U.S. patent application number 10/038899 was filed with the patent office on 2003-10-02 for peptide capable of inducing immune response to human gastric cancer and agent for preventing or treating human gastric cancer, containing the peptide.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Hamuro, Junji, Kikuchi, Kokichi, Sahara, Hiromitsu, Sato, Noriyuki, Suzuki, Manabu, Wada, Yoshimasa, Yasojima, Takahiro.
Application Number | 20030186406 10/038899 |
Document ID | / |
Family ID | 26521838 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186406 |
Kind Code |
A1 |
Kikuchi, Kokichi ; et
al. |
October 2, 2003 |
Peptide capable of inducing immune response to human gastric cancer
and agent for preventing or treating human gastric cancer,
containing the peptide
Abstract
A peptide that induces CTL against human gastric cancer cells is
provided. A peptide having a specific amino-acid sequence and
induces cytotoxic T cells that targets gastric cancer cells may be
used as an agent for preventing or treating gastric cancer.
Inventors: |
Kikuchi, Kokichi;
(Sapporo-shi, JP) ; Sato, Noriyuki; (Sapporo-shi,
JP) ; Sahara, Hiromitsu; (Rishiri-gun, JP) ;
Yasojima, Takahiro; (Setana-gun, JP) ; Wada,
Yoshimasa; (Sapporo-shi, JP) ; Suzuki, Manabu;
(Kawasaki-shi, JP) ; Hamuro, Junji; (Kawasaki-shi,
JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
No. 15-1, Kyobashi 1-chome, Chuo-ku
Tokyo
JP
|
Family ID: |
26521838 |
Appl. No.: |
10/038899 |
Filed: |
January 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10038899 |
Jan 8, 2002 |
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09103808 |
Jun 24, 1998 |
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6368852 |
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09103808 |
Jun 24, 1998 |
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08723116 |
Sep 30, 1996 |
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5837248 |
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Current U.S.
Class: |
435/183 ;
424/185.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 39/00 20130101; A61P 43/00 20180101; C07K 14/4748 20130101;
C07K 14/705 20130101; A61P 1/00 20180101; A61P 37/00 20180101; C07H
21/04 20130101; C07K 7/06 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/183 ;
424/185.1 |
International
Class: |
A61K 039/00; C12N
009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 29, 1995 |
JP |
7-253491 |
Aug 19, 1996 |
JP |
8-217140 |
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. A peptide which is a fragment of a gastric cancer antigen
protein present in a human gastric cancer cell, said fragment being
bound to an HLA molecule and capable of inducing a cytotoxic T cell
that targets said gastric cancer cell.
2. The peptide of claim 1, wherein the HLA molecule is HLA-A31.
3. The peptide of claim 1, wherein said peptide has an amino-acid
sequence represented by SEQ ID NO: 1 of the Sequence Listing.
4. The peptide of claim 1, which has an amino-acid sequence
obtained by modifying the amino-acid sequence represented by SEQ ID
NO: 1 of the Sequence Listing in order that said peptide can induce
more efficiently the cytotoxic T cell that targets the gastric
cancer cell.
5. The peptide of claim 4 which has an amino-acid sequence
represented by SEQ ID NO: 2 of the Sequence Listing.
6. A composition for preventing or treating human gastric cancer,
said composition containing a peptide which is a fragment of a
gastric cancer antigen protein present in a human gastric cancer
cell, said fragment being bound to an HLA molecule and capable of
inducing a cytotoxic T cell that targets the gastric cancer
cell.
7. The composition of claim 6, wherein the HLA molecule is
HLA-A31.
8. The composition of claim 6, wherein said peptide has an
amino-acid sequence represented by SEQ ID NO: 1 of the Sequence
Listing.
9. The composition of claim 6, wherein said peptide has an
amino-acid sequence represented by SEQ ID NO: 2 of the Sequence
Listing.
10. A DNA encoding a peptide which is a fragment of a gastric
cancer antigen protein present in a human gastric cancer cell, said
fragment being bound to an HLA molecule and capable of inducing a
cytotoxic T cell that targets said gastric cancer cell.
11. The DNA of claim 10, wherein the HLA molecule is HLA-A31.
12. The DNA of claim 10, wherein said peptide has an amino-acid
sequence represented by SEQ ID NO: 1 of the Sequence Listing.
13. The DNA of claim 10, wherein said peptide has an amino-acid
sequence represented by SEQ ID NO: 2 of the Sequence Listing.
14. A vaccine for preventing or treating human gastric cancer, said
vaccine containing a recombinant virus or a recombinant bacterium
having a DNA encoding a peptide which is a fragment of a gastric
cancer antigen protein present in a human gastric cancer cell, said
fragment being bound to an HLA molecule and capable of inducing a
cytotoxic T cell that targets said gastric cancer cell.
15. The vaccine of claim 14, wherein the HLA molecule is
HLA-A31.
16. The vaccine of claim 14, wherein said peptide has an amino-acid
sequence represented by SEQ ID NO: 1 of the Sequence Listing.
