U.S. patent application number 13/432915 was filed with the patent office on 2012-09-20 for emulsified composition for dilution and cancer vaccine composition.
This patent application is currently assigned to NOF CORPORATION. Invention is credited to Yusuke OKAWA, Koichi SAITO.
Application Number | 20120237569 13/432915 |
Document ID | / |
Family ID | 36692424 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237569 |
Kind Code |
A1 |
SAITO; Koichi ; et
al. |
September 20, 2012 |
EMULSIFIED COMPOSITION FOR DILUTION AND CANCER VACCINE
COMPOSITION
Abstract
Provided is an emulsified composition for diluting a cancer
antigen peptide or a dimer thereof. Also provided is a novel cancer
vaccine composition or specific CTL inducer that efficiently
induces CTL irrespective of the type of cancer antigen peptide when
mixing the emulsified composition for dilution and a water phase
comprising a cancer antigen peptide or a dimer thereof. The present
invention relates to an emulsified composition for diluting a
cancer antigen peptide or a dimer thereof, comprising a particular
ester, a particular surfactant, a particular emulsifier, and a
water phase. The present invention also relates to a cancer vaccine
composition or specific CTL inducer obtained by freshly diluting
and mixing a water phase comprising a cancer antigen peptide or a
dimer thereof with the emulsified composition for dilution.
Inventors: |
SAITO; Koichi; (Osaka,
JP) ; OKAWA; Yusuke; (Kawasaki-shi, JP) |
Assignee: |
NOF CORPORATION
Tokyo
JP
DAINIPPON SUMITOMO PHARMA CO., LTD.
Osaka
JP
|
Family ID: |
36692424 |
Appl. No.: |
13/432915 |
Filed: |
March 28, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11814270 |
Sep 20, 2007 |
8178102 |
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PCT/JP2006/301171 |
Jan 19, 2006 |
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13432915 |
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Current U.S.
Class: |
424/400 ;
424/277.1; 424/278.1; 424/283.1 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61K 9/107 20130101; A61K 9/0019 20130101; A61P 37/04 20180101;
A61P 43/00 20180101; C07K 7/08 20130101; A61K 47/14 20130101; A61K
2039/55566 20130101; C07K 7/06 20130101; A61P 35/02 20180101; A61P
35/00 20180101 |
Class at
Publication: |
424/400 ;
424/278.1; 424/277.1; 424/283.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/04 20060101 A61P037/04; A61K 47/44 20060101
A61K047/44; A61P 35/00 20060101 A61P035/00; A61K 47/14 20060101
A61K047/14; A61K 9/107 20060101 A61K009/107 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 19, 2005 |
JP |
2005-012140 |
Claims
1. An emulsified composition for diluting a cancer antigen peptide
having 8 to 12 amino acids or a dimer thereof, comprising: A) an
ester of a fatty acid having 8 to 22 carbon atoms and an alcohol
having 2 to 24 carbon atoms, the ester having a solidification
point of not more than 10.degree. C., at 50 to 90% by weight; B) a
nonionic surfactant consisting of a hydroxy fatty acid triglyceride
adduct with 5 to 20 mol of ethylene oxide, at 0.5 to 20% by weight;
C) an emulsifier that is a partial ester of a polyhydric alcohol
and a fatty acid, the partial ester being liquid at 40.degree. C.,
at 0 to 20% by weight; and D) water at 5 to 20% by weight.
2. The emulsified composition for dilution of claim 1, wherein the
average particle diameter of the dispersion phase of the emulsified
composition for dilution is 50 to 500 nm.
3. A method of preparing a cancer vaccine composition, comprising
diluting 0.25 to 1 part by volume of a water phase comprising a
cancer antigen peptide having 8 to 12 amino acids or a dimer
thereof with 1 part by volume of the emulsified composition for
dilution of claim 1.
4. The emulsified composition for dilution of claim 1, wherein the
fatty acid that constitutes ingredient A is oleic acid, myristic
acid or 2-ethylhexanoic acid.
5. The emulsified composition for dilution of claim 1, wherein the
ester of ingredient A is ethyl oleate, octyldodecyl myristate or
cetyl 2-ethylhexanoate.
6. The emulsified composition for dilution of claim 1, wherein the
hydroxy fatty acid triglyceride that constitutes the ingredient B
nonionic surfactant is castor oil or hardened castor oil.
7. A cancer vaccine composition comprising: A) an ester of a fatty
acid having 8 to 22 carbon atoms and an alcohol having 2 to 24
carbon atoms, the solidification point of the ester being not more
than 10.degree. C., at 30 to 80% by weight; B) a nonionic
surfactant consisting of a hydroxy fatty acid triglyceride adduct
with 5 to 20 mol of ethylene oxide, at 0.5 to 20% by weight; C) an
emulsifier that is a partial ester of a polyhydric alcohol and a
fatty acid, the partial ester being liquid at 40.degree. C., at 0
to 20% by weight; and E) a water phase comprising a cancer antigen
peptide having 8 to 12 amino acids or a dimer thereof, at 10 to 60%
by weight; wherein the composition is a W/O emulsion.
8. The cancer vaccine composition of claim 7, comprising ingredient
A at 40 to 60% by weight; ingredient B at 1.0 to 5.0% by weight;
ingredient C at 5.0 to 10.0% by weight; and ingredient E at 30 to
50% by weight.
9. A specific cytotoxic T cell inducer comprising: A) an ester of a
fatty acid having 8 to 22 carbon atoms and an alcohol having 2 to
24 carbon atoms, the solidification point of the ester being not
more than 10.degree. C., at 30 to 80% by weight; B) a nonionic
surfactant consisting of a hydroxy fatty acid triglyceride with 5
to 20 mol of ethylene oxide added thereto, at 0.5 to 20% by weight;
C) an emulsifier that is a partial ester of a polyhydric alcohol
and a fatty acid, the partial ester being liquid at 40.degree. C.,
at 0 to 20% by weight; and E) a water phase comprising a cancer
antigen peptide having 8 to 12 amino acids or a dimer thereof, at
10 to 60% by weight; wherein the composition is a W/O emulsion.
10. The specific cytotoxic T cell inducer of claim 9, comprising
ingredient A at 40 to 60% by weight; ingredient B at 1.0 to 5.0% by
weight; ingredient C at 5.0 to 10.0% by weight; and ingredient E at
30 to 50% by weight.
11. A kit for freshly preparing a cancer vaccine composition or
specific CTL inducer, comprising a water phase comprising a cancer
antigen peptide having 8 to 12 amino acids or a dimer thereof, and
the emulsified composition for dilution for diluting the water
phase, of claim 1.
12. A method of preparing a cancer vaccine composition, comprising
diluting 0.25 to 1 part by volume of a water phase comprising a
cancer antigen peptide having 8 to 12 amino acids or a dimer
thereof with 1 part by volume of the emulsified composition for
dilution of claim 2.
Description
TECHNICAL FIELD
[0001] The present invention belongs to the field of cancer
immunotherapy and relates to an emulsified composition for diluting
a cancer antigen peptide or a dimer thereof, having cytotoxic T
cell induction activity. More specifically, the present invention
relates to an emulsified composition for diluting a cancer antigen
peptide or a dimer thereof, comprising a particular ester, a
surfactant, an emulsifier, and a water phase, and occurring in the
form of a stable W/O emulsion. The present invention also relates
to a cancer vaccine composition or specific cytotoxic T cell
inducer, comprising a particular ester, a surfactant, an
emulsifier, and a water phase comprising a cancer antigen peptide
consisting of a particular amino acid sequence or a dimer thereof,
and occurring in the form of a stable W/O emulsion.
BACKGROUND ART
[0002] In the elimination of cancer cells, virus-infected cells and
the like by the living body, cellular immunity, particularly
cytotoxic T cells (hereinafter referred to as CTL) play an
important role. CTL recognizes the complex formed on cancer cells
by a cancer antigen protein-derived antigen peptide (cancer antigen
peptide) and MHC (Major Histocompatibility Complex) class I antigen
(in the case of humans, referred to as HLA antigen), and
attacks/destroys the cancer cells.
[0003] As a representative example of the cancer antigen protein,
those described in the table of Immunity, Vol. 10, page 281, 1999
can be mentioned. Specifically, gp100 and MART-1, which are
melanocyte tissue specific proteins, and melanosome proteins such
as tyrosinase can be mentioned. As the cancer antigen protein other
than melanoma, cancer markers such as CEA, PSA, and HER2/neu can be
mentioned. TERT, whose expression increases in cancer, and the like
can also be mentioned. Furthermore, WT1, which is highly expressed
in many types of cancer, is also known as a novel cancer antigen
protein in leukemia and solid cancers.
[0004] The cancer antigen peptide is a peptide resulting from
processing of a cancer antigen protein by intracellular protease.
As stated above, a complex of this resulting cancer antigen peptide
and MHC class I antigen (HLA antigen) is presented to the cell
surface and recognized by CTL. However, in developing a cancer
immunotherapeutic agent (cancer vaccine) utilizing cancer cell
destruction by CTL, it has been difficult to efficiently induce CTL
using a cancer antigen peptide alone, because of the generally low
immunogenicity. Therefore, there is a demand for the development of
a preparation capable of stimulating CTL induction.
[0005] To this end, many investigations have been conducted
regarding preparations that stimulate CTL induction. Particularly,
many investigations have been conducted regarding emulsion type
preparations.
[0006] Generally, there are different types of emulsion
preparations: O/W emulsions and W/O emulsions.
[0007] In the case of O/W emulsions, the peptide that serves as the
antigen cannot be retained in the inner phase, and therapeutically
effective specific CTL induction activity is not exhibited.
[0008] In the case of W/O emulsions, in which the water phase
serves as the inner phase, excellent CTL induction activity is
expected because the peptide that serves as the antigen is easy to
retain in the inner phase, but there are many problems to be solved
before they are brought into practical applications.
[0009] For example, as a W/O emulsion for cancer antigen peptide
carrier, incomplete Freund's adjuvant is known, but effective
activity cannot be obtained in some cases due to insufficient
stability. There have been other problems such as difficulty in
administration due to high viscosity.
[0010] Incomplete Freund's adjuvant has long been known as an
immunopotentiating adjuvant that causes inactivated bacteria or
inactivated virus to be included to induce antibody production, and
alternative W/O emulsion type vaccine compositions were
investigated; for example, the compositions described in Japanese
Patent Kohyo publication No. HEI-7-509733, the pamphlet for
International Patent Publication No. 94/20071, and Japanese Patent
Kokai Publication No. HEI-9-268130 were proposed.
[0011] As a substitute for incomplete Freund's adjuvant, an
application of W/O/W composite emulsion to vaccine is proposed in
Japanese Patent Kokai Publication No. 2001-131087.
