U.S. patent application number 11/171365 was filed with the patent office on 2006-04-06 for cytotoxic t lymphocyte.
This patent application is currently assigned to Mie University. Invention is credited to Atsunori Hiasa, Yoshihiro Miyahara, Hiroaki Naota, Satoshi Okumura, Hiroshi Shiku.
Application Number | 20060073126 11/171365 |
Document ID | / |
Family ID | 36125792 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073126 |
Kind Code |
A1 |
Shiku; Hiroshi ; et
al. |
April 6, 2006 |
Cytotoxic T lymphocyte
Abstract
The present invention relates to a T lymphocyte having an
activity to induce a T lymphocyte recognizing an antigen and a
technique to use the T lymphocyte.
Inventors: |
Shiku; Hiroshi; (Tsu-shi,
JP) ; Hiasa; Atsunori; (Tsu-shi, JP) ;
Okumura; Satoshi; (Tsu-shi, JP) ; Naota; Hiroaki;
(Tsu-shi, JP) ; Miyahara; Yoshihiro; (Tsu-shi,
JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Mie University
TAKARA BIO INC.
|
Family ID: |
36125792 |
Appl. No.: |
11/171365 |
Filed: |
July 1, 2005 |
Current U.S.
Class: |
424/93.21 ;
424/185.1; 435/372 |
Current CPC
Class: |
A61K 39/0011 20130101;
A61K 2039/5154 20130101; A61P 35/00 20180101; A61K 48/00 20130101;
A61K 2039/5156 20130101; C12N 5/0636 20130101; A61P 31/00 20180101;
A61K 39/00 20130101; A61P 37/04 20180101 |
Class at
Publication: |
424/093.21 ;
435/372; 424/185.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 39/00 20060101 A61K039/00; C12N 5/08 20060101
C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 2004 |
JP |
2004-290785 |
Claims
1. An RNA-introduced T lymphocyte into which an RNA encoding an
antigen of interest is introduced, which has an activity to induce
a T lymphocyte which recognizes the antigen.
2. The RNA-introduced T lymphocyte according to claim 1, which is a
CD4-positive cell activated with phytohemagglutinin.
3. A method for inducing a T lymphocyte, comprising using, as an
antigen-presenting cell, an RNA-introduced T lymphocyte into which
an RNA encoding an antigen of interest is introduced, to induce a T
lymphocyte which recognizes the antigen.
4. The method according to claim 3, wherein the RNA-introduced T
lymphocyte is a CD4-positive cell activated with
phytohemagglutinin.
5. The method according to claim 3, wherein the T lymphocyte which
recognizes the antigen is a cytotoxic T lymphocyte positive to
CD8.
6. The method according to any one of claims 3 to 5, wherein the
antigen is a tumor-associated antigen or an antigen of an
infectious microorganism.
7. The method according to claim 6, wherein the RNA is an RNA
prepared from a cancer tissue.
8. The method according to claim 6, wherein the antigen is EBNA3A
and/or CMVpp65.
9. An immune inducer containing the RNA-introduced T lymphocyte as
defined in claim 1 or 2.
10. A therapeutic agent for cancer or infectious disease,
containing the T lymphocyte obtained by the method of any one of
claims 3 to 8 as an active ingredient.
11. A cytotoxic T lymphocyte which recognizes a cell presenting on
the surface thereof a complex of a human major histo-compatibility
antigen (HLA)-A24-restricted antigen peptide represented by SEQ ID
NO: 1 or 2 and an HLA-A24 molecule or a complex of a functional
derivative of the HLA-A24-restricted antigen peptide and an HLA-A24
molecule, and which is CD8-positive.
12. A therapeutic agent for cancer, containing the cytotoxic T
lymphocyte of claim 11.
13. An inducer of the cytotoxic T lymphocyte of claim 11,
containing at least one peptide selected from the group consisting
of a human major histo-compatibility antigen (HLA)-A24-restricted
antigen peptide represented by SEQ ID NO: 1 or 2 and a functional
derivative thereof as an active ingredient.