17. The vaccine of claim 14, wherein said peptide has an amino-acid
sequence represented by SEQ ID NO: 2 of the Sequence Listing.
18. A method for preventing or treating gastric cancer comprising
administering to a patient in need thereof an effective amount of a
peptide which is a fragment of a gastric cancer antigen protein
present in a human gastric cancer cell, said fragment being bound
to an HLA molecule and capable of inducing a cytotoxic T cell that
targets said gastric cancer cell.
19. A method for preventing or treating gastric cancer comprising
administering to a patient in need thereof an effective amount of
CTL which have been activated with a peptide which is a fragment of
a gastric cancer antigen protein present in a human gastric cancer
cell, said fragment being bound to an HLA molecule and capable of
inducing a cytotoxic T cell that targets said gastric cancer
cell.
20. A method for preventing or treating gastric cancer, comprising
administering to a patient in need thereof an effective amount of a
vaccine containing a recombinant virus or a recombinant bacterium
having a DNA encoding a peptide which is a fragment of a gastric
cancer antigen protein present in a human gastric cancer cell, said
fragment being bound to an HLA molecule and capable of inducing a
cytotoxic T cell that targets said gastric cancer cell.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a peptide capable of
inducing CTL (Cytotoxic T Lymphocytes; refer to Medical Immunology,
revised 3rd edition, compiled by Kikuchi Kokichi) to human gastric
cells in vivo or in vitro, and to a DNA encoding this peptide. More
specifically, the present invention relates to a peptide capable of
presenting CTL to human gastric cells by being bound to HLA-A31
antigen (Human Leucocyte Antigen; refer to Modern Immunology, 2nd
edition, compiled by Yamamura Yuichi and Tada Tomio), and to a DNA
encoding the peptide.
[0003] The present invention further relates to an agent useful for
preventing or treating human gastric cancer, the agent containing a
peptide capable of inducing CTL to a human gastric cancer cell in
vivo or in vitro, and to a vaccine for preventing or treating human
gastric cancer, the vaccine containing a recombinant virus or a
recombinant bacterium having a DNA encoding such a peptide.
[0004] 2. Discussion of the Background
[0005] For pharmacotherapy of malignant tumors, a chemotherapeutic
agent which directly impairs tumor cells or an immunotherapeutic
agent with which treatment is conducted by non-specifically
activating an immunity of a host and enhancing a bioprotective
function of the host has been used. However, there is currently no
agent with which malignant tumors, above all, tumors of the
digestive tract can completely be cured.
[0006] In recent years, researchers using tumors of animals, mainly
mice, have revealed that tumors can completely be cured by
enhancing an antigen-specific immune response to tumor-related
antigens and tumor-specific antigens present in various tumor
cells. Clinically, the treatment has been conducted by enhancing
the antigen-specific immune response to these tumor-specific
antigens.
[0007] With respect to agents or approaches by which tumors are
treated upon enhancing the tumor antigen-specific immune response,
it has been reported that a monoclonal antibody against an antigen
which is expressed in tumor cells and mainly recognized by B cells
is used, that a tumor-specific immune response is induced by
administering to the patients their own tumor cells or solubilized
fractions thereof which have been inactivated with radiation or
medicines as a vaccine, and that in order to increase an
immunogenicity of tumor cells, viruses or various cytokine genes
are introduced into tumor cells and the thus-treated tumor cells
are administered to the patients themselves, whereby a
tumor-specific immune response is induced.
[0008] However, it is being made clear that T cells including CTL
mainly act on tumor rejection in vivo. It has been currently
clarified that the treatment using an antibody against a
B-cell-recognition antigen is limited.
[0009] Further, it has been clarified that the immune response with
the T cells acts either intensively or suppressively in a state
where two functional sub-populations (Th1 and Th2 subsets) present
in T cell populations are activated, and that the immune of tumor
cells themselves containing plural antigens sometimes rather
suppresses the immune response to the tumor cells.
[0010] Still further, with respect to the inoculation of
inactivated tumor cells, the possibility of re-growth of tumors
through re-activation in vivo cannot completely be denied. Thus,
the problem of safety remains.
[0011] In order to sidestep the above-mentioned problems, a method
is considered in which an antigen protein to activate CTL which is
deemed to play a central role in tumor rejection is identified and
CTL is activated using this antigen protein.
[0012] It has been clarified through advances of researches in
recent years that CTL is induced such that a peptide which is
fragmented by a protease in cells and which comprises from 8 to 12
antigen-protein-derived amino acids acts as an antigen presenting
molecule to CTL by being bound to an HLA antigen.
[0013] Accordingly, regarding the cancer antigen peptide, it is
considered that if a peptide which is bound to an HLA molecule to
induce CTL to tumor cells can be discovered, this peptide can he
used as an agent for preventing or treating cancers.