[0012] However, as a cancer antigen peptide carrier to substitute
for incomplete Freund's adjuvant, no W/O emulsion capable of
therapeutically effectively activating CTL induction is known.
[0013] Generally, because cancer antigen peptides are low-molecular
compounds, it is not easy to keep them stable in W/O emulsion.
Furthermore, it is feared that the cancer antigen peptide undergoes
degradation, aggregation and the like due to the energy during
emulsification. Furthermore, cancer antigen peptides range from
highly water-soluble ones to slightly soluble ones depending on the
amino acid sequence thereof. Therefore, there is a demand for the
development of a cancer vaccine based on a highly versatile W/O
emulsion that therapeutically effectively stimulates CTL induction,
irrespective of the properties of the cancer antigen peptide.
[0014] An object of the present invention is to provide an
emulsified composition for dilution for preparing a cancer vaccine
composition that exhibits effective CTL induction activity in vivo
in various cancer antigen peptides, and is of low viscosity and
excellent stability. Another object of the present invention is to
provide a method of preparing a novel cancer vaccine that
specifically stimulates CTL induction in vivo according to the
cancer antigen peptide contained, using the above-described
emulsified composition for dilution, and the composition.
DISCLOSURE OF THE INVENTION
[0015] The present inventors found that by diluting various
peptides known as cancer antigens with a particular emulsified
composition comprising a particular ester and a particular
surfactant, CTL induction activity specific for each antigen can be
exhibited, irrespective of the type and properties of the peptide,
conducted further diligent investigations, and completed the
present invention.
[0016] Accordingly, the present invention encompasses the following
modes of embodiment:
[1] An emulsified composition for diluting a cancer antigen peptide
having 8 to 12 amino acids or a dimer thereof, comprising: A) an
ester of a fatty acid having 8 to 22 carbon atoms and an alcohol
having 2 to 24 carbon atoms, the ester having a solidification
point of not more than 10.degree. C., at 50 to 90% by weight; B) a
nonionic surfactant consisting of a hydroxy fatty acid triglyceride
adduct with 5 to 20 mol of ethylene oxide, at 0.5 to 20% by weight;
D) water at 5 to 20% by weight. [2] An emulsified composition for
diluting a cancer antigen peptide having 8 to 12 amino acids or a
dimer thereof, comprising: A) an ester of a fatty acid having 8 to
22 carbon atoms and an alcohol having 2 to 24 carbon atoms, the
ester having a solidification point of not more than 10.degree. C.,
at 50 to 90% by weight; B) a nonionic surfactant consisting of a
hydroxy fatty acid triglyceride adduct with 5 to 20 mol of ethylene
oxide, at 0.5 to 20% by weight; C) an emulsifier that is a partial
ester of a polyhydric alcohol and a fatty acid, the partial ester
being liquid at 40.degree. C., at 0 to 20% by weight; and D) water
at 5 to 20% by weight. [3] The emulsified composition for dilution
described in [2], comprising: ingredient A at 60 to 80% by weight;
ingredient B at 1 to 10% by weight; ingredient C at 5 to 15% by
weight; and ingredient D at 5 to 20% by weight. [4] The emulsified
composition for dilution described in any of [1] to [3], wherein
the average particle diameter of the dispersion phase of the
emulsified composition for dilution is 50 to 500 nm. [5] The
emulsified composition for dilution described in any of [1] to [4],
wherein the fatty acid that constitutes ingredient A is oleic acid,
myristic acid or 2-ethylhexanoic acid. [6] The emulsified
composition for dilution described in any of [1] to [5], wherein
the ester of ingredient A is ethyl oleate, octyldodecyl myristate
or cetyl 2-ethylhexanoate. [7] The emulsified composition for
dilution described in any of [1] to [6], wherein the hydroxy fatty
acid triglyceride that constitutes the ingredient B nonionic
surfactant is castor oil or hardened castor oil. [8] A method of
preparing a cancer vaccine composition, comprising diluting 0.25 to
1 part by volume of a water phase comprising a cancer antigen
peptide having 8 to 12 amino acids or a dimer thereof with 1 part
by volume of the emulsified composition for dilution described in
any of [1] to [7]. [9] A cancer vaccine composition comprising: A)
an ester of a fatty acid having 8 to 22 carbon atoms and an alcohol
having 2 to 24 carbon atoms, the solidification point of the ester
being not more than 10.degree. C., at 30 to 80% by weight; B) a
nonionic surfactant consisting of a hydroxy fatty acid triglyceride
adduct with 5 to 20 mol of ethylene oxide, at 0.5 to 20% by weight;
C) an emulsifier that is a partial ester of a polyhydric alcohol
and a fatty acid, the partial ester being liquid at 40.degree. C.,
at 0 to 20% by weight; and E) a water phase comprising a cancer
antigen peptide having 8 to 12 amino acids or a dimer thereof, at
10 to 60% by weight; wherein the composition is a W/O emulsion.
[10] The cancer vaccine composition described in [9], comprising
ingredient A at 40 to 60% by weight; ingredient B at 1.0 to 5.0% by
weight; ingredient C at 5.0 to 10.0% by weight; and ingredient E at
30 to 50% by weight. [11] A specific CTL inducer comprising: A) an
ester of a fatty acid having 8 to 22 carbon atoms and an alcohol
having 2 to 24 carbon atoms, the solidification point of the ester
being not more than 10.degree. C., at 30 to 80% by weight; B) a
nonionic surfactant consisting of a hydroxy fatty acid triglyceride
adduct with 5 to 20 mol of ethylene oxide, at 0.5 to 20% by weight;
C) an emulsifier that is a partial ester of a polyhydric alcohol
and a fatty acid, the partial ester being liquid at 40.degree. C.,
at 0 to 20% by weight; and E) a water phase comprising a cancer
antigen peptide having 8 to 12 amino acids or a dimer thereof, at
10 to 60% by weight; wherein the composition is a W/O emulsion.
[12] The specific CTL inducer described in [11], comprising
ingredient A at 40 to 60% by weight; ingredient B at 1.0 to 5.0% by
weight; ingredient C at 5.0 to 10.0% by weight; and ingredient E at
30 to 50% by weight. [13] A kit for freshly preparing a cancer
vaccine composition or specific CTL inducer, comprising a water
phase comprising a cancer antigen peptide having 8 to 12 amino
acids or a dimer thereof, and the emulsified composition for
dilution for diluting the water phase, described in any of [1] to
[7]. [14] A commercial package comprising a water phase comprising
a cancer antigen peptide having 8 to 12 amino acids or a dimer
thereof, the emulsified composition for dilution described in any
of [1] to [7], and a printed matter bearing the statement that a
W/O emulsion obtained by diluting the water phase with an
emulsified composition for dilution can be used, or should be used,
for the treatment or prevention of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising the TERT
peptide.
[0018] FIG. 2 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising the MAGE-1
peptide.
[0019] FIG. 3 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising the PSA
peptide.
[0020] FIG. 4 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising the PSA
peptide.
[0021] FIG. 5 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising the CEA
peptide.
[0022] FIG. 6 is a graph showing the results of specific CTL
induction using cancer vaccine compositions comprising various
cancer antigen peptides.
[0023] FIG. 7 shows the particle size distribution of emulsified
composition for dilution 8.
[0024] FIG. 8 shows the particle size distribution of emulsified
composition for dilution 29.
[0025] FIG. 9 is a graph showing the results of specific CTL
induction to each peptide using cancer vaccine compositions
comprising a mixture of two kinds of cancer antigen peptide.
BEST MODE FOR EMBODYING THE INVENTION
[0026] The emulsified composition for dilution of the present
invention is hereinafter described in detail.
[0027] The "emulsified composition for dilution" of the present
invention refers to a composition used to dilute a cancer antigen
peptide or a dimer thereof. The "cancer vaccine composition" of the
present invention refers to a composition obtained by diluting a
cancer antigen peptide or a dimer thereof with the emulsified
composition for dilution of the present invention.
[0028] The emulsified composition for dilution of the present
invention is an emulsion pre-emulsified into the w/o type. The
emulsified composition for dilution of the present invention is a
composition for diluting a water phase comprising a cancer antigen
peptide having 8 to 12 amino acids or a dimer thereof; by so
diluting the water phase, a cancer vaccine composition that
stimulates CTL induction specific for each cancer antigen peptide
can be prepared.
[0029] In the emulsified composition for dilution of the present
invention, ingredient A is an ester of a fatty acid and an alcohol,
preferably an ester of a fatty acid having 8 to 22 carbon atoms and
an alcohol having 2 to 24 carbon atoms, the solidification point of
the ester being not more than 10.degree. C.
[0030] The ester of a fatty acid and an alcohol, used as ingredient
A, may be chosen from among those that have a solidification point
of not more than 10.degree. C. If the solidification point of
ingredient A exceeds 10.degree. C., it is feared that the
stability, particularly the stability at low temperature, of the
emulsified composition for dilution decreases, and this is
therefore undesirable.
[0031] As examples of the fatty acid having 8 to 22 carbon atoms,
saturated or unsaturated fatty acids having 8 to 22 carbon atoms
can be mentioned; specifically, caprylic acid, 2-ethylhexanoic
acid, nonanoic acid, capric acid, undecanoic acid, lauric acid,
tridecanoic acid, myristic acid, myristoleic acid, pentadecanoic
acid, palmitic acid, isopalmitic acid, palmitoleic acid, isostearic
acid, stearic acid, oleic acid, linoleic acid, nervonic acid and
the like can be mentioned. As examples of the alcohol having 2 to
24 carbon atoms, alkanols having 2 to 24 carbon atoms and alkenols
having 2 to 24 carbon atoms can be mentioned; specifically,
alcohols such as ethanol, propanol, isopropanol, ethylene glycol,
butanol, hexanol, octanol, 2-ethylhexanol, decanol, dodecanol,
myristyl alcohol, cetyl alcohol, isocetyl alcohol (2-hexyldecanol),
isostearyl alcohol, oleyl alcohol, icosenol, docosenol,
tetracosenol, and 2-octyldodecanol and the like can be
mentioned.
[0032] As examples of the ingredient A used in the present
invention, specifically, cetyl 2-ethylhexanoate, hexyl laurate,
butyl myristate, isopropyl myristate, isocetyl myristate,
octyldodecyl myristate, 2-ethylhexyl palmitate, isostearyl
palmitate, isopropyl isostearate, isocetyl isostearate,
2-ethylhexyl stearate, ethyl oleate, decyl oleate, oleyl oleate,
octyldodecyl oleate, ethyl linoleate, isopropyl linoleate and the
like can be mentioned.