14. A therapeutic agent for cancer, containing a human major
histo-compatibility antigen (HLA)-A24-restricted antigen peptide
represented by SEQ ID NO: 1 or 2 and a functional derivative
thereof as an active ingredient.
15. A tetramer for detecting a T cell receptor possessed by the
cytotoxic T lymphocyte of claim 11, comprising a human major
histo-compatibility antigen (HLA)-A24-restricted antigen peptide
represented by SEQ ID NO: 1 or 2 or a functional derivative
thereof.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to T lymphocytes having an
activity of inducing T lymphocytes which recognize an antigen, a
method of inducing specific antigen-specific T lymphocytes, use of
induced T lymphocytes as a therapeutic agent for cancer or an
infectious disease, HLA-A2402-restricted cytotoxic T lymphocytes
(CTLs) specific for a tumor-associated antigen, an antigen peptide
recognized by the CTL, use of the antigen peptide as a CTL inducer
and a therapeutic agent for cancer, and a tetramer formed by
tetramerizing MHC/antigen peptide complexes which is useful for
detection of the CTL.
[0003] 2. Discussion of Related Art
[0004] Among cytotoxic T lymphocytes (CTLs), there is a CTL capable
of recognizing, by a specific T cell receptor (abbreviated
hereinafter as "TCR"), a complex wherein an antigen peptide and a
major histo-compatibility antigen MHC molecule encoded by a major
histo-compatibility gene complex (abbreviated hereinafter as "MHC")
are bound to each other, thus injuring cells presenting the complex
on the cell surface thereof. The major histo-compatibility antigen
MHC molecule, in the case of humans, is called human leukocyte
antigen (abbreviated hereinafter as "HLA"). The CTL recognizes and
injures only a target cell having the same HLA molecule as in the
CTL itself. Accordingly, the CTL is called "HLA-restricted
CTL".
[0005] The cytotoxicity reaction can be generated by: [0006] 1) the
presence of a CTL having a specific TCR, and [0007] 2) the presence
of an antigen peptide not only capable of binding to an HLA
molecule but also forming a complex recognized by the TCR in order
to become an antigen peptide presented by an HLA and recognized by
the CTL.
[0008] Such antigen peptide is generated, for example, by
processing, in an endoplasmic reticulum, of an antigen and the like
such as a protein synthesized intracellularly in a mammalian cell,
to degrade the antigen and the like into smaller epitope peptides.
The antigen peptide is further associated with an HLA molecule and
presented on the surface of a cell. I.e., the protein is degraded
into peptides consisting of from 8 to 15 amino acid residues in a
proteosome complex consisting of many subunits, and some of the
generated peptides are transported from the cytosol to an
endoplasmic reticulum by a TAP transporter. The peptides, when
bound to a class I/.beta.2 microglobulin heterodimer in the
endoplasmic reticulum, are stabilized as a 3-molecule complex and
transported through a Golgi apparatus into the surface of a
cell.
[0009] Furthermore, it has been revealed that upon infection with
organisms such as viruses, microorganisms, protozoa and fungi, a
CTL against an antigen possessed by these organisms plays an
important role in protection against infection.
[0010] The HLA class I molecules are roughly divided into HLA-A,
HLA-B and HLA-C. It is known that an antigen peptide presented upon
binding to the HLA class I molecule is composed of from 8 to 10
amino acid residues and has certain structural features which vary
depending on the respective HLA molecules. For example, a peptide
consisting of from 9 to 10 amino acid residues having a Leu residue
at the second position from the N-terminal thereof and a Leu
residue or a Val residue at the C-terminal is best known worldwide
as a peptide binding to the HLA-A2.1 molecule found most
frequently. A peptide consisting of from 9 to 10 amino acid
residues having any one of a Tyr residue, a Phe residue, a Met
residue and a Trp residue at the second position from the
N-terminal thereof and any one of a Leu residue, an Ile residue, a
Trp residue and a Phe residue at the C-terminal is best known as a
peptide binding to an HLA-A24 molecule abundant in Asians races
including Japanese (J. Immunol., 155, p.4307-4312 (1995)).