[0014] On the basis of these considerations, a cancer antigen
peptide capable of inducing CTL has been studied. However, only a
peptide derived from a protein, i.e. MAGE family, Mart-1,
Tyrosinase, gp100, which is a cancer antigen present in melanoma
tumors has been made clear to date. With respect to cancers of the
digestive tract including gastric cancer, the presence of a cancer
antigen peptide capable of inducing CTL and the structure of the
cancer antigen peptide are currently unknown.
SUMMARY OF THE INVENTION
[0015] Accordingly, it is one object of the present invention to
provide novel peptides capable of inducing an immune response to
human gastric cancer.
[0016] It is another object of the present invention to provide
novel DNA encoding such a peptide.
[0017] It is another object of the present invention to provide
novel agents or compositions useful for treating or preventing
human gastric cancer, which contain such a peptide.
[0018] It is another object of the present invention to provide
novel recombinant viruses and novel recombinant bacteria which
contain DNA encoding such a peptide.
[0019] If is another object of the present invention to provide
novel vaccines useful for preventing human gastric cancer, which
contain such a recombinant virus or recombinant bacterium
containing DNA encoding such a peptide.
[0020] If is another object of the present invention to provide a
novel method for preparing such a peptide by culturing such a
recombinant virus or recombinant bacteria.
[0021] To achieve these objects, the present inventors have focused
on the fact that CTL which recognizes the tumor antigen plays an
important role as a bioprotective mechanism to tumor cells. That
is, they have conducted studies in consideration of the fact that
the efficient induction of CTL with a peptide which can be used as
a part of a vaccine or with a transformant containing a DNA
encoding the peptide is effective for preventing or treating
cancers.
[0022] It has been known that the HLA antigen is bound to the
cancer antigen peptide and acts as an antigen-presenting molecule
to CTL whereby CTL is induced. Accordingly, it is considered that
if a peptide which can activate CTL reactive with gastric cancer
cells can be found, it is possible to use the same as an agent for
preventing or treating the gastric cancer.
[0023] The present inventors have succeeded in establishing a CTL
cell strain (Tc-HST-2) which is specifically reacted with gastric
cancer cells restrained to HLA-A31, this strain being derived from
the patient suffering from a gastric cancer, and they have
succeeded in establishing a gastric cancer cell strain (HST-2) on
which this CTL acts, this strain being derived from the same
patient (J. I. Meth., vol. 154, pp. 235-243 (1992) and Cancer, vol.
75, pp. 1484-1489 (1995)).
[0024] However, it has been unclear whether this CTL recognizes the
tumor antigen and what antigen this CTL recognizes. On top of that,
the presence of the cancer antigen peptide which exists
specifically in cancers of the digestive tract including gastric
cancer and which is capable of inducing CTL has not been clarified
at all; much less has the essence thereof been clarified.
[0025] Accordingly, the present inventors have purified an
HLA-bound peptide present on the cell surface of gastric cancer
cell HST-2, and have identified a peptide that activates the CTL
cell strain Tc-HST-2. Further, they have chemically synthesized
this identified peptide in a conventional manner. Thus, it has been
clarified for the first time that this peptide actually activates
the CTL cell strain Tc-HST-2. Still further, it has been found that
this peptide is expressed in gastric cancer cells other than
HST-2.
[0026] From these results, it has been clarified that the
above-mentioned peptide can be utilized well as an agent for
preventing or treating gastric cancer. These findings have led to
the completion of the present invention.
[0027] Thus, the present invention provides: (1) a peptide which is
a fragment of a gastric cancer antigen protein present in a human
gastric cancer cell, the fragment being bound to an HLA molecule
and capable of inducing a cytotoxic T cell that targets the gastric
cancer cell; (2) an agent for preventing or treating human gastric
cancer, the agent containing the above-mentioned peptide; (3) a DNA
encoding the above-mentioned peptide; and (4) a vaccine for
preventing or treating human gastric cancer, the vaccine containing
a recombinant virus or a recombinant bacterium having this DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0029] FIG. 1 is an elution pattern of an HLA-bound peptide from
HST-2 gastric cancer cells in a reversed-phase HPLC column. The
line indicates the acetonitrile concentration, and the curve
indicates the absorbance at OD210 in each concentration;
[0030] FIG. 2 is a graph showing the specific cytotoxicity of
Tc-HST-2 when using an HST-2-derived HLA-bound peptide.
.largecircle. indicates the specific cytotoxicity when using HST-2.