[0033] The ingredient A used in the present invention may be
synthesized as an ester, and may be extracted/purified from a
natural oil or fat. As examples of the naturally derived ester oil,
jojoba oil, orange roughy oil and the like can be mentioned. These
esters are generally commercially available, from among which one
suitable for pharmaceutical use may be chosen according to the
purpose.
[0034] The ingredient A used in the present invention is preferably
an oleic acid ester, a myristic acid ester or a 2-ethylhexanoeic
acid ester, more preferably ethyl oleate, octyldodecyl myristate,
or cetyl 2-ethylhexanoate, still more preferably ethyl oleate.
[0035] In the emulsified composition for dilution of the present
invention, the content of ingredient A is 50 to 90% by weight,
preferably 55 to 85% by weight, more preferably 60 to 80% by
weight. If the content of ingredient A is less than 50% by weight,
the finished cancer vaccine composition obtained by diluting a
cancer antigen peptide possibly becomes unstable, and this is
therefore undesirable.
[0036] In the emulsified composition for dilution of the present
invention, the ingredient B is a nonionic surfactant consisting of
a hydroxy fatty acid triglyceride adduct with 5 to 20 mol of
ethylene oxide.
[0037] The hydroxy fatty acid triglyceride adduct with ethylene
oxide is a surfactant widely used for emulsification,
solubilization and the like, and 5 to 100 mol of ethylene oxide
adducts are commercially available. A surfactant consisting of less
than 5 mol of ethylene oxide adduct has a low surfactant activity
such that an emulsified composition for dilution showing a good
emulsified state cannot be formed, and this is therefore
undesirable. Because a surfactant consisting of more than 20 mol of
ethylene oxide adduct is highly hydrophilic, not only it is feared
that the emulsification stability decreases due to aggregation of
water phase particles and the like in the cancer vaccine
composition obtained by diluting the emulsified composition, but
also the CTL induction activity for the cancer vaccine composition
can decrease, and this is therefore undesirable.
[0038] The nonionic surfactant used as the ingredient B is
preferably constituted by a castor oil or hardened castor oil
adduct with 5 to 20 mol of ethylene oxide. By using such a nonionic
surfactant, an emulsified composition for dilution of excellent
stability can be obtained, and, furthermore, a cancer vaccine
composition that exhibits excellent stability and high CTL
induction activity when a cancer antigen peptide is diluted with
the emulsified composition for dilution can be prepared. By using a
nonionic surfactant constituted by a castor oil or hardened castor
oil adduct with 5 to 20 mol of ethylene oxide, a cancer vaccine
composition of excellent safety to the administration site can be
finally prepared. Of these, preferable as the ingredient B of the
present invention is a hardened castor oil adduct with 10 to 20 mol
of ethylene oxide. Furthermore, in the present invention, it is
most preferable to use, out of these, a hardened castor oil adduct
with 10 mol of ethylene oxide.
[0039] In the emulsified composition for dilution of the present
invention, the content of ingredient B is 0.5 to 20% by weight,
preferably 1 to 15% by weight, more preferably 1 to 10% by weight.
If the content of ingredient B is less than 0.5% by weight, the
surfactant activity decreases in excess and, as a result, it is
possible that no stable emulsified composition for dilution is
obtained, and this is therefore undesirable. Conversely, if the
content exceeds 20% by weight, not only phase conversion occurs
during dilution of the cancer antigen peptide, which hampers the
final obtainment of a good cancer vaccine composition, but also it
is feared that the safety of the cancer vaccine composition to the
administration site decreases, and this is therefore again
undesirable.
[0040] The ingredient C optionally contained in the emulsified
composition for dilution of the present invention is an emulsifier
that is a partial ester of a polyhydric alcohol and a fatty acid,
the partial ester being liquid at 40.degree. C.
[0041] In the present invention, in addition to the above-described
ingredient B, by containing ingredient C, a cancer vaccine
composition that exhibits a better emulsified state and has
excellent stability when used to dilute a cancer antigen peptide
can be prepared and, as a result, a cancer vaccine composition that
exhibits excellent CTL induction activity in diversified kinds of
peptides can be obtained.
[0042] As examples of the polyhydric alcohol that constitutes the
ingredient C of the present invention, glycerin, diglycerin,
sorbitan, sorbitol, sorbide, mannitan, mannitol, sucrose and the
like can be mentioned. As examples of the fatty acid that
constitutes the ingredient C, lauric acid, myristic acid,
isostearic acid, oleic acid, linoleic acid and the like can be
mentioned. As the ingredient C of the present invention, one being
liquid at 40.degree. C. may be chosen from among these partial
esters of a polyhydric alcohol and a fatty acid.
[0043] As specific suitable examples of ingredient C, glycerin
monooleate, glycerin dioleate, diglycerin monooleate, diglycerin
dioleate, sorbitan monooleate, sorbitan sesquioleate, sorbitan
dioleate, sorbitan trioleate, mixtures thereof and the like can be
mentioned. Particularly suitable ones are glycerin monooleate,
glycerin dioleate, sorbitan monooleate, sorbitan sesquioleate,
sorbitan dioleate, and mixtures thereof. These are generally
commercially available, from among which those suitable for
pharmaceutical use can be chosen.
[0044] In the emulsified composition for dilution of the present
invention, the content of ingredient C is chosen from the range of
0 to 20% by weight. In particular, the range of 1 to 20% by weight
is preferable, and the range of 5 to 15% by weight is more
preferable. At this time, if the content exceeds 20% by weight, not
only the viscosity for the emulsified composition for dilution
increases so that a good cancer vaccine composition cannot be
obtained when the cancer antigen peptide of the present invention
is diluted, but also it is feared that the safety of the cancer
vaccine composition to the administration site decreases, and this
is therefore undesirable. If the content of ingredient C is not
less than 1% by weight, a sufficient additive effect is exhibited,
and this is therefore preferable.
[0045] The ingredient D that constitutes the emulsified composition
for dilution of the present invention is a water phase for forming
an emulsion. The ingredient D of the present invention may be
chosen from among aqueous ingredients in common use in
pharmaceuticals; for example, purified water, water for injection,
phosphate-buffered solution, physiological saline,
phosphate-buffered saline and the like can be mentioned. The
content of ingredient D in the emulsified composition for dilution
of the present invention is 5 to 20% by weight, preferably 8 to 16%
by weight.
[0046] In the emulsified composition for dilution of the present
invention, in addition to the above-described ingredients, an
antioxidant, a stabilizer and the like can also be added, as long
as the emulsion stability is not affected.
[0047] As examples of the antioxidant, tocopherols such as
.alpha.-tocopherol and .delta.-tocopherol, gallic acid esters,
dibutylhydroxytoluene and the like can be mentioned.
[0048] As the stabilizer, alcohols such as glycerin, propylene
glycol, 1,3-butylene glycol, polyethylene glycol and the like can
be mentioned.
[0049] The emulsified composition for dilution of the present
invention can be produced by an ordinary method of producing an
emulsified composition adopted as appropriate. Usually, a method is
chosen in which ingredient A, ingredient B and ingredient C are
previously mixed, ingredient D is gradually added while stirring
the mixture, and the mixture is finally stirred and emulsified
using an emulsifying apparatus such as a homogenizer.
[0050] The emulsified composition for dilution of the present
invention is preferably obtained in the form of a W/O emulsion
having a very low viscosity and a fine particle diameter.
[0051] The average particle diameter of the dispersion phase of the
emulsified composition for dilution of the present invention is
preferably not more than 1000 nm, more preferably not more than 500
nm, still more preferably 50 to 500 nm, particularly preferably 50
to 300 nm.
[0052] In the present invention, the average particle diameter of
the dispersion phase of the emulsified composition for dilution is
expressed as the mean of the particle diameters of all particles
(dispersion phase) measured by the dynamic light scattering method.
Specifically, particle diameters can be measured by applying a
sample obtained by diluting as appropriate an emulsified
composition as the subject of measurement with ingredient A to, for
example, ZETASIZER NANO-S manufactured by MALVERN INSTRUMENTS
Company and the like.
[0053] If the average particle diameter of the dispersion phase
exceeds 1000 nm, not only the stability for the emulsified
composition for dilution decreases, but also it is feared that the
homogeneity, stability, and CTL induction activity of the cancer
vaccine composition finally obtained by diluting a water phase
comprising a cancer antigen peptide decrease, and this is therefore
undesirable. Provided that the average particle diameter of the
dispersion phase is 50 to 300 nm, the emulsified composition can
also be filter-sterilized in the state of a W/O emulsion. The
filter used for the filter sterilization may be chosen as
appropriate to the filtration of oily liquid. Specifically, a
hydrophobic membrane filter having a pore diameter of 0.2 .mu.m
(made of polytetrafluoroethylene) and the like can be
mentioned.
[0054] The emulsified composition for dilution of the present
invention is used to obtain a cancer vaccine or specific CTL
inducer that stimulates CTL induction specific for each antigen
when combined with a cancer antigen peptide having 8 to 12 amino
acids or a dimer thereof. In so preparing a cancer vaccine
composition or specific CTL inducer, 0.25 to 1 part by volume of a
water phase comprising a cancer antigen peptide having 8 to 12
amino acids or a dimer thereof may be diluted with 1 part by volume
of an emulsified composition for dilution. At this time, the cancer
antigen peptide or dimer thereof may be dissolved or suspended in
the water phase.
[0055] The emulsified composition for dilution of the present
invention is a W/O emulsion having water particles finely dispersed
therein, and is of very low viscosity. Therefore, when a
peptide-containing water phase is diluted with this emulsified
composition for dilution, a stable W/O emulsion can be easily
obtained. The emulsified composition of the present invention is a
stable W/O emulsion per se. The cancer vaccine composition or
specific CTL inducer obtained by diluting a water phase comprising
a cancer antigen peptide with the emulsified composition of the
present invention as described above is also a stable W/O
emulsion.
[0056] Therefore, as the stirring methods for diluting a water
phase comprising a cancer antigen peptide with the emulsified
composition for dilution of the present invention, an ordinary
emulsifying apparatus such as a homogenizer or homomixer may be
used, and a simple stirring apparatus not in common use for
emulsification, for example, a shikenkan mixer and the like may be
used. The above-described dilution can also be performed by the
syringe connection method, which permits simple operation in the
laboratory. Hence, the dilution can also be freshly prepared just
before administration according to the intended use.