[0011] Tumor antigens of which antigen peptides have been
identified up to now include MAGE-1 and MAGE-3 against HLA-A1;
MAGE-3, MART1, tyrosinase, gp100, HER2/neu, CEA and the like
against HLA-A2.1; MAGE-3 against HLA-Cw1; MAGE-3 against HLA-B44;
MAGE-A4 against HLA-B37; MAGE-1, MAGE-2, MAGE-3, CEA, HER2/neu,
tyrosinase and .beta.-catenin against HLA-A24, and the like.
[0012] Many of the antigen peptides have been found by establishing
a tumor cell-recognizing class I-restricted CTL, to identify a
tumor antigen recognized by the CTL, finding the minimum unit in a
protein serving as the tumor antigen by a genetic engineering
method and selecting a peptide in the minimum unit, on the basis of
information on a binding motif to HLA class I molecule (Proc. Natl.
Acad. Sci. USA, 91, p.3515-3519 (1994)).
[0013] The antigen peptide is determined by finding HLA class I
molecule-binding peptides consisting of a sequence in a tumor
antigen protein, on the basis of a motif structure common in the
HLA class I molecule-binding peptide, selecting a CTL-inducible
peptide from the found HLA class I molecule-binding peptides, by
using antigen-presenting cells, and evaluating whether a CTL having
cytotoxicity on tumor cells can be finally induced or not (Proc.
Natl. Acad. Sci. USA, 91, p.2105-2109 (1994); J. Exp. Med., 179,
p.921-930 (1994)).
[0014] The HLA class I molecules are classified into some subtypes.
The subtype possessed by humans varies significantly among races.
The proportion of humans having HLA-A2 is highest in the world and
accounts for 45% of the Caucasoid, for example. Identification of
the HLA-A2-restricted antigen peptide has advanced most. On the
other hand, in the Japanese, the proportion possessing HLA-A2
accounts for 40%. 20% of the Japanese have HLA-A*0201 which is the
same subtype as in the Caucasoid, and many of the rest of the
Japanese have A*0206. Peptides binding to these subtypes vary
depending on the subtype. For example, a mainly studied peptide
binding to HLA-A2 is HLA-A*0201. On the other hand, the proportion
of the Japanese possessing HLA-A24 accounts for 60% or more. The
proportion possessing the HLA-A24 is higher in Asian races than in
other races.
[0015] The antigen peptide, even if the antigen is the same, varies
depending on the type of HLA, and induction of a CTL utilizing the
antigen peptide is thus troublesome. To solve this problem, various
devices have been made, but satisfactory results have not been
obtained yet at present. One of the devices is a method of inducing
T lymphocytes by utilizing cells obtained by transducing an antigen
gene into antigen-presenting cells derived from a patient himself
(autologous). As the antigen-presenting cells, use of B cells,
macrophages or dendritic cells, known as professional
antigen-presenting cells, have been examined, and a clinical test
of using the dendritic cells mainly for vaccine adjuvant and the
like is conducted (J. Immunotherapy, 21, p.41-47 (1998)). In these
antigen-presenting cells, however, there are some disadvantages
that much labor is required to prepare the cells in a necessary
amount to induce immunization. In addition, the B cells have the
advantage that the cells can be prepared in a large amount by
immortalization with EB virus. Owing to use of the virus, however,
there is the disadvantage of lacked generality. With respect to the
method of introducing the gene, introduction of the gene using a
virus vector or a plasmid DNA has the disadvantage of generating a
new variant in some cases by insertion of the gene into a
chromosome.