.tangle-solidup. indicates the cytotoxicity when using an
HLA-A31-positive KG-1 cell as a target cell. All of the tests were
conducted such that the ratio of target cells to CTL was 1:100;
[0031] FIG. 3 is a elution pattern of a 12th fraction showing a CTL
inducibility shown in FIG. 2 by a reversed-phase HPLC column. The
line indicates the acetonitrile concentration, and the curve
indicates the absorbance at OD210 in each concentration;
[0032] FIG. 4 is a graph showing the specific cytotoxicity of
Tc-HST-2 when using an HST-2-derived HLA-bound peptide. .gradient.
indicates the specific cytotoxicity when using HST-2 as the target
cell. .tangle-solidup. indicates the cytotoxicity when using an
HLA-A31-positive CCRF-CEM cell as the target cell. All of the tests
were conducted such that the ratio of target cells to CTL was
1:100;
[0033] FIG. 5 is a graph showing the CTL inducibility of Tc-HST-2
measured by the TNF-releasing method when using Peptide 1 (the
peptide represented by SEQ ID NO: 1 of the Sequence Listing);
Peptide 1-9 (the peptide represented by SEQ ID NO: 2 of the
Sequence Listing); Peptide 1-8 (the peptide represented by SEQ ID
NO: 3 of the Sequence Listing); and Peptide 1-7 (the peptide
represented by SEQ ID NO: 4 of the Sequence Listing).
[0034] The TNF-sensitive Wehi cells were impaired by the release of
TNF, and incorporation of MTT by the residual cells is decreased.
The peptide concentrations are 10 mM, 1 mM and 0.1 mM. In all of
the tests, HST-2 was used as a target cell, and the ratio of target
cells to CTL was 1:100.
[0035] .box-solid. indicates the cytotoxicity when adding Peptide
1. .gradient. indicates the cytotoxicity when adding Peptide 1-9.
.DELTA. indicates the cytotoxicity when adding Peptide 1-8.
.largecircle. indicates the cytotoxicity when adding Peptide 1-7. A
broken line indicates a cytotoxicity when not adding a peptide;
and
[0036] FIG. 6 is a graph showing the cytotoxicity of TC-HST-2 when
using MKN28 A31(+)cl-2 cells and MKN28 A31(+)cl-5 cells, which are
sub-lines obtained by artificially expressing HLA-A31 and parental
MKN28 gastric cancer cells. All of the tests were conducted such
that the ratio of target cells to CTL was 1:100.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] The present invention will be described in detail below.
[0038] In order to identify a peptide which is a fragment of a
gastric cancer antigen protein present in a human gastric cancer
cell, the fragment being bound to an HLA molecule and capable of
inducing a cytotoxic T cell that targets the gastric cancer cell,
it is indispensable to establish (1) a HLA-type human cancer cell
strain that can be grown in vitro in a large amount and (2) a CTL
cell strain which reacts with the above-mentioned cancer cell
strain to restrain the HLA molecule and the T cell receptor,
exhibiting a cytotoxicity and which can be grown in a large amount
in vitro.
[0039] As an HLA-type human gastric cancer cell strain that can be
grown in vitro in a large amount, for example, the HST-2 cell line
established by the present inventors [J. Immunol. Meth., vol. 154,
pp. 235-243 (1992); and Jpn. J. Cancer Res., vol. 84, pp. 906-913
(1993), which are incorporated herein by reference] can be used.
This cell strain is a gastric signet ring cell carcinoma strain
formed from carcinomatous ascites of a patient suffering from a
gastric cancer.
[0040] Further, as a CTL cell strain which is reactive with the
above-mentioned cancer cell strain to restrain the HLA molecule and
the T cell receptor, exhibiting the cytotoxicity and which can be
grown in a large amount in vitro, the Tc-HST-2 cell line
established by the present inventors [J. Immunol. Meth., vol. 154,
pp. 235-243 (1992); and Jpn. J. Cancer Res., vol. 84, pp. 906-913
(1993), which are incorporated herein by reference] can be
used.
[0041] It is described in Cancer, vol. 75, pp. 1484-1489 (1995)
that this CTL cell strain is specifically reactive with HST-2 cell
and recognizes the HST-2 antigen and the HLA-A31 antigen at the
same time.
[0042] HST-2 cells can be used to obtain the HLA-bound peptide
containing the gastric cancer antigen peptide capable of inducing
the CTL activity of the Tc-HST-2 cell strain. That is, the HST-2
cell is incubated under ordinary incubation conditions, for
example, in an RPMI1640 medium containing fetal bovine serum. Then,
the culture is washed with phosphate-buffer saline (PBS).
Thereafter, an HLA molecule-bound peptide present on the cell
surface is extracted efficiently with a 0.1% trifluoroacetic acid
(TFA) solution. The extraction of the HLA-bound peptide using TFA
is described in Science, vol. 249, pp. 283-287 (1990), which is
incorporated herein by reference.
[0043] The gastric cancer antigen peptide capable of inducing the
CTL activity of the Tc-HST-2 cell can be purified from the
above-mentioned HLA-bound peptide through reversed-phase
chromatography. The purification of this gastric cancer antigen
peptide will be described later in the Examples in detail.
[0044] The Tc-HST-2-reactive gastric cancer antigen peptide in the
HLA-bound peptide fraction which is separated through
reversed-phase chromatography can be identified by measuring the
CTL inducibility of each fraction in the same manner as described
in Science, vol. 249, pp. 283-287 (1990), which is incorporated
herein by reference.
