-774 VMNAQAKKSL 26 0 0 11 26 0 12
Example 6
Results of Immunohistochemical Analysis of hsp105 in Tissues
[0107] The expression of hsp105 in various tissues was
immunohistochemically analyzed. Specifically, thin sections with a
size of 3 mm were prepared from blocks obtained by immobilizing
various tissues with formalin and embedding them in paraffin.
Thereafter, using VECTOR stain ABC-PO (rabbit IgG) kit (Vector
Laboratories, Inc. Burlingame, Calif.), these sections were
subjected to immunohistochemical analysis by the ABC method
(avidin-biotin complex immune peroxidase technique). Rabbit
polyclonal anti-human HSP105 Ab (SANTACRUZ, Santa Cruz, Calif.) was
purchased, and it was then 200 times diluted. The obtained product
was used as a primary antibody.
[0108] FIG. 5 is a microphotograph showing the results of the above
immunohistochemical analysis. In FIG. 5, symbols have the following
meanings. A: colon cancer, B: colon cancer in colon polyp, C: liver
metastasis of colon cancer, D: pancreatic cancer, E: insulinoma, F:
papillary adenocarcinoma in breast cancer, G: scirrhus cancer in
breast cancer, H: esophageal cancer, I: thyroid cancer, J: gastric
malignant lymphoma, K: pheochromocytoma, L: bladder cancer, M:
testis, and N: seminoma. As is clear from the results shown in FIG.
5, a high level of expression of hsp105 was observed in A, B, C, D,
E, F, H, I, J, K, L, M, and N, that is, tumors other than G, and
also in the testis.
Example 7
In Vivo Antitumor Activity of Mouse CD4 Positive Helper T Cell Line
Induced by hsp105
[0109] The spleen of a BALB/c mouse was collected and ground to
separate spleen cells. 200,000 spleen cells per well on a 96-well
flat plate were cultured in 200 .mu.l of a culture solution
containing IL-2 (100 IU/ml) and a 2 .mu.g/ml recombinant hsp105
protein for 1 week. Thereafter, every week, the culture product was
repeatedly stimulated with 200,000 spleen cells, which had been
pulsed with a 2 .mu.g/ml recombinant hsp105 protein and then
exposed to radioactive rays, so as to establish multiple CD4
positive helper T cell lines (Th). The expression of CD4 and CD8 on
the surface of cells was confirmed by performing immunofluorescent
staining with Monoclonal Antibody MOUSE CD4-FITC, CD8-FITC
(IMMUNOTECH, Marseille, France), and then analyzed with FACS (FIG.
6A). It was examined by the intake of [.sup.3H] thymidine, whether
or not Th specifically reacts with the hsp105 protein and grows.
Specifically, 150,000 spleen cells were placed in each well of a
96-well flat plate, and several wells were pulsed with the hsp105
protein overnight, and the other wells were not pulsed therewith.
To both types of wells, 30,000 Th cells were added, followed by the
reaction for 72 hours (1 .mu.Ci of [.sup.3H] thymidine was added to
each well for the last 16 hours). Thereafter, the intake of
[.sup.3H] thymidine was measured with a liquid scintillation
counter. The Th cells specifically reacted with the hsp105 protein
and grew (FIG. 6B). On the other hand, 24 hours after the addition
of Th, the supernatant was kept. Thus, IFN-.gamma. and IL-4
secreted from Th as a result of the reaction were measured with
Mouse IFN-.gamma., IL-4 ELISA Ready-SET-Go! (eBioscience). The Th
was of Th1 type, which generates a large amount of IFN-.gamma. as a
result of the specific reaction with the hsp105 protein (FIG. 6C).
Colon-26 was subcutaneously implanted into the back of a BALB/c
mouse to form a tumor with a size of 3 mm. Thereafter, the above Th
was injected into the local site, followed by observation of the
progression. As a result, after such a treatment, the growth of the
Colon-26 tumor was clearly retarded (FIG. 6D).
[0110] From the above-described results, it was found that the
BALB/c mouse CD4 positive helper T cell line induced by the hsp105
protein grows hsp105 protein-specifically, and that it delays the
growth of a tumor mass of the colon cancer cell line Colon-26 that
highly expresses hsp105.