SUMMARY OF THE INVENTION
[0016] A first aspect of the present invention relates to provide
RNA-introduced T lymphocytes which enable induction of cytotoxic T
lymphocytes which can recognize an antigen, for example act
specifically on organisms causing an infectious disease or tumor
cells in individuals, especially Asian races, particularly
Japanese, exhibit a therapeutic action on a tumor, causing specific
cytolysis of target cells or cells presenting a specific antigen in
the individuals, or cause cytokine release reaction. A second
aspect of the present invention relates to provide an immune
inducer capable of inducing the cytotoxic T lymphocytes or inducing
immunity effective against cancer or an infectious disease in the
individuals. A third aspect of the present invention relates to
provide a method of inducing T lymphocytes, which at least enables
induction of T lymphocytes recognizing an antigen of interest. A
fourth aspect of the present invention relates to provide a
therapeutic agent for cancer or an infectious disease, which acts
specifically on organisms causing an infectious disease or tumor
cells in the individuals. A fifth aspect of the present invention
relates to provide cytotoxic T lymphocytes which enable recognition
of an antigen, causing specific cytolysis of target cells or cells
presenting a specific antigen, or causing cytokine release
reaction, in the individuals. A sixth aspect of the present
invention relates to provide an inducer of the cytotoxic T
lymphocytes, which enables any one of induction of the cytotoxic T
lymphocytes or exhibiting a specific action on organisms causing an
infectious disease or tumor cells in the individuals. A seventh
aspect of the present invention relates to provide a tetramer for
detecting T cell receptors possessed by cytotoxic T lymphocytes,
which enables any one of monitoring of the cytotoxic T lymphocytes
in the individuals or samples derived from the individuals.
[0017] A first embodiment of the present invention relates to
RNA-introduced T lymphocytes into which an RNA encoding an antigen
of interest is introduced, the RNA-introduced T lymphocytes having
an activity of inducing T lymphocytes which recognize the antigen.
Such RNA-introduced T lymphocytes include, for example,
CD4-positive cells activated by phytohemagglutinin. Such
RNA-introduced T lymphocytes have an excellent effect that, for
example, cytotoxic T lymphocytes which recognize an antigen can be
induced by a simple technique. Here, such cytotoxic T lymphocytes
can act specifically on organisms causing an infectious disease or
tumor cells in individuals, especially Asian races, particularly
Japanese, can exhibit a therapeutic action on a tumor, and can
specifically cause cytolysis of target cells or cells presenting a
specific antigen, cytokine release reaction and the like, in the
individuals.
[0018] A second embodiment of the present invention relates to a
method of inducing T lymphocytes which recognize the antigen, which
comprises using the RNA-introduced T lymphocytes in the first
embodiment as antigen-presenting cells to induce T lymphocytes
which recognize the antigen. The RNA-introduced T lymphocytes
include, for example, CD4-positive cells activated with
phytohemagglutinin. In addition, the T lymphocytes which recognize
an antigen include, for example, CD8-positive cytotoxic T
lymphocytes. The antigen includes a tumor-associated antigen or an
antigen of an infectious microorganism. The RNA includes, for
example, an RNA prepared from cancerous tissues. The antigen
includes, for example, EBNA3A, CMVpp65 and the like. This induction
method exhibits an excellent effect that T lymphocytes recognizing
the antigen of interest can be simply induced.
[0019] A third embodiment of the present invention relates to an
immune inducer, which comprises the RNA-introduced T lymphocytes in
the first embodiment. Such immune inducer exhibits an excellent
effect that, for example, the cytotoxic T lymphocytes can be
induced to induce immunity effective against cancer or an
infectious disease in the individuals.
[0020] A fourth embodiment of the present invention relates to a
therapeutic agent for cancer or an infectious disease, which
comprises, as an active ingredient, T lymphocytes obtained by the
method of inducing T lymphocytes in the second embodiment. Such
therapeutic agent for cancer or an infectious disease exhibits an
excellent effect that the agent can act specifically on, for
example, organisms causing an infectious disease or tumor cells in
the individuals.