[0057] Preferably, by adding an emulsified composition for dilution
and a water phase comprising a cancer antigen peptide or a dimer
thereof in a ratio by volume of 1:0.25 to 1:1, and then stirring
the mixture using a shikenkan mixer for about 30 seconds to 3
minutes according to stirring speed, a stable and good cancer
vaccine composition or specific CTL inducer can be obtained. At
this time, it is preferable that while the emulsified composition
for dilution is stirred using the shikenkan mixer, the water phase
comprising the cancer antigen peptide is gradually added, and after
the entire quantity to be added has been added, the mixture is
further stirred for not less than 30 seconds.
[0058] The cancer vaccine composition or specific CTL inducer in
the present invention is obtained by diluting and mixing a water
phase comprising a cancer antigen peptide having 8 to 12 amino
acids or a dimer thereof with the emulsified composition for
dilution of the present invention as described above, and, as
described above, occurs in the form of a stable W/O emulsion.
[0059] Peptides are generally susceptible to heat, and are often
difficult to keep stable in water for a long time. As described
above, in the present invention, because the dilution can be
prepared before use by a simple method, even when using a peptide
of relatively low stability to heat and in water, the degradation
and aggregation of the peptide during emulsifying operation and
storage are reduced. Hence, because undesirable deterioration of a
cancer antigen peptide having 8 to 12 amino acids or a dimer
thereof is avoided by using the emulsified composition for dilution
of the present invention, it becomes possible to prepare a cancer
vaccine composition or specific CTL inducer that stably retains
various peptides (including dimers thereof), irrespective of the
kind and properties thereof.
[0060] In the present invention, the cancer antigen peptide is a
cancer antigen peptide having 8 to 12 amino acids or a dimer
thereof, presented to MHC class I antigen, and comprising a peptide
recognized as an antigen by CTL.
[0061] In the present invention, the cancer antigen peptide having
8 to 12 amino acids can be chosen from among commonly known ones.
For example, partial peptides derived from gp100 and MART-1, which
are melanocyte tissue specific proteins, and melanosome proteins
such as tyrosinase, can be mentioned. As other cancer antigen
peptides, partial peptides derived from cancer markers such as CEA,
PSA, and HER2/neu; partial peptides derived from cancer.testis
antigens such as MAGE-1, MAGE-2, MAGE-3 and NY-ESO-1; partial
peptides derived from epithelial cancer cell protein such as MUC-1,
SART-1, and SART-3, and the like can be mentioned. Furthermore,
partial peptides derived from TERT, whose expression is increased
in cancer, WT1-derived partial peptides, Survivin-2B-derived
partial peptides and the like can also be mentioned as suitable
cancer antigen peptides.
[0062] These may be natural substances or modifications thereof
wherein some amino acids have been modified, as long as they do not
interfere with therapeutically effective specific CTL induction for
each cancer antigen in the finally obtained cancer vaccine
composition or specific CTL inducer.
[0063] These cancer antigen peptides may be contained in the cancer
vaccine composition or specific CTL inducer not only singly, but
also in combination of two or more kinds.
[0064] These cancer antigen peptides can be obtained by solid phase
synthesis based on the Fmox method and the like, and other commonly
known methods.
[0065] As specific examples of the amino acid sequence of each
cancer antigen peptide, the following can be mentioned. Cancer
antigen peptides having the amino acid sequences shown below are
commonly known.
(1) For gp100-derived peptides, KTWGQYWQV (SEQ ID NO:1), AMLGTHTMEV
(SEQ ID NO:2), MLGTHTMEV (SEQ ID NO:3), ITDQVPFSV (SEQ ID NO:4),
YLEPGPVTA (SEQ ID NO:5), LLDGTATLRL (SEQ ID NO:6), VLYRYGSFSV (SEQ
ID NO:7), SLADTNSLAV (SEQ ID NO:8), RLMKQDFSV (SEQ ID NO:9),
RLPRIFCSC (SEQ ID NO:10), VYFFLPDHL (SEQ ID NO:11) and the like;
(2) for MART-1-derived peptides, AAGIGILTV (SEQ ID NO:12),
EAAGIGILTV (SEQ ID NO:13), ILTVILGVL (SEQ ID NO:14) and the like;
(3) for tyrosinase-derived peptides, MLLAVLYCL (SEQ ID NO:15),
YMDGTMSQV (SEQ ID NO:16), AFLPWHRLF (SEQ ID NO:17), AFLPWHRLFL (SEQ
ID NO:18) and the like; (4) for CEA-derived peptides, YLSGANLNL
(SEQ ID NO:19), IMIGVLVGV (SEQ ID NO:20), LLTFWNPPT (SEQ ID NO:21),
QYSWFVNGTF (SEQ ID NO:22), TYACFVSNL (SEQ ID NO:23) and the like;
(5) for PSA-derived peptides, FLTPKKLQCV (SEQ ID NO:24), VSHSFPHPLY
(SEQ ID NO:25), VISNDVCAQV (SEQ ID NO:26), CYASGWGSI (SEQ ID NO:27)
and the like; (6) for MUC-1-derived peptides, STAPPVHNV (SEQ ID
NO:28) and the like; (7) for HER2/neu-derived peptides, QIISAVVGIL
(SEQ ID NO:29), QLFEDNYAL (SEQ ID NO:30), KIFGSLAFL (SEQ ID NO:31),
CLTSVQLV (SEQ ID NO:32), VMAGVGSPYV (SEQ ID NO:33), RLLQETELV (SEQ
ID NO:34), PYVSRLLGI (SEQ ID NO:35), TYLPTNASL (SEQ ID NO:36) and
the like; (8) for MAGE-1-derived peptides, NYKHCFPEI (SEQ ID NO:37)
and the like; (9) for MAGE-3-derived peptides, FLWGPRALV (SEQ ID
NO:38), KVAELVHFL (SEQ ID NO:39), IMPKAGLLI (SEQ ID NO:40),
TFPDLESEF (SEQ ID NO:41) and the like; (10) for NY-ESO-1-derived
peptides, SLLMWITQC (SEQ ID NO:42), SLLMWITQCFL (SEQ ID NO:43),
QLSLLMWIT (SEQ ID NO:44) and the like; (11) for SART-1-derived
peptides, EYRGFTQDF (SEQ ID NO:45) and the like; (12) for
SART-3-derived peptides, VYDYNCHVDL (SEQ ID NO:46) and the like;
(13) for TERT-derived peptides, ILAKFLHWL (SEQ ID NO:47), VYAETKHFL
(SEQ ID NO:48), VYGFVRACL (SEQ ID NO:49) and the like; (14) for
WT1-derived peptides, RMFPNAPYL (SEQ ID NO:50), CMTWNQMNL (SEQ ID
NO:51), RWPSCQKKF (SEQ ID NO:52) and the like; (15) for
Survivin-2B-derived peptides, AYACNTSTL (SEQ ID NO:53) and the
like:
[0066] In this description, the left end of each of the
above-described peptides indicates the N terminus thereof, and the
individual amino acid symbols show the following amino acid
residues, respectively.
A: Alanine residue G: Glycine residue M: Methionine residue S:
Serine residue C: Cysteine residue H: Histidine residue N:
Asparagine residue T: Threonine residue D: Aspartic acid residue I:
Isoleucine residue P: Proline residue V: Valine residue E: Glutamic
acid residue K: Lysine residue Q: Glutamine residue W: Tryptophan
residue F: Phenylalanine residue L: Leucine residue R: Arginine
residue Y: Tyrosine residue
[0067] These cancer antigen peptides are partial peptides of cancer
antigen proteins, and some of them have cysteine at the N terminus
of the amino acid sequence thereof. Such a cancer antigen peptide
is likely to dimerize in water, and part or almost all of its
content often has dimerized at the time of administration. In the
present invention, even if a peptide having 8 to 12 amino acids
dimerizes as described above, it exhibits similar CTL induction
activity; therefore, a cancer antigen peptide can be chosen from
among cancer antigen peptides having 8 to 12 amino acids or a dimer
thereof according to the intended use. To efficiently induce CTL in
vivo, the cancer antigen peptide in the present invention is
preferably an epitope sequence restricted by HLA-A2402 (also simply
referred to as HLA-A24) or HLA-A0201 (also simply referred to as
HLA-A2) out of the cancer antigen peptides having 8 to 12 amino
acids. By using such a cancer antigen peptide, an effective cancer
vaccine composition that eventually induces and activates cytotoxic
T cells in vivo can be prepared.
[0068] The concentration of the cancer antigen peptide (including a
dimer thereof; the same applies below) in the water phase when
mixed with the emulsified composition for dilution of the present
invention may be chosen according to the kind of peptide used so
that the content in the finally obtained cancer vaccine composition
will fall in the appropriate range for activating CTL induction.
The cancer vaccine composition or specific CTL inducer of the
present invention (hereinafter also simply referred to as cancer
vaccine composition) can be kept stable irrespective of the kind
and properties of the peptide by using an emulsified composition
for dilution, and the peptide concentration to be contained can be
set in a broad range. Specifically, although it varies depending on
the route of administration and methods of administration, usually,
the peptide concentration in the finally obtained cancer vaccine
composition is 0.01 to 100 mg/mL.
[0069] Regarding the route of administration of the cancer vaccine
composition of the present invention, a method of administration in
common use may be chosen, as long as activation of CTL induction
specific for the cancer antigen peptide used is possible. As
examples of the route of administration, subcutaneous, intradermal,
intramuscular and the like can be mentioned.
[0070] The dose of the cancer antigen peptide can be adjusted as
appropriate according to the disease to be treated, the patient's
symptoms, age, body weight, sex and the like, and is 0.001 to 100
mg, more preferably 0.01 to 10 mg; this dose is preferably
administered once per several days to several months.
[0071] The cancer vaccine composition of the present invention is a
W/O emulsion. The cancer vaccine composition of the present
invention is a W/O emulsion comprising ingredient A at 30 to 80% by
weight, ingredient B at 0.5 to 20% by weight, ingredient C at 0 to
20% by weight, and ingredient E at 10 to 60% by weight, preferably
comprising ingredient A at 40 to 60% by weight, ingredient B at 1.0
to 5.0% by weight, ingredient C at 5.0 to 10.0% by weight, and
ingredient E at 30 to 50% by weight. Considering the preservation
and administration using a syringe for injection, the cancer
vaccine composition of the present invention is preferably liquid
at 5.degree. C., and preferably has a viscosity at 25.degree. C. of
not more than 300 mPas, more preferably not more than 200 mPas.
[0072] In the water phase that is the ingredient E of the present
invention, a stabilizer, a solvent, a solubilizer, a suspending
agent, a diluent, a buffering agent, a tonicity agent, an
acidifying agent, an alkalizing agent and the like may be contained
according to the properties of the cancer antigen peptide, as long
as they do not influence the stability of the finally obtained
cancer vaccine composition. Therefore, when a water phase
comprising an antigen peptide is diluted with an emulsified
composition for dilution, each of the above-described excipients
may be chosen as appropriate according to the properties of the
antigen peptide.