Example 8
CD4 Positive Helper T Cell Line of Colon Cancer Patient Induced by
hsp105
[0111] Monocytes were separated from the peripheral blood. 200,000
monocytes per well of a 96-well flat plate were cultured in 200
.mu.l of a culture solution containing IL-2 (100 IU/ml) and a 2
.mu.g/ml recombinant hsp105 protein for 1 week. Thereafter, every
week, the culture product was repeatedly stimulated with 200,000
monocytes, which had been pulsed with a 2 .mu.g/ml recombinant
hsp105 protein and then exposed to radioactive rays, so as to
establish multiple CD4 positive helper T cell lines (Th). The
expression of CD4 and CD8 on the surface of cells was confirmed by
performing immunofluorescent staining using Pharmingen anti-human
CD4, CD8-FITC, and then analyzed with FACS. It was examined in the
same manner as in Example 7, whether or not Th specifically reacts
with the hsp105 protein and grows. The Th cells specifically
reacted with the hsp105 protein and grew (FIG. 7A). In addition, in
the same manner as in Example 7, IFN-.gamma. and IL-4 secreted from
Th as a result of the reaction were measured using Human
IFN-.gamma., IL-4 US ELISA Kit (BIOSOURCE, Camarillo, Calif.). The
Th was of Th1 type, which generates a large amount of IFN-.gamma.
as a result of the specific reaction with the hsp105 protein (FIG.
7B). In general, it has been known that Th1 acts favorably for the
induction of CTL and antitumor immunity. It was found that such Th1
can be induced also in humans by stimulating peripheral blood
lympocytes with the hsp105 protein.
Example 9
In Vivo Antitumor Activity of Cytotoxic T Lymphocytes Stimulated
with hsp105 Peptide
[0112] It was examined whether or not BALB/c mouse cytotoxic T
lymphocytes (CTL) induced by an hsp105-derived peptide
Asn-Tyr-Gly-Ile-Tyr-Lys-Gln-Asp-Leu (SEQ ID NO: 3) reduce a tumor
mass of the colon cancer cell line Colon-26 that highly expresses
hsp105. Specifically, Colon-26 was subcutaneously implanted into
the back of a BALB/c mouse to form a tumor with a size of 5 mm.
Thereafter, CTL was injected into the local site. 1 week later, the
mouse was subjected to anatomy, and the site was pathologically
observed by HE staining. The results are shown in FIG. 8. As is
apparent from the results shown in FIG. 8, the tumor was clearly
reduced by administration of the CTL induced by the hsp105-derived
peptide.
[0113] Also, it was examined whether or not the cytotoxic T
lymphocytes (CTL) of a colon cancer patient induced by an
hsp105-derived peptide Lys-Leu-Met-Ser-Ser-Asn-Ser-Thr-Asp-Leu (SEQ
ID NO: 14) reduce a tumor mass of the colon cancer cell line sw620
that highly expresses hsp105. Specifically, sw620 was
subcutaneously implanted into the back of a nude mouse to form a
tumor with a size of 5 mm, and thereafter, CTL was injected into
the local site.
[0114] 1 week after injection of the CTL, the tumor was reduced. 2
weeks after the treatment, the mouse was subjected to anatomy, and
the site was pathologically observed by HE staining. The results
are shown in FIG. 9. As is apparent from the results shown in FIG.
9, the increase of the tumor was clearly retarded by administration
of the CTL.
INDUSTRIAL APPLICABILITY
[0115] The cancer antigen protein and antigen peptide of the
present invention, or DNA encoding the protein or peptide of the
present invention, can be used as an excellent anti-cancer vaccine
having few side effects such as self-injury. In addition, an
antibody can be used as a diagnostic agent. Moreover, helper T
cells, cytotoxic T lymphocytes, or an immunocyte population
containing these cells, which are stimulated and activated with the
antigen of the present invention, can be used as anticancer agents.