[0021] A fifth embodiment of the present invention relates to
cytotoxic T lymphocytes which recognize cells presenting, on the
surfaces of the cells, a complex of a human major
histo-compatibility antigen (HLA)-A24-restricted antigen peptide
represented by SEQ ID NO: 1 or 2 and an HLA-A24 molecule, or a
complex of a functional derivative of the HLA-A24-restricted
antigen peptide and an HLA-A24 molecule and which are positive to
CD8. Such cytotoxic T lymphocytes exhibit an excellent effect of
specific recognition of an antigen. The cytotoxic T lymphocytes of
the present invention also exhibit an excellent effect that the
cytotoxic T lymphocytes can act specifically on, for example,
organisms causing an infectious disease or tumor cells in the
individuals, exhibit a therapeutic action on tumor, and cause
specific cytolysis of target cells or cells presenting a specific
antigen, cytokine release reaction and the like, in the
individuals.
[0022] A sixth embodiment of the present invention relates to a
therapeutic agent for cancer, which comprises the cytotoxic T
lymphocytes in the fifth embodiment as an active ingredient. Such
therapeutic agent for cancer exhibits an excellent effect that, for
example, it can act specifically on tumor cells in the
individuals.
[0023] A seventh embodiment of the present invention relates to an
inducer for the cytotoxic T lymphocytes in the fifth embodiment,
which comprises, as an active ingredient, at least one antigen
peptide selected from the group consisting of a human major
histo-compatibility antigen (HLA)-A24-restricted antigen peptide
represented by SEQ ID NO: 1 or 2 and the functional derivative
thereof. Such inducer exhibits an excellent effect that, for
example, the cytotoxic T lymphocytes can be induced by a simple
technique to exhibit a specific action on organisms causing an
infectious disease or tumor cells in the individuals.
[0024] An eighth embodiment of the present invention relates to a
therapeutic agent for cancer, which comprises, as an active
ingredient, at least one antigen peptide selected from the group
consisting of a human major histo-compatibility antigen
(HLA)-A24-restricted antigen peptide represented by SEQ ID NO: 1 or
2 and the functional derivative thereof. Such therapeutic agent for
cancer exhibits an excellent effect that, for example, the agent
can act specifically on tumor cells in the individuals.
[0025] A ninth embodiment of the present invention relates to a
tetramer for detecting T cell receptors possessed by the cytotoxic
T lymphocytes in the fifth embodiment, which comprises a human
major histo-compatibility antigen (HLA)-A24-restricted antigen
peptide represented by SEQ ID NO: 1 or 2 or the functional
derivative thereof. Such tetramer exhibits an excellent effect
that, for example, the tetramer can monitor the cytotoxic T
lymphocytes in the individuals.
[0026] According to the present invention, there is provided a
method of inducing a CTL, wherein easily obtainable T lymphocytes
are used as antigen-presenting cells. Further, there is provided a
novel HLA-A24-restricted antigen peptide. The CTL induced by using
the antigen-presenting cells or the antigen peptide is useful, for
example, in treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A to 1C are graphs showing immunogenicity of peptides
in HHDA2402.+-..beta.2m-/- mice. FIG. 1A shows the results of
EBNA3A, FIG. 1B shows the results of MAGE-A4, and FIG. 1C shows the
results of SAGE.
[0028] FIG. 2 is a graph showing immunogenicity of MAGE-A4-derived
peptides in humans. Panel A is a graph showing the results of
IFN-.gamma. ELISPOT assay of human bulk CTLs sensitized twice with
autologous CD4+ PHA blast cells into which a mRNA corresponding to
MAGE-A4 or SAGE was introduced. In panel A, the black bar indicates
LCL into which MAGE-A4 mRNA was introduced, and the shaded bar
indicates LCL into which SAGE mRNA was introduced. Panel B is a
graph showing the results of expansion of well #2 with autologous
LCL into which mRNA corresponding to MAGE-A4 or SAGE was
introduced, or with autologous PBMC and IL-2 (20
IU/milliliter).
[0029] FIG. 3 is a graph showing the results of presentation of
cell surfaces with MAGE-A4.sub.143-151 peptide together with
HLA-A2402 after intracellular processing. Panel A shows the result
of staining with MAGE-A4.sub.143-151 A24 tetramer, and panel B
shows the results of staining with a control tetramer. Panel C
shows the cytotoxicity of MAGE-A4.sub.143-151-specific CTL #2-28
cells on tumor cells. The black triangle shows T2A24 pulsed with
MAGE-A4.sub.143-151; the black square shows T2 pulsed with
MAGE-A4.sub.143-151; the lozenge shows T2A24 pulsed with
SAGE.sub.715-723; and the black circle shows T2A24 not pulsed.