[0073] The cancer vaccine composition of the present invention
stimulates CTL induction specific for the cancer antigen peptide
contained, and attacks cancer cells.
[0074] CTL induction activity can be confirmed by counting the
number of CTLs by the HLA tetramer method (Int. J. Cancer: 100,
565-570 (2002)) or limited dilution method (Nat. Med.: 4, 321-327
(1998)). Alternatively, for example, HLA-A24-restricted CTL
induction activity can be confirmed by using the HLA-A24 model
mouse described in the pamphlet for International Patent
Publication No. 02/47474 and Int. J. Cancer: 100, 565-570 (2002),
and the like.
[0075] The cancer vaccine composition of the present invention has
the capability of inducing specific CTL according to the cancer
antigen peptide contained; the CTL induced exhibits cytotoxic
action and anticancer action via lymphokine production. Therefore,
the cancer vaccine composition of the present invention can be used
as a cancer immunotherapy preparation for the treatment or
prevention of cancer.
EXAMPLES
[0076] The present invention is hereinafter described in more
detail by means of the following Examples, which, however, are not
to be construed as limiting the scope of the invention.
Preparation of Emulsified Composition for Dilution
Example 1
[0077] Ethyl oleate (a supply conforming to the Japanese
Pharmaceutical Excipients) as ingredient A, polyoxyethylene (10)
hardened castor oil (a supply conforming to the Japanese
Pharmaceutical Excipients; 10 mol of ethylene oxide adduct) as
ingredient B, and sorbitan sesquioleate (a supply conforming to the
Japanese Pharmacopoeia) as ingredient C were mixed in the amounts
shown in Table 1, respectively. Next, while stirring this mixture,
water for injection (a supply conforming to the Japanese
Pharmacopoeia) as ingredient D was gradually added in the amounts
shown in Table 1. Emulsification was performed by stirring using
CLEARMIX 1.5S (manufactured by M-TECHNIQUE Co., Ltd.) at 10,000 rpm
for 5 minutes at room temperature. Thereby, an emulsified
composition for dilution 1 was obtained. When this emulsified
composition was allowed to stand at room temperature for 24 hours
after preparation, no change in the appearance was observed.
Examples 2 to 35
[0078] In the same manner as Example 1, the ingredients other than
ingredient D were previously mixed. While stirring this mixture,
ingredient D was gradually added and emulsification was performed.
In all cases, emulsification was performed by stirring using
CLEARMIX 1.5S (manufactured by M-TECHNIQUE Co., Ltd.) at 10,000 rpm
for 5 minutes at room temperature. As ingredient D, water for
injection, 10 mM phosphate-buffered solution (pH 7.4), and 10 mM
phosphate-buffered saline (pH 7.4) were used respectively according
to Tables 1 to 7. Each ingredient shown in Tables 1 to 7 were
supplies conforming to the Japanese Phaunacopoeia, the Japanese
Pharmaceutical Excipients, or Japanese Standards of Cosmetic
Ingredients. Thereby, emulsified compositions 2 to 35 were
obtained. When the emulsified compositions for dilution 2 to 35
obtained were allowed to stand at room temperature for 24 hours
after preparation, and examined for appearance change; no change
such as separation was observed in any of the emulsified
compositions.
Comparative Examples 1 to 25
[0079] In the same manner as Example 1, ethyl oleate (a supply
conforming to the Japanese Pharmaceutical Excipients), the various
polyoxyethylene hardened castor oils shown in Tables 8 to 12
(supplies conforming to the Japanese Pharmaceutical Excipients),
and sorbitan sesquioleate (a supply conforming to the Japanese
Pharmacopoeia) were mixed to obtain the amounts shown in Tables 8
to 12, respectively. Polyoxyethylene (40) hardened castor oil,
polyoxyethylene (60) hardened castor oil and the like were mixed in
a state molten by heating at 50.degree. C. Because the
polyoxyethylene (160) polyoxypropylene (30) glycol used to prepare
an emulsified composition 46 (Comparative Example 11) is not easy
to mix, it was previously dissolved in a water phase and mixed at
the time of emulsification. Next, while stirring this, water for
injection, 10 mM phosphate-buffered solution (pH 7.4), and 10 mM
phosphate-buffered saline (pH 7.4) were gradually added according
to Tables 8 to 12, respectively. Emulsification was performed by
stirring using CLEARMIX 1.5S (manufactured by M-TECHNIQUE Co.,
Ltd.) at 10,000 rpm for 5 minutes at room temperature. Thereby,
emulsified compositions for dilution 36 to 60 were obtained. Each
ingredient shown in the Tables was supplies conforming to the
Japanese Pharmacopoeia, the Japanese Pharmaceutical Excipients, or
Japanese Standards of Cosmetic Ingredients. For the ingredients not
listed in any of these compendia, supplies of pharmaceutical grade
were used. When each emulsified composition obtained was allowed to
stand for 24 hours after preparation, no change such as separation
was observed in the emulsified compositions 36 to 39, 56 and 57,
but separation of the oil phase and the water phase (including
creaming) was observed in the emulsified compositions 40 to 55 and
58 to 60.
TABLE-US-00001 TABLE 1 Example 1 2 3 4 5 Emulsified composition
number 1 2 3 4 5 Ethyl oleate 70.0 70.0 -- 70.0 70.0 Cetyl
2-ethylhexanoate -- -- 70.0 -- -- Polyoxyethylene (10) 3.0 3.0 3.0
3.0 3.0 hardened castor oil Sorbitan sesquioleate 12.0 -- -- 12.0
-- Sorbitan monooleate -- 12.0 12.0 -- 12.0 Water for injection
15.0 15.0 15.0 -- -- Phosphate-buffered saline -- -- -- 15.0 15.0
(pH 7.4) Change in state after None None None None None being
allowed to stand at room temperature for 24 hours
TABLE-US-00002 TABLE 2 Example 6 7 8 9 10 Emulsified composition
number 6 7 8 9 10 Ethyl oleate 70.0 70.0 70.0 70.0 70.0
Polyoxyethylene (5) -- 3.0 -- -- -- hardened castor oil
Polyoxyethylene (10) 3.0 -- 3.0 -- -- hardened castor oil
Polyoxyethylene (10) -- -- -- 3.0 -- castor oil Polyoxyethylene
(20) -- -- -- -- 3.0 hardened castor oil Sorbitan sesquioleate --
6.0 6.0 6.0 6.0 Glycerin monooleate 12.0 6.0 6.0 6.0 6.0
Phosphate-buffered 15.0 15.0 15.0 15.0 15.0 solution (pH 7.4)
Change in state after None None None None None being allowed to
stand at room temperature for 24 hours
TABLE-US-00003 TABLE 3 Example 11 12 13 14 15 Emulsified
composition number 11 12 13 14 15 Ethyl oleate 70.0 -- -- -- --
Cetyl 2-ethylhexanoate -- 70.0 -- -- -- Octyldodecyl myristate --
-- 70.0 -- -- 2-ethylhexyl palmitate -- -- -- 70.0 -- Oleyl oleate
-- -- -- -- 70.0 Polyoxyethylene (10) 2.0 3.0 3.0 -- -- hardened
castor oil Polyoxyethylene (20) 1.0 -- -- 3.0 3.0 hardened castor
oil Sorbitan sesquioleate 12.0 6.0 6.0 6.0 6.0 Glycerin monooleate
-- 6.0 6.0 6.0 6.0 Water for injection 15.0 15.0 15.0 15.0 15.0
Change in state after None None None None None being allowed to
stand at room temperature for 24 hours
TABLE-US-00004 TABLE 4 Example 16 17 18 19 20 Emulsified
composition number 16 17 18 19 20 Cetyl 2-ethylhexanoate 70.0 70.0
-- -- -- Ethyl oleate -- -- 70.0 70.0 Octyldodecyl myristate -- --
-- -- 70.0 Polyoxyethylene (10) 3.0 -- 2.0 5.0 3.0 hardened castor
oil Polyoxyethylene (20) -- 3.0 1.0 -- -- hardened castor oil
Sorbitan sesquioleate 6.0 12.0 6.0 6.0 11.0 Glycerin monooleate 5.0
-- 5.0 5.0 -- Glycerin 1.0 -- 1.0 1.0 1.0 Phosphate-buffered 15.0
15.0 15.0 15.0 15.0 solution (pH 7.4) Change in state after None
None None None None being allowed to stand at room temperature for
24 hours
TABLE-US-00005 TABLE 5 Example 21 22 23 24 25 Emulsified
composition number 21 22 23 24 25 Ethyl oleate 70.0 -- -- 70.0 70.0
Cetyl 2-ethylhexanoate -- 70.0 -- -- -- Octyldodecyl myristate --
-- 70.0 -- -- Polyoxyethylene (10) 15.0 15.0 15.0 15.0 15.0
hardened castor oil Water for injection 15.0 15.0 15.0 -- --
Phosphate-buffered water -- -- -- 15.0 15.0 (pH 7.4) Change in
state after None None None None None being allowed to stand at room
temperature for 24 hours
TABLE-US-00006 TABLE 6 Example 26 27 28 29 30 Emulsified
composition number 26 27 28 29 30 Cetyl 2-ethylhexanoate 75.0 75.0
-- -- -- Ethyl oleate -- -- 75.0 75.0 75.0 Polyoxyethylene (5) --
-- 3.5 -- -- hardened castor oil Polyoxyethylene (10) 14.0 3.0 --
3.0 -- hardened castor oil Polyoxyethylene (20) -- -- -- -- 2.5
hardened castor oil Sorbitan sesquioleate -- 6.0 6.5 12.0 11.5
Glycerin monooleate -- 6.0 5.0 -- -- Glycerin 1.0 -- -- -- 1.0
Phosphate-buffered water 10.0 10.0 10.0 10.0 10.0 (pH 7.4) Change
in state after None None None None None being allowed to stand at
room temperature for 24 hours
TABLE-US-00007 TABLE 7 Example 31 32 33 34 35 Emulsified
composition number 31 32 33 34 35 Ethyl oleate 72.0 78.0 80.0 74.0
-- Jojoba oil -- -- -- -- 70.0 Polyoxyethylene (10) 3.0 3.5 3.0 3.0
3.0 hardened castor oil Sorbitan sesquioleate 15.0 7.5 5.0 15.0
12.0 Glycerin 1.0 1.0 -- 1.0 -- Phosphate-buffered water 10.0 10.0
12.0 7.0 15.0 (pH 7.4) Change in state after None None None None
None being allowed to stand at room temperature for 24 hours
TABLE-US-00008 TABLE 8 Comparative Example 1 2 3 4 5 Emulsified
composition number 36 37 38 39 40 Ethyl oleate 70.0 70.0 -- 70.0
70.0 Cetyl 2-ethylhexanoate -- -- 70.0 -- -- Polyoxyethylene (30)
-- 3.0 -- -- -- hardened castor oil Polyoxyethylene (40) 3.0 -- 3.0