Sequence CWU 1
1
23 1 858 PRT Homo sapiens 1 Met Ser Val Val Gly Leu Asp Val Gly Ser
Gln Ser Cys Tyr Ile Ala 1 5 10 15 Val Ala Arg Ala Gly Gly Ile Glu
Thr Ile Ala Asn Glu Phe Ser Asp 20 25 30 Arg Cys Thr Pro Ser Val
Ile Ser Phe Gly Ser Lys Asn Arg Thr Ile 35 40 45 Gly Val Ala Ala
Lys Asn Gln Gln Ile Thr His Ala Asn Asn Thr Val 50 55 60 Ser Asn
Phe Lys Arg Phe His Gly Arg Ala Phe Asn Asp Pro Phe Ile 65 70 75 80
Gln Lys Glu Lys Glu Asn Leu Ser Tyr Asp Leu Val Pro Leu Lys Asn 85
90 95 Gly Gly Val Gly Ile Lys Val Met Tyr Met Gly Glu Glu His Leu
Phe 100 105 110 Ser Val Glu Gln Ile Thr Ala Met Leu Leu Thr Lys Leu
Lys Glu Thr 115 120 125 Ala Glu Asn Ser Leu Lys Lys Pro Val Thr Asp
Cys Val Ile Ser Val 130 135 140 Pro Ser Phe Phe Thr Asp Ala Glu Arg
Arg Ser Val Leu Asp Ala Ala 145 150 155 160 Gln Ile Val Gly Leu Asn
Cys Leu Arg Leu Met Asn Asp Met Thr Ala 165 170 175 Val Ala Leu Asn
Tyr Gly Ile Tyr Lys Gln Asp Leu Pro Ser Leu Asp 180 185 190 Glu Lys
Pro Arg Ile Val Val Phe Val Asp Met Gly His Ser Ala Phe 195 200 205
Gln Val Ser Ala Cys Ala Phe Asn Lys Gly Lys Leu Lys Val Leu Gly 210
215 220 Thr Ala Phe Asp Pro Phe Leu Gly Gly Lys Asn Phe Asp Glu Lys
Leu 225 230 235 240 Val Glu His Phe Cys Ala Glu Phe Lys Thr Lys Tyr
Lys Leu Asp Ala 245 250 255 Lys Ser Lys Ile Arg Ala Leu Leu Arg Leu
Tyr Gln Glu Cys Glu Lys 260 265 270 Leu Lys Lys Leu Met Ser Ser Asn
Ser Thr Asp Leu Pro Leu Asn Ile 275 280 285 Glu Cys Phe Met Asn Asp
Lys Asp Val Ser Gly Lys Met Asn Arg Ser 290 295 300 Gln Phe Glu Glu
Leu Cys Ala Glu Leu Leu Gln Lys Ile Glu Val Pro 305 310 315 320 Leu
Tyr Ser Leu Leu Glu Gln Thr His Leu Lys Val Glu Asp Val Ser 325 330
335 Ala Val Glu Ile Val Gly Gly Ala Thr Arg Ile Pro Ala Val Lys Glu
340 345 350 Arg Ile Ala Lys Phe Phe Gly Lys Asp Ile Ser Thr Thr Leu
Asn Ala 355 360 365 Asp Glu Ala Val Ala Arg Gly Cys Ala Leu Gln Cys
Ala Ile Leu Ser 370 375 380 Pro Ala Phe Lys Val Arg Glu Phe Ser Val
Thr Asp Ala Val Pro Phe 385 390 395 400 Pro Ile Ser Leu Ile Trp Asn
His Asp Ser Glu Asp Thr Glu Gly Val 405 410 415 His Glu Val Phe Ser
Arg Asn His Ala Ala Pro Phe Ser Lys Val Leu 420 425 430 Thr Phe Leu
Arg Arg Gly Pro Phe Glu Leu Glu Ala Phe Tyr Ser Asp 435 440 445 Pro
Gln Gly Val Pro Tyr Pro Glu Ala Lys Ile Gly Arg Phe Val Val 450 455
460 Gln Asn Val Ser Ala Gln Lys Asp Gly Glu Lys Ser Arg Val Lys Val
465 470 475 480 Lys Val Arg Val Asn Thr His Gly Ile Phe Thr Ile Ser
Thr Ala Ser 485 490 495 Met Val Glu Lys Val Pro Thr Glu Glu Asn Glu
Met Ser Ser Glu Ala 500 505 510 Asp Met Glu Cys Leu Asn Gln Arg Pro
Pro Glu Asn Pro Asp Thr Asp 515 520 525 Lys Asn Val Gln Gln Asp Asn
Ser Glu Ala Gly Thr Gln Pro Gln Val 530 535 540 Gln Thr Asp Ala Gln