[0045] That is, HLA-A31-positive HST-2 or KG-1 cells are
radio-labeled with sodium chromate containing .sup.51cr, and are
then mixed with the HLA-bound peptide separated through reversed
phase chromatography. The mixture is incubated at 37.degree. C. for
3 hours, and Tc-HST-2 is then added thereto. The resulting mixture
is further incubated for 12 hours. The CTL inducibility can be
detected by measuring the radio-activity of .sup.51Cr released in
the supernatant.
[0046] With respect to the gastric cancer antigen peptide capable
of inducing the CTL activity of the Tc-HST-2 cell separated through
the reversed-phase chromatography, the amino-acid sequence thereof
can be determined in the usual manner using a solid-phase-technique
protein sequencer. For example, a peptide represented by SEQ ID NO:
1 of the Sequence Listing can be mentioned as a peptide exhibiting
the CTL inducibility.
[0047] Needless-to-say, the peptide having the amino-acid sequence
represented by SEQ ID NO: 1 of the Sequence Listing can be used in
the present invention. Further, a peptide having an amino-acid
sequence obtained by modifying a part of the amino-acid sequence
represented by SEQ ID NO: 1 of the Sequence Listing can also be
used, this fragment being bound to an HLA molecule and capable of
inducing a cytotoxic T cell that targets a gastric cancer cell.
[0048] Specific examples of a peptide having an amino-acid sequence
obtained by modifying a part of the amino-acid sequence represented
by SEQ ID NO: 1 of the Sequence Listing in the present invention
include (1) an amino-acid sequence obtained by replacing at least
one amino acid in the amino-acid sequence represented by SEQ ID NO:
1 of the Sequence Listing with another amino acid; (2) an
amino-acid sequence obtained by deleting at least one amino acid
from the N-terminus or C-terminus of the peptide having the
amino-acid sequence represented by SEQ ID NO: 1 of the Sequence
Listing; and (3) a peptide which contains the amino-acid sequence
represented by SEQ ID NO: 1 of the Sequence Listing as a part of
the overall amino-acid sequence.
[0049] The peptide of the present invention ranges from a peptide
formed by binding a few amino acids to a peptide formed by binding
many amino acids.
[0050] Even a peptide having an amino-acid sequence obtained by
modifying a part of the amino-acid sequence represented by SEQ ID
NO: 1 of the Sequence Listing cannot be used in the present
invention unless it is capable of inducing a cytotoxic T cell that
targets a gastric cancer cell.
[0051] For example, the peptide having the amino-acid sequence
represented by SEQ ID NO: 2 of the Sequence Listing can be used in
the present invention, but the peptides having the amino-acid
sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 of the
Sequence Listing cannot be used, because they exhibit no CTL
inducibility (refer to Example which will be later described).
[0052] The method of producing the above-mentioned peptide is not
particularly limited. The peptide may be produced using a
solid-phase peptide synthesis technique or a recombinant DNA
technique.
[0053] Specifically, a CTL-inducible peptide that targets a gastric
cancer cell as represented by SEQ ID NO: 1 or SEQ ID NO: 2 of the
Sequence Listing may be synthesized using the usual solid-phase
peptide synthesis technique. It may also be produced by
incorporating a DNA encoding amino acids constituting this peptide
into an appropriate expression vector, and incubating the
thus-transformed BCG bacteria or bacteria belonging to the genus
Escherichia coli.
[0054] The DNA encoding the peptide having the amino-acid sequence
represented by SEQ ID NO: 1 or SEQ ID NO: 2 of the Sequence Listing
can be deduced from the amino-acid sequence. Codons corresponding
to each amino acid sequence are naturally well known to those
skilled in the art.
[0055] All of the peptides of the present invention were purified
by reversed-phase HPLC, and it was confirmed through reversed-phase
HPLC that these are single components. Then, the peptides were
identified through mass analysis, and were used in CTL assay.
[0056] The peptides of the present invention can induce the CTL
activity to the gastric cell-specific CTL strain, Tc-HST-2. The CTL
inducibility can be measured by the above-mentioned Cr-releasing
method or by the TNF-releasing method described in Immunogenetics,
vol. 35, p. 145 (1992), which is incorporated herein by
reference.
[0057] That is, in the TNF-releasing method, the HLA-A31-positive
HST-2 or KG-1 cell is mixed with the peptide of the present
invention, and the mixture is incubated at 37.degree. C. for 3
hours. Then, Tc-HST-2 cells are added thereto, and the resulting
mixture is further incubated for 12 hours. The activity of any TNF
released in the supernatant is measured to detect the CTL
inducibility.
[0058] The TNF activity can be measured by the method described in
Immunogenetics, vol. 35, p. 145 (1992).
[0059] That is, TNF-sensitive Wehil64 cells are mixed with a test
sample, and the mixture is incubated for 24 hours. Then, an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
solution is added to the culture, and the resulting mixture is
further incubated for 3 hours. After any formazan converted within
mitochondria is dissolved in acid propanol, the amount thereof is
measured in terms of an absorbance at OD570 to detect the TNF
activity.