-- -- hardened castor oil Polyoxyethylene (60) -- -- -- 3.0 --
hardened castor oil Sorbitan sesquioleate 12.0 6.0 6.0 6.0 7.5
Glycerin monooleate -- 6.0 6.0 6.0 7.5 Water for injection 15.0
15.0 15.0 15.0 15.0 Change in state after None None None None
Separated being allowed to stand at room temperature for 24
hours
TABLE-US-00009 TABLE 9 Comparative Example 6 7 8 9 10 Emulsified
composition number 41 42 43 44 45 Cetyl 2-ethylhexanoate 70.0 -- --
-- -- Diethyl sebacate -- 70.0 -- -- -- Purified soybean oil -- --
70.0 -- -- Squalane -- -- -- 70.0 -- Light liquid paraffin -- -- --
-- 70.0 Polyoxyethylene (10) -- 3.0 3.0 3.0 3.0 hardened castor oil
Sorbitan sesquioleate 7.5 6.0 6.0 6.0 6.0 Glycerin monooleate 7.5
6.0 6.0 6.0 6.0 Water for injection 15.0 15.0 15.0 15.0 15.0 Change
in state after Sepa- Sepa- Sepa- Sepa- Slightly being allowed to
stand at rated rated rated rated sepa- room temperature for 24
rated hours
TABLE-US-00010 TABLE 10 Comparative Example 11 12 13 14 15
Emulsified composition number 46 47 48 49 50 Ethyl oleate 70.0 70.0
70.0 70.0 70.0 Polyoxyethylene (160) 3.0 -- -- -- --
polyoxypropylene (30) glycol Polyoxyethylene (20) -- 3.0 -- -- --
polyoxypropylene (20) glycol Polyoxyethylene (6) -- -- 3.0 -- --
sorbitan monooleate Polyoxyethylene (6) -- -- -- 3.0 -- sorbitol
tetraoleate Polyoxyethylene (30) -- -- -- -- 3.0 sorbitol
tetraoleate Sorbitan sesquioleate 6.0 6.0 6.0 6.0 6.0 Glycerin
monooleate 6.0 6.0 6.0 6.0 6.0 Phosphate-buffered water 15.0 15.0
15.0 15.0 15.0 (pH 7.4) Change in state after Sepa- Sepa- Sepa-
Sepa- Sepa- being allowed to stand at rated rated rated rated rated
room temperature for 24 hours
TABLE-US-00011 TABLE 11 Comparative Example 16 17 18 19 20
Emulsified composition number 51 52 53 54 55 Ethyl oleate 70.0 70.0
70.0 -- 70.0 Cetyl 2-ethylhexanoate -- -- -- 70.0 --
Polyoxyethylene (60) -- -- 15.0 15.0 -- hardened castor oil
Polysorbate 80 3.0 -- -- -- 15.0 Castor oil -- 3.0 -- -- --
Sorbitan sesquioleate 6.0 6.0 -- -- -- Glycerin monooleate 6.0 6.0
-- -- -- Water for injection 15.0 15.0 15.0 15.0 15.0 Change in
state after Sepa- Sepa- Sepa- Sepa- Sepa- being allowed to stand at
rated rated rated rated rated room temperature for 24 hours
TABLE-US-00012 TABLE 12 Comparative Example 21 22 23 24 25
Emulsified composition number 56 57 58 59 60 Cetyl 2-ethylhexanoate
75.0 -- -- -- -- Ethyl oleate -- 75.0 -- -- -- Squalane -- -- 75.0
-- -- Purified soybean oil -- -- -- 75.0 -- Tri(caprylate/caprate)
-- -- -- -- 75.0 glycerin Polyoxyethylene (10) -- -- 3.0 3.0 3.0
hardened castor oil Polyoxyethylene (40) 3.0 -- -- -- -- hardened
castor oil Polyoxyethylene (60) -- 3.0 -- -- -- hardened castor oil
Sorbitan sesquioleate 11.0 -- 4.0 11.0 11.0 Glycerin monooleate --
11.0 4.0 -- -- Glycerin 1.0 1.0 -- 1.0 1.0 Phosphate-buffered water
10.0 10.0 10.0 10.0 10.0 (pH 7.4) Change in state after None None
Sepa- Sepa- Sepa- being allowed to stand at rated rated rated room
temperature for 24 hours
Test Example 1
<Preparation of Cancer Vaccine Compositions>
[0080] Of the emulsified compositions for dilution obtained in the
Examples, those showing no apparent separation when allowed to
stand for 24 hours after preparation were used to prepare cancer
vaccine compositions comprising various cancer antigen peptides.
The cancer antigen peptides used for the preparation are shown in
Table 13. All these peptides are HLA-A24-restricted.
TABLE-US-00013 TABLE 13 Name of antigen Source Amino acid peptide
protein positions Amino acid sequence TERT Telomerase 324-332
VYAETKHFL (SEQ ID NO: 48) MAGE-1 Melanoma 135-143 NYKHCFPEI antigen
(SEQ ID NO: 37) CEA Carcino- 652-660 TYACFVSNL embryonic (SEQ ID
NO: 23) antigen PSA Prostate- 152-160 CYASGWGSI specific (SEQ ID
NO: 27) antigen
[0081] For TERT, MAGE-1, and CEA out of the cancer antigen peptides
shown in Table 13, 1.2 mg of synthetic peptide was dissolved in
10.8 .mu.L of DMSO, after which the solution was diluted with 349.2
.mu.L of water for injection, and the absence of precipitate was
visually confirmed (DMSO concentration 3%). For PSA, 1.2 mg of
synthetic peptide was diluted with water for injection to make 360
.mu.L, and the absence of precipitate was visually continued.
Separately, the emulsified composition obtained in each Example was
thoroughly mixed using a shikenkan mixer (Touch Mixer MT-51,
manufactured by Yamato Scientific Co., Ltd.) (fully stirred and
homogenized before use).
[0082] Next, 700 .mu.L of the emulsified composition was collected
in cryogenic vial inner cap type of 5 mL capacity (manufactured by
Sumitomo Bakelite Co., Ltd.) using a 1000 .mu.L Eppendorf pipette.
Next, while stirring the tube using a shikenkan mixer, 300 .mu.L of
the above-described peptide-containing water phase was added drop
by drop, and mixed. The stirring speed of the shikenkan mixer was
set at the maximum level. After the peptide-containing water phase
was added drop by drop, the mixture was further stirred using the
shikenkan mixer for 2 minutes to yield a cancer vaccine
composition.
[0083] In the experiments below, CTL induction activity was
actually evaluated using prepared cancer vaccine compositions;
unless otherwise stated, the cancer vaccine compositions used for
the evaluation were prepared according to the above-described
method.
Test Example 2
[0084] <CTL Induction with Cancer Vaccine Composition Comprising
Tert Peptide>
[0085] The specific CTL induction potential of the cancer vaccine
composition comprising the TERT peptide, prepared in Test Example
1, was evaluated using HLA-A24 transgenic mice (Int. J. Cancer:
100, 565, 2002).
[0086] To prepare cancer vaccine compositions, emulsified
compositions 7, 8, 37, 38, 39, and 45 were used. The cancer vaccine
compositions prepared were given the same numbers as the emulsified
compositions. In the evaluation, separately from Test Example 1,
groups receiving an emulsion comprising the TERT peptide, prepared
using incomplete Freund's adjuvant (IFA, purchased from Wako Pure
Chemical Industries, Ltd.), or the peptide water phase alone, as
the dosing liquid, were established, and compared with groups
receiving each cancer vaccine composition as the dosing liquid
(matched to ensure that all dosing groups would receive the same
dose of peptide). 200 .mu.L of each dosing liquid (dose of each
peptide 200 .mu.g) was subcutaneously administered to the tail root
of each HLA-A24/K.sup.b transgenic mouse. Three mice were used for
each group. Seven to eight days after administration, the spleen
was extirpated and splenocytes were prepared. Some of the
splenocytes were pulsed with 100 .mu.g/mL peptide for 1 hour.
"Pulse" refers to adding a peptide to splenocytes to bind the
antigen peptide to HLA on the cell surface. The splenocytes not
pulsed with the peptide were sown to a 24-well plate at
7.times.10.sup.6 cells/well, and the above splenocytes pulsed with
the peptide were further added at 7.times.10.sup.5 cells/well and
cultured. The culture broth comprised an RPMI1640 medium
supplemented with 10% FCS, 10 mM HEPES, 20 mM L-glutamine, 1 mM
sodium pyruvate, 1 mM MEM non-essential amino acids, 1% MEM
vitamin, and 55 .mu.M 2-mercaptoethanol, and the cells were
cultured for 5 to 6 days. The CTL activity specific for the peptide
administered in the cultured splenocytes was measured by .sup.51Cr
release assay (J. Immunol.: 159, 4753, 1997). The target cells used
were cells of the cell line EL4-A2402/K.sup.b prepared by
transferring a gene to mouse lymphoma-derived cell line EL-4 cells
(ATCC line number TIB-39) so that the HLA-A24 and H-2K.sup.b
chimeric MHC class I molecule (Int. J. Cancer: 100, 565, 2002)
would be stably expressed. The target cells were labeled with
.sup.51Cr at 3.7 Mbq/10.sup.6 cells, after which the aforementioned
peptide was added to obtain a concentration of 100 .mu.g/mL, and
the cells were further pulsed for 1 hour. The target cells, but not
pulsed with the peptide (non-pulsed), were labeled with .sup.51Cr
for 2 hours and used as control target cells. These labeled target
cells and previously prepared splenocytes were mixed in a ratio of
1:80 and cultured for 4 hours, and CTL activity was calculated from
the ratio of target cells injured. The results are shown in FIG.
1.
[0087] As shown in FIG. 1, in the groups receiving the cancer
vaccine composition of the present invention, the target cells
pulsed with the peptide were severely injured, but the control
target cells not pulsed with the peptide were less injured;
therefore, it was demonstrated that peptide specific CTL was
induced. On the other hand, in the groups receiving the cancer
vaccines 37, 38, 39, and 45, the group receiving the peptide
emulsion prepared with IFA, and the group receiving the peptide
alone, the cytotoxicity was low, and the CTL induction activity was
low. From this finding, it is evident that the emulsified
composition for dilution of the present invention activates in vivo
CTL induction when combined with cancer antigen peptides. It is
also shown that the choice of the ingredient A of the present
invention and the molar number of ethylene oxide adduct to the
ingredient B influence CTL induction activity.