Gln Thr Ser Gln Ser Pro Pro Ser Pro Glu Leu 545 550 555 560 Thr Ser
Glu Glu Asn Lys Ile Pro Asp Ala Asp Lys Ala Asn Glu Lys 565 570 575
Lys Val Asp Gln Pro Pro Glu Ala Lys Lys Pro Lys Ile Lys Val Val 580
585 590 Asn Val Glu Leu Pro Ile Glu Ala Asn Leu Val Trp Gln Leu Gly
Lys 595 600 605 Asp Leu Leu Asn Met Tyr Ile Glu Thr Glu Gly Lys Met
Ile Met Gln 610 615 620 Asp Lys Leu Glu Lys Glu Arg Asn Asp Ala Lys
Asn Ala Val Glu Glu 625 630 635 640 Tyr Val Tyr Glu Phe Arg Asp Lys
Leu Cys Gly Pro Tyr Glu Lys Phe 645 650 655 Ile Cys Glu Gln Asp His
Gln Asn Phe Leu Arg Leu Leu Thr Glu Thr 660 665 670 Glu Asp Trp Leu
Tyr Glu Glu Gly Glu Asp Gln Ala Lys Gln Ala Tyr 675 680 685 Val Asp
Lys Leu Glu Glu Leu Met Lys Ile Gly Thr Pro Val Lys Val 690 695 700
Arg Phe Gln Glu Ala Glu Glu Arg Pro Lys Met Phe Glu Glu Leu Gly 705
710 715 720 Gln Arg Leu Gln His Tyr Ala Lys Ile Ala Ala Asp Phe Arg
Asn Lys 725 730 735 Asp Glu Lys Tyr Asn His Ile Asp Glu Ser Glu Met
Lys Lys Val Glu 740 745 750 Lys Ser Val Asn Glu Val Met Glu Trp Met
Asn Asn Val Met Asn Ala 755 760 765 Gln Ala Lys Lys Ser Leu Asp Gln
Asp Pro Val Val Arg Ala Gln Glu 770 775 780 Ile Lys Thr Lys Ile Lys
Glu Leu Asn Asn Thr Cys Glu Pro Val Val 785 790 795 800 Thr Gln Pro
Lys Pro Lys Ile Glu Ser Pro Lys Leu Glu Arg Thr Pro 805 810 815 Asn
Gly Pro Asn Ile Asp Lys Lys Glu Glu Asp Leu Glu Asp Lys Asn 820 825
830 Asn Phe Gly Ala Glu Pro Pro His Gln Asn Gly Glu Cys Tyr Pro Asn
835 840 845 Glu Lys Asn Ser Val Asn Met Asp Leu Asp 850 855 2 3611
DNA Homo sapiens 2 gaggaagtgg gacctcccct tttgggtcgg tagttcagcg
ccggcgccgg tgtgcgagcc 60 gcggcagagt gaggcaggca acccgaggtg
cggagcgacc tgcggaggct gagccccgct 120 ttctcccagg gtttcttatc
agccagccgc cgctgtcccc gggggagtag gaggctcctg 180 acaggccgcg
gctgtctgtg tgtccttctg agtgtcagag gaacggccag accccgcggg 240
ccggagcaga acgcggccag ggcagaaagc ggcggcagga gaagcaggca gggggccgga
300 ggacgcagac cgagacccga ggcggaggcg gaccgcgagc cggccatgtc
ggtggtgggg 360 ttggacgtgg gctcgcagag ctgctacatc gcggtagccc
gggccggggg catcgagacc 420 atcgccaatg agttcagcga ccggtgcacc
ccgtcagtca tatcatttgg atcaaaaaat 480 agaacaatcg gagttgcagc
caaaaatcag caaatcactc atgcaaacaa tacggtgtct 540 aacttcaaaa
gatttcatgg ccgagcattc aacgacccct tcattcaaaa ggagaaggaa 600
aacttgagtt acgatttggt tccattgaaa aatggtggag ttggaataaa ggtaatgtac
660 atgggtgaag aacatctatt tagtgtggag cagataacag ccatgttgtt
gactaagctg 720 aaggaaactg ctgaaaacag cctcaagaaa ccagtaacag
attgtgttat ttcagtcccc 780 tccttcttta cagatgctga gaggcgatct
gtgttagatg ctgcacagat tgttggccta 840 aactgtttaa gacttatgaa
tgacatgaca gctgttgctt tgaattacgg aatttataag 900 caggatctcc
caagcctgga tgagaaacct cggatagtgg tttttgttga tatgggacat 960