[0060] The peptides of the present invention, for example, the
peptide having the amino-acid sequence represented by SEQ ID NO: 1
of the Sequence Listing can induce gastric cancer cell-specific CTL
as a T cell epitope. Accordingly, it is expected to be effective as
an agent for preventing or treating human gastric cancer.
[0061] In actual use, the peptides of the present invention can be
administered: (1) as such; (2) along with a pharmaceutically
acceptable carrier and/or diluent; or (3) further along with the
following adjuvants as required, using a syringe or through
subcutaneous absorption from a mucous membrane by spraying or the
like. The carrier may be a human serum albumin or the like, and the
diluent may be PBS, distilled water, or the like.
[0062] In the present invention, the agent for preventing or curing
the human gastric cancer contains from 0.01 to 100% by weight,
preferably from 0.1 to 95% by weight of a peptide which is a
fragment of a gastric cancer antigen protein present in a human
gastric cancer cell, the fragment being bound to an HLA molecule,
specifically, an HLA-A31 molecule, and capable of inducing a
cytotoxic T cell that targets the gastric cancer cell.
[0063] This agent is administered to an adult person at such a dose
that the amount of the peptide of the present invention is between
0.01 mg and 100 mg in one administration. However, this range is
only a standard, and it is not critical.
[0064] The formulation of the preparation is not particularly
limited. A freeze-dried agent or a granular agent containing a
vehicle such as saccharide may also be used.
[0065] This preparation has no problematic acute toxicity in the
above-mentioned administration.
[0066] The adjuvants which may be added to the agent of the present
invention to increase the CTL inducibility include microorganisms
such as BCG, ISCOM described in Nature, vol. 344, p. 873 (1990),
which is incorporated herein by reference, saponin-type QS-21
described in J. Immunol., vol. 148, p. 1438 (1992), which is
incorporated herein by reference, liposomes and aluminum
hydroxide.
[0067] Further, an immunological activator can be used as an
adjuvant. Examples thereof include lentinan, schizophyllan and
picibanil. Cytokines that enhance growth or differentiation of T
cells, such as IL-2, IL-4, IL-12, IL-1, IL-6 and TNF can also be
used as adjuvants.
[0068] It is described in the above-mentioned literature and in
Science, vol. 255, p. 333 (1992), which is incorporated herein by
reference, that the immune response of CTL or the like can be
induced in vivo by the above-mentioned method.
[0069] Further, it is possible that the above-mentioned antigen
peptide may be added in vitro to cells collected from a patient or
cells having the same HLA haplotype in order to present the
antigen, after which these cells are administered to the blood
vessel of the patient to effectively induce CTL within the body of
the patient. Still further, it is possible that the above-mentioned
peptide may be added to peripheral blood lymphocytes of the patient
and the peptide-containing lymphocytes are incubated in vitro to
induce CTL in vitro, after which the resulting lymphocytes are
returned to the blood vessel of the patient. The treatment through
such a cell transfer has been already practiced as a cancer
therapy, and it is well known to those skilled in the art.
[0070] The DNA encoding the gastric cancer antigen peptides of the
present invention is inserted into an appropriate vector; viruses
such as vaccinia virus or bacteria such as BCG, which contain this
recombinant DNA, can effectively be used as a live vaccine for
preventing or treating human gastric cancer.
[0071] That is, when: (1) a DNA encoding the peptide having the
amino-acid sequence represented by SEQ ID NO: 1 of the Sequence
Listing or (2) a DNA encoding a peptide having the amino-acid
sequence obtained by modifying a part of the amino-acid sequence
represented by SEQ ID NO: 1 of the Sequence Listing (for example, a
peptide having an amino-acid sequence represented by SEQ ID NO: 2
of the Sequence Listing) is inserted into an antigen protein gene
which is expressed in the recombinant viruses or the recombinant
bacteria, this peptide sequence is expressed as a part of the
antigen protein, then processed in the cells, and presented to the
HLA antigen, making it possible to induce CTL that recognizes the
same.
[0072] The dose and the administration method of the agent are the
same as those in usual vaccination or administration of BCG
vaccine.
[0073] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLES
Example 1
[0074] Purification of Tc-HST-2 response peptide from HST-2 and
identification thereof:
[0075] HST-2 cells (2.times.10.sup.5 cells/ml) were inoculated in
50 ml of a 10% FCS-containing RPMI1640 medium (containing 2 mM
glutamine, 5.times.10.sup.31 5M 2ME, 100 units/ml of penicillin,
100 .mu.g/ml of streptomycin and 16 mM NaHCO.sub.3) in a 175
cm.sup.2 plastic incubator (cat. No. 3028, manufactured by Falcon),
and were incubated in the presence of a 5% CO.sub.2 gas in air at
37.degree. C. for 4 days.
[0076] After the completion of the incubation, the supernatant
formed was removed, and the residue was washed with PBS (-). Then,
20 ml of 0.1% TFA solution were added thereto, and the mixture was
incubated at 4.degree. C. for 30 minutes. An HLA-bound peptide was
released from the HLA molecule. The HLA-bound peptide released was
centrifuged at 10,000 g for 15 minutes to separate the cell residue
therefrom.