Test Example 3
[0088] <CTL Induction with Cancer Vaccine Composition Comprising
MAGE-1 Peptide>
[0089] The specific CTL induction potential of the cancer vaccine
composition comprising the MAGE-1 peptide, prepared in Test Example
1, was evaluated in the same manner as Test Example 2.
[0090] To prepare cancer vaccine compositions, the emulsified
compositions 8, 37, 38, and 45 were used. The cancer vaccine
compositions prepared were given the same numbers as the emulsified
compositions. In the evaluation, separately from Test Example 1,
groups receiving an emulsion comprising the MAGE-1 peptide,
prepared using incomplete Freund's adjuvant (IFA, purchased from
Wako Pure Chemical Industries, Ltd.), or the peptide water phase
alone, as the dosing liquid, were established, and compared with
groups receiving each cancer vaccine composition as the dosing
liquid. The results are shown in FIG. 2.
[0091] As shown in FIG. 2, in the group receiving the cancer
vaccine composition of the present invention, the target cells
pulsed with the peptide were severely injured, but the control
target cells not pulsed with the peptide were less injured;
therefore, it was demonstrated that peptide specific CTL was
induced. On the other hand, in the groups receiving the cancer
vaccines 37, 38, and 45, the group receiving the peptide emulsions
prepared with IFA, and the group receiving the peptide alone, the
cytotoxicity was low, and the CTL induction activity was low. These
results are similar to those of Test Example 2, demonstrating the
utility of the emulsified composition for dilution of the present
invention.
Test Example 4
[0092] <CTL Induction with Cancer Vaccine Composition Comprising
the PSA Peptide (1)>
[0093] The specific CTL induction potential of the cancer vaccine
composition comprising the PSA peptide, prepared in Test Example 1,
was evaluated in the same manner as Test Example 2.
[0094] To prepare cancer vaccine compositions, the emulsified
compositions 2, 7, 10, 29, and 38 were used. The cancer vaccine
compositions prepared were given the same numbers as the emulsified
compositions. Also, using the emulsified composition 7, 700 .mu.L
of the emulsified composition and 300 .mu.L of water for injection
were mixed according to Test Example 1 to prepare a peptide-free
composition, and a comparison was performed. The results are shown
in FIG. 3.
[0095] As shown in FIG. 3, in the groups receiving the cancer
vaccine composition of the present invention (2, 7, 10, 29), the
target cells pulsed with the peptide were severely injured, but the
control target cells not pulsed with the peptide were less injured;
therefore, it was demonstrated that peptide specific CTL was
induced. On the other hand, in the group receiving the cancer
vaccine 38, the cytotoxicity was low, and the CTL induction
activity was low. In the group receiving the peptide-free
composition, absolutely no cytotoxicity was observed, demonstrating
that the emulsified composition alone does not have the activity to
induce CTL. These results are similar to those of Test Example 2,
demonstrating the utility of the emulsified composition for
dilution of the present invention.
Test Example 5
[0096] <CTL Induction with Cancer Vaccine Composition Comprising
PSA Peptide (2)>
[0097] The specific CTL induction potential of the cancer vaccine
composition comprising the PSA peptide, prepared in Test Example 1,
was evaluated in the same manner as Test Example 4.
[0098] To prepare cancer vaccine compositions, the emulsified
compositions 16, 17, and 20 were used. The cancer vaccine
compositions prepared were given the same numbers as the emulsified
compositions. In the evaluation, separately from Test Example 1, a
group receiving an emulsion comprising the PSA peptide, prepared
using incomplete Freund's adjuvant (IFA, purchased from Wako Pure
Chemical Industries, Ltd.) as the dosing liquid, was established,
and compared with groups receiving each cancer vaccine composition
as the dosing liquid. The results are shown in FIG. 4.
[0099] As shown in FIG. 4, in the group receiving the cancer
vaccine composition of the present invention, the target cells
pulsed with the peptide were severely injured, but the control
target cells not pulsed with the peptide were less injured;
therefore, it was demonstrated that peptide specific CTL was
induced. When the PSA peptide was used, even in the group receiving
the peptide emulsion prepared with IFA, peptide specific CTL was
induced. From this result and the results of Test Examples 2 and 3,
it was shown that IFA, which is conventionally known, activated CTL
induction in some cancer antigen peptides, but that this activation
was significantly influenced by the kind of peptide, and that IFA
is insufficient in versatility.
Test Example 6
[0100] <CTL Induction with Cancer Vaccine Composition Comprising
CEA Peptide>
[0101] The specific CTL induction potential of the CEA-containing
cancer vaccine composition prepared in the Test Example 1 was
evaluated in the same manner as Test Example 2.
[0102] To prepare cancer vaccine compositions, the emulsified
compositions 12, 13, 28, 29, 30, 37, and 38 were used. The cancer
vaccine compositions prepared were given the same numbers as the
emulsified compositions. The results are shown in FIG. 5. As shown
in FIG. 5, in the groups receiving a cancer vaccine composition
prepared using the emulsified composition of the present invention
(12, 13, 28, 29), the target cells pulsed with the peptide were
severely injured, but the control target cells not pulsed with the
peptide were less injured; therefore, it was demonstrated that
peptide specific CTL was induced. On the other hand, in the groups
receiving the cancer vaccines 37 and 38, the cytotoxicity was low,
and the CTL induction activity was low. These results are similar
to those of Test Example 2, demonstrating the utility of the
emulsified composition for dilution of the present invention.
Test Example 7
[0103] <CTL Induction with Cancer Vaccine Compositions
Comprising Various Cancer Antigen Peptides>
[0104] Using the emulsified composition 21, the specific CTL
induction potentials of the cancer vaccine compositions comprising
each of the TERT, MAGE-1, and CEA peptides, prepared according to
Test Example 1, were evaluated in the same manner as Test Example
2. The results are shown in FIG. 6. According to FIG. 6, in the
group receiving a cancer vaccine composition prepared using the
emulsified composition of the present invention, the target cells
pulsed with the peptide were severely injured, but the control
target cells not pulsed with the peptide were less injured;
therefore, it was demonstrated that peptide specific CTL was
induced.
[0105] These results show that the emulsified composition for
dilution of the present invention activates CTL induction specific
for each cancer antigen in vivo when combined with various cancer
antigen peptides.
Test Example 8
<Evaluation of Stability of Emulsified Compositions for
Dilution>
[0106] Emulsified compositions for dilution prepared in Examples
were tested for stability. For the test, the emulsified
compositions 1 to 39, 56, and 57, which showed no apparent
separation when allowed to stand for 24 hours after preparation,
were used. Specifically, 2 mL of each emulsified composition was
filled in a glass vial of 4 mL capacity, and the vial was tightly
closed and stored at 25.degree. C. for 1 month, 3 months, and 6
months, after which each composition was examined for apparent
change. As a result, no apparent change was observed in any of the
emulsified compositions.
Test Example 9
<Microscopic Examination of Emulsified Compositions for
Dilution>
[0107] In the same manner as Test Example 8, the emulsified
compositions showing no apparent separation when allowed to stand
for 24 hours after preparation (1 to 39, 56, 57) were used to
microscopically examine the emulsified state both before and after
dilution. First, the emulsified state of each emulsified
composition was examined using a phase contrast microscope at
200-fold magnification. As a result, all emulsified compositions
exhibited a fine good emulsified state. Hence, 700 .mu.L of each
emulsified composition and 300 .mu.L of water for injection were
taken in cryogenic vial inner cap type of 5 mL capacity
(manufactured by Sumitomo Bakelite Co., Ltd.), and stirred using a
shikenkan mixer (Touch Mixer MT-51, manufactured by Yamato
Scientific Co., Ltd.) for 2 minutes, after which each composition
was examined for emulsified state under microscope in the same
manner as above. As a result, the emulsified compositions 1 to 35
exhibited a fine good emulsified state, whereas the emulsified
compositions 36 to 39, 56 and 57 exhibited evident aggregation of
water phase particles.
Test Example 10
<Evaluation of Average Particle Diameters of Emulsified
Compositions for Dilution>
[0108] Of the emulsified compositions for dilution obtained in the
Examples and Comparative Examples, the emulsified compositions 1,
2, 4, 6, 7, 8, 10, 21, 29, 36, and 39 were tested to determine the
average particle diameters thereof. Measurements were performed by
the dynamic light scattering method using a particle size
distribution analyzer (ZETA-SIZER NANO-S, manufactured by MALVERN
INSTRUMENTS Company). To ensure accurate measurements, each
emulsified composition was diluted as appropriate with a continual
phase of ethyl oleate at the time of measurement. The results are
shown in Table 14. Each average particle diameter is the mean of
all particle diameters calculated from the light scattering
intensity. As examples of the measurement results, the particle
size distributions of the emulsified compositions 8 and 29 at the
time of preparation (initial) are shown in FIGS. 7 and 8,
respectively.
TABLE-US-00014 TABLE 14 Average particle diameter (nm) At time of
After storage Emulsified preparation at 5.degree. C. composition
(initial) for 3 months 1 181 179 2 195 203 4 233 252 6 266 275 7
388 420 8 192 189 10 163 171 21 78 81 29 150 132 36 208 39 160
[0109] As a result of the measurements, all emulsified compositions
had an average particle diameter in the range of 50 to 500 nm at
the time of preparation. From this finding, it is seen that simply
exhibiting a good emulsified state is insufficient to use for
diluting cancer antigen peptides. Hence, this data showed that what
is essential to the CTL induction potential of the emulsified
composition for dilution of the present invention is not "to be
capable of preparing a good emulsified composition", but for the
molar number of ethylene oxide adduct to the ingredient B nonionic
surfactant to fall in a particular range.
[0110] For the emulsified compositions 1, 2, 4, 6, 7, 8, 10, 21,
and 29 out of the above-described emulsified compositions for
dilution, average particle diameters after storage at 5.degree. C.
for 3 months were measured in the same manner. As a result, no
major change in average particle diameter was observed in any of
the emulsified compositions; therefore, it is seen that the
emulsified composition for dilution of the present invention is
also excellent in low-temperature stability.
Test Example 11
[0111] <CTL Induction with Cancer Vaccine Compositions
Comprising a Mixture of Two Kinds of Cancer Antigen Peptide>
[0112] Cancer vaccine compositions comprising each of mixtures of
two kinds of peptides, i.e., MAGE-1 and CEA, TERT and CEA, and CEA
and PSA, out of the cancer antigen peptides described in Table 13,
were prepared, and the specific CTL induction potentials thereof
were evaluated.