tcagcttttc aagtgtctgc ttgtgctttt aacaagggaa aattgaaggt actgggaaca
1020 gcttttgatc ctttcttagg aggaaaaaac ttcgatgaaa agttagtgga
acatttctgt 1080 gcagaattta aaactaagta caagttggat gcaaaatcca
aaatacgagc actcctacgt 1140 ctgtatcagg aatgtgaaaa actgaaaaag
ctaatgagct ctaacagcac agaccttcca 1200 ctgaatatcg aatgctttat
gaatgataaa gatgtttccg gaaagatgaa caggtcacaa 1260 tttgaagaac
tctgtgctga acttctgcaa aagatagaag taccccttta ttcactgttg 1320
gaacaaactc atctcaaagt agaagatgtg agtgcagttg agattgttgg aggcgctaca
1380 cgaattccag ctgtgaagga aagaattgcc aaattctttg gaaaagatat
tagcacaaca 1440 ctcaatgcag atgaagcagt agccagagga tgtgcattac
agtgtgcaat actttccccg 1500 gcatttaaag ttagagaatt ttccgtcaca
gatgcagttc cttttccaat atctctgatc 1560 tggaaccatg attcagaaga
tactgaaggt gttcatgaag tctttagtcg aaaccatgct 1620 gctcctttct
ccaaagttct cacctttctg agaagggggc cttttgagct agaagctttc 1680
tattctgatc cccaaggagt tccatatcca gaagcaaaaa taggccgctt tgtagttcag
1740 aatgtttctg cacagaaaga tggagaaaaa tctagagtaa aagtcaaagt
gcgagtcaac 1800 acccatggca ttttcaccat ctctacggca tctatggtgg
agaaagtccc aactgaggag 1860 aatgaaatgt cttctgaagc tgacatggag
tgtctgaatc agagaccacc agaaaaccca 1920 gacactgata aaaatgtcca
gcaagacaac agtgaagctg gaacacagcc ccaggtacaa 1980 actgatgctc
aacaaacctc acagtctccc ccttcacctg aacttacctc agaagaaaac 2040
aaaatcccag atgctgacaa agcaaatgaa aaaaaagttg accagcctcc agaagctaaa
2100 aagcccaaaa taaaggtggt gaatgttgag ctgcctattg aagccaactt
ggtctggcag 2160 ttagggaaag accttcttaa catgtatatt gagacagagg
gtaagatgat aatgcaagat 2220 aaattggaaa aagaaaggaa tgatgctaaa
aatgcagttg aggaatatgt gtatgagttc 2280 agagacaagc tgtgtggacc
atatgaaaaa tttatatgtg agcaggatca tcaaaatttt 2340 ttgagactcc
tcacagaaac tgaagactgg ctgtatgaag aaggagagga ccaagctaaa 2400
caagcatatg ttgacaagtt ggaagaatta atgaaaattg gcactccagt taaagttcgg
2460 tttcaggaag ctgaagaacg gccaaaaatg tttgaagaac taggacagag
gctgcagcat 2520 tatgccaaga tagcagctga cttcagaaat aaggatgaga
aatacaacca tattgatgag 2580 tctgaaatga aaaaagtgga gaagtctgtt
aatgaagtga tggaatggat gaataatgtc 2640 atgaatgctc aggctaaaaa
gagtcttgat caggatccag ttgtacgtgc tcaggaaatt 2700 aaaacaaaaa
tcaaggaatt gaacaacaca tgtgaacccg ttgtaacaca accgaaacca 2760
aaaattgaat cacccaaact ggaaagaact ccaaatggcc caaatattga taaaaaggaa
2820 gaagatttag aagacaaaaa caattttggt gctgaacctc cacatcagaa
tggtgaatgt 2880 taccctaatg agaaaaattc tgttaatatg gacttggact
agataacctt aaattggcct 2940 attccttcaa ttaataaaat atttttgcca
tagtatgtga ctctacataa catactgaaa 3000 ctatttatat tttctttttt
aaggatattt agaaattttg tgtattatat ggaaaaagaa 3060 aaaaagctta
agtctgtagt ctttatgatc ctaaaaggga aaattgcctt ggtaactttc 3120
agattcctgt ggaattgtga attcatacta agctttctgt gcagtctcac catttgcatc
3180 actgaggatg aaactgactt ttgtcttttg gagaaaaaaa actgtactgt