[0077] Twenty milliliters of the HLA-bound peptide solution
obtained by incubating HST-2 as mentioned above were freeze-dried
and dissolved in 5 ml of a 0.1% TFA solution. One milliliter of
this HLA-bound peptide solution was treated with a reversed-phase
HPLC column (.mu. BONDSHERE, 5.mu. C18-300 A, 19 mm.times.150 mm).
The reversed-phase HPLC column had been equilibrated with 0.1%
TFA-containing distilled water in advance, and then the targeted
substance was eluted by increasing the concentration of
acetonitrile in 0.1% TFA linearly from 0 to 80%. This procedure was
conducted at a flow rate of 1 ml/min. The eluent was collected in
amounts of 1 ml each.
[0078] The above procedure was repeated for a total of 10 times.
Fractions that showed the same retention time were collected, and
freeze-dried. No significant difference was found in elution
patters of HPLC in the above-mentioned procedures. The elution
patterns of HPLC are shown in FIG. 1.
[0079] The CTL inducibility of the HPLC elution fractions against
Tc-HST-2 cells was measured as follows. That is, 1.times.10.sup.6
HST-2 or KG-1 cells expressing HLA-A31 antigen as target cells were
suspended in a 10% FCS-containing RPIM medium such that the number
of cells reached 1.times.10.sup.7 cells/ml. To the suspension were
added 100 .mu.l of a mixture of sodium chromate containing
.sup.51Cr and PBS, and the resulting mixture was incubated in the
presence of 5% CO.sub.2 at 37.degree. C. for 3 hours for
.sup.51Cr-labeling. The .sup.51Cr-labeled cells (1.times.10.sup.4
cells) were suspended in 100 .mu.l of the solution. Three
microliters of the solution containing the HLA-bound peptide
separated through HPLC were added thereto, and the mixture was
further incubated under the same conditions for 3 hours, whereby
the HLA-bound peptide was bound to the HLA molecule.
[0080] Subsequently, Tc-HST-2 cells (2.5.times.10.sup.5 cells/ml)
were added thereto in an amount of 100 .mu.l, and the mixture was
incubated under the same conditions for 12 hours. After 12 hours of
incubation, 100 .mu.l of the supernatant formed were collected, and
the radioactivity of .sup.51Cr released in this supernatant was
measured using a .gamma.-counter to measure the CTL activity.
[0081] The specific cytotoxicity was calculated using the following
formula. 1 Specific Cytotoxicity = ( measured valueof eachwell ) -
( minimum release value ) ( maximum release value ) - ( minimum
release value ) .times. 100
[0082] wherein the minimum release value indicates a measured value
of a well filled with target cells alone and a value of .sup.51Cr
naturally released from target cells, and the maximum release value
indicates a release value when the cells are destroyed by adding a
surfactant, 1% Triton x-100, to target cells.
[0083] The results are shown in FIG. 2. As is made clear from FIG.
2, the CTL inducibility was observed in the 12th fraction eluted
through HPLC.
[0084] The 12th fraction of which the CTL inducibility of Tc-HST-2
had been identified was further treated with a reversed-phase HPLC
column (.mu. BONDSHERE, 5.mu. C18-300A, 19 mm.times.150 mm). The
reversed-phase HPLC column had been equilibrated with 0.1%
TFA-containing distilled water in advance, and then the targeted
substance was eluted by increasing the concentration of
acetonitrile in 0.1% TFA-containing eluent linearly from 0 to 40%.
This procedure was conducted at a flow rate of 1 ml/min. The eluent
was collected in amounts of 1 ml each.
[0085] The above-described procedure was repeated for a total of 10
times. Fractions that showed the same retention time were collected
and freeze-dried. No significant difference was found in elution
patterns of HPLC in the above-mentioned procedures. The elution
patterns of HPLC are shown in FIG. 3.
[0086] The CTL inducibility of Tc-HST-2 cells in the HPLC elution
fractions was measured by the above-mentioned method. The results
are shown in FIG. 4. As is made clear from FIG. 4, the CTL
inducibility was identified in the 17th fraction eluted through
HPLC.
[0087] The 17th fraction of which the CTL inducibility had been
identified was separated with a reversed-phase HPLC column for
analysis (ZORBAX, ODS column, 4 mm.times.250 mm). The
reversed-phase HPLC column had been equilibrated with 0.1%
TFA-containing distilled water in advance, and then the targeted
substance was eluted by increasing the concentration of
acetonitrile in 0.1% TFA-containing elution linearly from 0 to 60%.
This procedure was conducted at a flow rate of 1 ml/min to obtain a
main peak.
[0088] This peak was separated and introduced into a protein
sequencer (477A, manufactured by ABI). The amino-acid sequence was
determined by the Edman degradation method. The amino-acid sequence
from the N-terminus is represented by SEQ ID NO: 1 of the Sequence
Listing.