[0113] Each cancer vaccine composition was prepared using the
emulsified composition 4, specifically as described below.
[0114] First, each of the MAGE-1, CEA, TERT, and PSA peptides was
prepared as a 100 mg/mL DMSO solution. Next, each of the
combinations of MAGE-1 and CEA, TERT and CEA, CEA and PSA in DMSO
solution was mixed, and each mixture was diluted using water for
injection to obtain a concentration of 2 mg/600 .mu.L for each
peptide to yield peptide mixed solutions (4 mg/600 .mu.L as the
total amount of peptide). After preparation of each solution, the
absence of precipitate was visually confirmed. Separately,
emulsified compositions obtained in Examples were thoroughly mixed
using a shikenkan mixer (Touch Mixer MT-51, manufactured by Yamato
Scientific Co., Ltd.) (fully stirred and homogenized before
use).
[0115] Next, 700 .mu.L of the emulsified composition was collected
in cryogenic vial inner cap type of 5 mL capacity (manufactured by
Sumitomo Bakelite Co., Ltd.) using a 1000 .mu.L Eppendorf pipette.
Next, while stirring the tube using the shikenkan mixer, 300 .mu.L
of the above-described peptide mixed solution was added drop by
drop, and mixed. The stirring speed of the shikenkan mixer was set
at the maximum level. After the peptide mixed solution was added
drop by drop, the mixture was further stirred for 2 minutes using
the shikenkan mixer to yield three kinds of cancer vaccine
compositions (MAGE-1/CEA mixture, TERT/CEA mixture, CEA/PSA
mixture).
[0116] The specific CTL induction potentials of the three kinds of
cancer vaccine composition prepared were evaluated using HLA-A24
transgenic mice (Int. J. Cancer: 100, 565, 2002).
[0117] 200 .mu.L of each dosing liquid (each peptide dose of 200
.mu.g.times.2 kinds) was subcutaneously administered to the tail
root of each HLA-A24/K.sup.b transgenic mouse. One mouse was used
for each group. Seven days after administration, the spleen was
extirpated and splenocytes were prepared. Some of the splenocytes
were pulsed with 50 .mu.g/ml, of each of two kinds of subject
peptides (100 .mu.g/mL in total) for 1 hour. Splenocytes not pulsed
with the peptides were sown to a 24-well plate at 7.times.10.sup.6
cells/well, and the above-mentioned splenocytes pulsed with the
peptide were further added at 7.times.10.sup.5 cells/well and
cultured. The culture broth comprised an RPMI1640 medium
supplemented with 10% FCS, 10 mM HEPES, 20 mM L-glutamine, 1 mM
sodium pyruvate, 1 mM MEM non-essential amino acids, 1% MEM
vitamin, and 55 .mu.M 2-mercaptoethanol, and the cells were
cultured for 5 days. The CTL activity specific for the peptide
administered in the cultured splenocytes was measured by .sup.51Cr
release assay (J. Immunol.: 159, 4753, 1997). The target cells used
were cells of the cell line EL4-A2402/K.sup.b prepared by
transferring a gene to mouse lymphoma-derived cell line EL-4 cells
(ATCC line number TIB-39) so that the HLA-A24 and H-2K.sup.b
chimeric MHC class I molecule (Int. J. Cancer: 100, 565, 2002)
would be stably expressed. The target cells were labeled with
.sup.51Cr at 3.7 Mbq/10.sup.6 cells, after which each peptide was
added to obtain a concentration of 100 .mu.g/mL, and the cells were
pulsed for 1 hour. Target cells not pulsed with the peptide
(non-pulsed) were labeled with .sup.51Cr for 2 hours and served as
control target cells. These labeled target cells and previously
prepared splenocytes were mixed in a ratio of 1:80 and cultured for
4 hours, and CTL activity was determined from the ratio of target
cells injured. The results are shown in FIG. 9.
[0118] As shown in FIG. 9, in the groups receiving cancer vaccine
compositions prepared using the emulsified composition for dilution
of the present invention, the target cells pulsed with the peptides
were severely injured. On the other hand, the control target cells
not pulsed with the peptides were less injured; therefore, it was
demonstrated that when two kinds of cancer antigen peptide were
mixed, CTL specific for each peptide was concurrently induced. From
this finding, it was demonstrated that the emulsified composition
for dilution of the present invention activates CTL induction in
vivo when combined with various cancer antigen peptides.
INDUSTRIAL APPLICABILITY
[0119] The emulsified composition for dilution of the present
invention is a stable W/O emulsion per se. By diluting a cancer
antigen peptide or a dimer thereof with the emulsified composition
for dilution of the present invention by a simple operation, there
is provided a cancer vaccine composition that is a stable W/O
emulsion exhibiting CTL induction activity specific for each cancer
antigen in vivo.
[0120] The present invention also provides an emulsified
composition for diluting a cancer antigen peptide or a dimer
thereof, intended to induce specific CTL. Also provided by this
emulsified composition for dilution is a cancer vaccine composition
having CTL induction activity specific for various cancer antigens
in vivo. The present invention is considered to be effective in
ameliorating conditions in many cancer patients.
[0121] This application is based on a patent application No.
2005-012140 filed in Japan on Jan. 19, 2005, the contents of which
are incorporated in full herein by this reference.
Sequence CWU 1
1
5319PRTHomo sapiens 1Lys Thr Trp Gly Gln Tyr Trp Gln Val1
5210PRTHomo sapiens 2Ala Met Leu Gly Thr His Thr Met Glu Val1 5
1039PRTHomo sapiens 3Met Leu Gly Thr His Thr Met Glu Val1
549PRTHomo sapiens 4Ile Thr Asp Gln Val Pro Phe Ser Val1 559PRTHomo
sapiens 5Tyr Leu Glu Pro Gly Pro Val Thr Ala1 5610PRTHomo sapiens
6Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu1 5 10710PRTHomo sapiens
7Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val1 5 10810PRTHomo sapiens
8Ser Leu Ala Asp Thr Asn Ser Leu Ala Val1 5 1099PRTHomo sapiens
9Arg Leu Met Lys Gln Asp Phe Ser Val1 5109PRTHomo sapiens 10Arg Leu
Pro Arg Ile Phe Cys Ser Cys1 5119PRTHomo sapiens 11Val Tyr Phe Phe
Leu Pro Asp His Leu1 5129PRTHomo sapiens 12Ala Ala Gly Ile Gly Ile
Leu Thr Val1 51310PRTHomo sapiens 13Glu Ala Ala Gly Ile Gly Ile Leu
Thr Val1 5 10149PRTHomo sapiens 14Ile Leu Thr Val Ile Leu Gly Val
Leu1 5159PRTHomo sapiens 15Met Leu Leu Ala Val Leu Tyr Cys Leu1
5169PRTHomo sapiens 16Tyr Met Asp Gly Thr Met Ser Gln Val1
5179PRTHomo sapiens 17Ala Phe Leu Pro Trp His Arg Leu Phe1
51810PRTHomo sapiens 18Ala Phe Leu Pro Trp His Arg Leu Phe Leu1 5
10199PRTHomo sapiens 19Tyr Leu Ser Gly Ala Asn Leu Asn Leu1
5209PRTHomo sapiens 20Ile Met Ile Gly Val Leu Val Gly Val1
5219PRTHomo sapiens 21Leu Leu Thr Phe Trp Asn Pro Pro Thr1
52210PRTHomo sapiens 22Gln Tyr Ser Trp Phe Val Asn Gly Thr Phe1 5
10239PRTHomo sapiens 23Thr Tyr Ala Cys Phe Val Ser Asn Leu1
52410PRTHomo sapiens 24Phe Leu Thr Pro Lys Lys Leu Gln Cys Val1 5
102510PRTHomo sapiens 25Val Ser His Ser Phe Pro His Pro Leu Tyr1 5
102610PRTHomo sapiens 26Val Ile Ser Asn Asp Val Cys Ala Gln Val1 5
10279PRTHomo sapiens 27Cys Tyr Ala Ser Gly Trp Gly Ser Ile1
5289PRTHomo sapiens 28Ser Thr Ala Pro Pro Val His Asn Val1
52910PRTHomo sapiens 29Gln Ile Ile Ser Ala Val Val Gly Ile Leu1 5
10309PRTHomo sapiens 30Gln Leu Phe Glu Asp Asn Tyr Ala Leu1
5319PRTHomo sapiens 31Lys Ile Phe Gly Ser Leu Ala Phe Leu1
5328PRTHomo sapiens 32Cys Leu Thr Ser Val Gln Leu Val1 53310PRTHomo
sapiens 33Val Met Ala Gly Val Gly Ser Pro Tyr Val1 5 10349PRTHomo
sapiens 34Arg Leu Leu Gln Glu Thr Glu Leu Val1 5359PRTHomo sapiens
35Pro Tyr Val Ser Arg Leu Leu Gly Ile1 5369PRTHomo sapiens 36Thr
Tyr Leu Pro Thr Asn Ala Ser Leu1 5379PRTHomo sapiens 37Asn Tyr Lys
His Cys Phe Pro Glu Ile1 5389PRTHomo sapiens 38Phe Leu Trp Gly Pro
Arg Ala Leu Val1 5399PRTHomo sapiens 39Lys Val Ala Glu Leu Val His
Phe Leu1 5409PRTHomo sapiens 40Ile Met Pro Lys Ala Gly Leu Leu Ile1
5419PRTHomo sapiens 41Thr Phe Pro Asp Leu Glu Ser Glu Phe1
5429PRTHomo sapiens 42Ser Leu Leu Met Trp Ile Thr Gln Cys1
54311PRTHomo sapiens 43Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu1
5 10449PRTHomo sapiens 44Gln Leu Ser Leu Leu Met Trp Ile Thr1
5459PRTHomo sapiens 45Glu Tyr Arg Gly Phe Thr Gln Asp Phe1
54610PRTHomo sapiens 46Val Tyr Asp Tyr Asn Cys His Val Asp Leu1 5
10479PRTHomo sapiens 47Ile Leu Ala Lys Phe Leu His Trp Leu1
5489PRTHomo sapiens 48Val Tyr Ala Glu Thr Lys His Phe Leu1
5499PRTHomo sapiens 49Val Tyr Gly Phe Val Arg Ala Cys Leu1
5509PRTHomo sapiens 50Arg Met Phe Pro Asn Ala Pro Tyr Leu1
5519PRTHomo sapiens 51Cys Met Thr Trp Asn Gln Met Asn Leu1
5529PRTHomo sapiens 52Arg Trp Pro Ser Cys Gln Lys Lys Phe1
5539PRTHomo sapiens 53Ala Tyr Ala Cys Asn Thr Ser Thr Leu1 5
* * * * *