tgttcaagag 3240 ggctgtgatt aaaatcttta agcatttgtt cctgccaagg
tagttttctt gcattttgct 3300 ctccattcag catgtgtgtg ggtgtggatg
tttataaaca agactaagtc tgacttcata 3360 agggctttct aaaaccattt
ctgtccaaga gaaaatgact ttttgctttg atattaaaaa 3420 ttcaatgagt
aaaacaaaag ctagtcaaat gtgttagcag catgcagaac aaaaacttta 3480
aactttctct ctcactatac agtatattgt caatgtgaaa gtgtggaatg gaagaaatgt
3540 cgatcctgtt gtaactgatt gtgaacactt ttatgagctt taaaataaag
ttcatcttat 3600 ggtgtcattt t 3611 3 9 PRT Artificial Sequence
immune-stimulating peptide 3 Asn Tyr Gly Ile Tyr Lys Gln Asp Leu 1
5 4 10 PRT Artificial Sequence immune-stimulating peptide 4 Ala Phe
Asn Lys Gly Lys Leu Lys Val Leu 1 5 10 5 10 PRT Artificial Sequence
immune-stimulating peptide 5 Lys Tyr Lys Leu Asp Ala Lys Ser Lys
Ile 1 5 10 6 9 PRT Artificial Sequence immune-stimulating peptide 6
Gln Phe Glu Glu Leu Cys Ala Glu Leu 1 5 7 10 PRT Artificial
Sequence immune-stimulating peptide 7 Met Tyr Ile Glu Thr Glu Gly
Lys Met Ile 1 5 10 8 10 PRT Artificial Sequence immune-stimulating
peptide 8 Thr Phe Leu Arg Arg Gly Pro Phe Glu Leu 1 5 10 9 10 PRT
Artificial Sequence immune-stimulating peptide 9 Glu Tyr Val Tyr
Glu Phe Arg Asp Lys Leu 1 5 10 10 9 PRT Artificial Sequence
immune-stimulating peptide 10 His Tyr Ala Lys Ile Ala Ala Asp Phe 1
5 11 10 PRT Artificial Sequence immune-stimulating peptide 11 Lys
Tyr Asn His Ile Asp Glu Ser Glu Met 1 5 10 12 10 PRT Artificial
Sequence immune-stimulating peptide 12 Ser Leu Asp Glu Lys Pro Arg
Ile Val Val 1 5 10 13 9 PRT Artificial Sequence immune-stimulating
peptide 13 Arg Leu Tyr Gln Glu Cys Glu Lys Leu 1 5 14 10 PRT
Artificial Sequence immune-stimulating peptide 14 Lys Leu Met Ser
Ser Asn Ser Thr Asp Leu 1 5 10 15 9 PRT Artificial Sequence
immune-stimulating peptide 15 Leu Met Ser Ser Asn Ser Thr Asp Leu 1
5 16 10 PRT Artificial Sequence immune-stimulating peptide 16 Ser
Gln Phe Glu Glu Leu Cys Ala Glu Leu 1 5 10 17 10 PRT Artificial
Sequence immune-stimulating peptide 17 Lys Ile Gly Arg Phe Val Val
Gln Asn Val 1 5 10 18 9 PRT Artificial Sequence immune-stimulating
peptide 18 Tyr Val Tyr Glu Phe Arg Asp Lys Leu 1 5 19 9 PRT
Artificial Sequence immune-stimulating peptide 19 Leu Leu Thr Glu
Thr Glu Asp Trp Leu 1 5 20 10 PRT Artificial Sequence
immune-stimulating peptide 20 Trp Leu Tyr Glu Glu Gly Glu Asp Gln
Ala 1 5 10 21 9 PRT Artificial Sequence immune-stimulating peptide
21 Glu Leu Met Lys Ile Gly Thr Pro Val 1 5 22 10 PRT Artificial
Sequence immune-stimulating peptide 22 Val Met Asn Ala Gln Ala Lys
Lys Ser Leu 1 5 10 23 10 PRT Artificial Sequence immune-stimulating
peptide 23 Glu Cys Val Tyr Glu Phe Arg Asp Lys Leu 1 5 10
* * * * *
References