Example 2
[0089] Synthesis of a Gastric Cancer Antigen Peptide and
Measurement of CTL Inducibility:
[0090] Synthetic Peptide 1 having the same amino-acid sequence as
that represented by SEQ ID NO: 1 of the Sequence Listing and three
types of peptides containing from 9 to 7 amino acids starting from
the N-terminus of this Synthetic Peptide 1, namely: Peptide 1-9
(the peptide having the amino-acid sequence represented by SEQ ID
NO: 2 of the Sequence Listing); Peptide 1-8 (the peptide having the
amino-acid sequence represented by SEQ ID NO: 3 of the Sequence
Listing); and Peptide 1-7 (the peptide having the amino-acid
sequence represented by SEQ ID NO: 4 of the Sequence Listing) were
synthesized using an automated peptide synthesizer (477A,
manufactured by ABI) according to a protocol in the manual of the
same machine.
[0091] These synthetic peptides were cut from the resin, and
re-precipitated with a mixture of TFA and dry ether. They were
identified to be single components through reversed phase HPLC, and
were further identified using a mass spectrometer. Subsequently,
they were subjected to CTL assay using a TNF-releasing method.
[0092] That is, 1.times.10.sup.4 HST-2 cells expressing HLA-A31
antigen were suspended as target cells in 100 .mu.l of the
solution, and 5 .mu.l of each of the above-mentioned four synthetic
peptides were added thereto such that the final concentration
reached 10 .mu.M, 1 .mu.M or 0.1 .mu.M. The mixture was further
incubated under the same conditions for 3 hours to bind the
HLA-bound peptide to the HLA molecule. Subsequently, Tc-HST-2 cells
(1.times.10.sup.6 cells/ml) were added thereto in an amount of 100
.mu.l, and the resulting mixture was further incubated for 12 hours
under the same conditions.
[0093] After 12 hours of incubation, 30 .mu.l of the supernatant
formed was collected, and the TNF activity released in the
supernatant was measured to detect the CTL inducibility. The TNF
activity was measured by the following method.
[0094] That is, 7.times.10.sup.4 Wehil64 cells which are
TNF-sensitive cells were suspended in 120 .mu.l of the solution,
and 30 .mu.l of the above-obtained supernatant were added thereto.
The mixture was incubated for 24 hours. After the completion of the
incubation, 5 mg/ml of MTT were added to each well in amounts of 10
.mu.l each, and the mixture was further incubated for 3 hours.
Subsequently, 150 .mu. of propanol containing 0.01% hydrochloric
acid were added thereto to solubilize any formazan formed during
the incubation. Then, the absorbance was measured at a wavelength
of 570 nm to measure the TNF activity.
[0095] The results are shown in FIG. 5. As is made clear from FIG.
5, the CTL inducibility of TC-HST-2 was identified in Synthetic
Peptides 1 and 1-9.
Example 3
[0096] Expression of a Gastric Cancer Antigen Peptide in the Human
MKN28 Gastric Cancer Cell Strain:
[0097] In order to identify whether or not a gastric cancer antigen
peptide recognized by TC-HST-2 was expressed in gastric cancer cell
strains other than HST-2, the cytotoxicity given by TC-HST-2 to
MKN28 which is an HLA-A31-negative human gastric cancer cell strain
was detected by the .sup.51Cr-releasing method. The detection of
the CTL activity by the .sup.51Cr-releasing method was conducted in
the same manner as in Example 1. The results are shown in FIG. 6.
TC-HST-2 did not exhibit a cytotoxicity to the MKN28 cells which
did not express HLA-A31. On the other hand, TC-HST-2 exhibited a
significant cytotoxicity to MKN28 A31(+) cl-2 and MKN28 A31(+)
cl-5, sub-lines of MNK28 formed by transfecting the plasmid pBJ-A31
containing the HLA-A31 gene into MKN28 through electroporation to
express HLA-A31, as shown in FIG. 6.
[0098] From the above-mentioned results, it was identified that the
gastric cancer antigen peptide recognized by TC-HST-2 was also
expressed in MKN28, and that this peptide was bound to the HLA-A31
molecule and recognized by TC-HST-2.
[0099] The peptide of the present invention can induce CTL against
human gastric cancer cells in vivo or in vitro, and this peptide
itself and an agent comprising this peptide can be expected to act
as an agent for preventing or treating human gastric cancer.
Further, a recombinant virus or a recombinant bacterium containing
DNA encoding the peptide capable of inducing CTL against human
gastric cancer cells in vivo or in vitro exhibits quite a
satisfactory pharmaceutical effect as a vaccine for preventing or
treating human gastric cancer.
[0100] The present application is based on Japanese Patent
Application Nos. 253491/1995 and 217140/1996, filed on Sep. 29,
1995, and Aug. 19, 1996, respectively, both of which are
incorporated herein by reference in their entirety.
[0101] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
Sequence CWU 1
1
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