Panel D shows the cytotoxicity of MAGE-A4.sub.143-151-specific CTL
#2-28 cells on tumor cells. The black triangle shows KE-4 cells
expressing HLA-A2402 and MAGE-A4; the black square shows TE-10
cells expressing HLA-A2402 and MAGE-A4; the lozenge shows 11-18
cells expressing HLA-A2402 and MAGE-A4; and the black circle shows
LB-23 cells expressing HLA-A2402, but not expressing MAGE-A4.
[0030] FIGS. 4A to 4C are graphs showing the immunogenicity of
SAGE.sub.715-723 peptide in humans. FIG. 4A is a graph showing the
results of IFN-.gamma. ELISPOT assay using human bulk CTL cells
obtained by sensitization twice with autologous CD4+ PHA blast
cells into which SAGE mRNA was introduced. In FIG. 4A, the black
bar indicates the results where T2A24 cells pulsed with
SAGE.sub.715-723 peptide were used as target cells, and the shaded
bar indicates the results where T2A24 cells pulsed with a control
peptide (HER2.sub.63-71) were used. FIG. 4B is a graph showing the
results of expansion of bulk CTLs in well #2. FIG. 4C is a graph
showing the results of IFN-.gamma. ELISPOT assay using human bulk
CTL cells obtained by sensitization twice with autologous CD4+ PHA
blast cells into which SAGE mRNA was introduced.
[0031] FIGS. 5A to 5C are graphs showing the results of
presentation of SAGE.sub.715-723 peptide on cell surfaces together
with HLA-A2402 after intracellular processing. FIG. 5A is a graph
showing the results of staining with SAGE.sub.715-723 A24 tetramer.
FIG. 5B is a graph showing the amount of IFN-.gamma. released by
SAGE.sub.715-723-specific CTL#22 cells in the presence of various
cells. FIG. 5C shows the cytotoxicity of SAGE.sub.715-723-specific
CTL #22 cells on various cells. In FIG. 5C, panel A shows K562
cells not expressing SAGE or HLA-A2402. In panel A, the lozenge
shows K562 cells, the black square shows K562A24 cells, and the
black triangle shows K564A24 pulsed with SAGE.sub.715-723. Panel B
shows R27 cells expressing SAGE but not expressing HLA-A2402. In
panel B, the lozenge shows R27 cells, the black square shows R27A24
cells, and the black triangle shows R27A24 pulsed with
SAGE.sub.715-723. Panel C shows LCL not expressing SAGE but
expressing HLA-A2402. In panel C, the black triangle shows LCL into
which SAGE mRNA was introduced, and the lozenge shows LCL into
which EBNA3A mRNA was introduced.
[0032] FIGS. 6A to 6D are graphs each showing one example of the
results of detection of SAGE.sub.715-723-specific precursor in
healthy normal human PBMC.
DETAILED DESCRIPTION OF THE INVENTION
[0033] In the present specification, the amino acid residue is
sometimes expressed in three-letter or one-letter designation of an
amino acid in accordance with conventional nomenclature in
biochemistry.
[0034] The first embodiment of the present invention relates to
RNA-introduced T lymphocytes into which an RNA encoding an antigen
of interest is introduced and having an activity of inducing T
lymphocytes recognizing the antigen. The RNA-introduced T
lymphocytes of the present invention have RNA encoding an antigen
of interest introduced into them, and thus exhibit an excellent
effect that T lymphocytes recognizing the antigen can be obtained.
Accordingly, the RNA-introduced T lymphocytes exhibit an excellent
effect that cytotoxic T lymphocytes acting specifically on
organisms causing an infectious disease or tumor cells in
individuals, especially Asian races, particularly Japanese,
exhibiting a therapeutic action on tumors, and causing specific
cytolysis of target cells or cells presenting a specific antigen,
and cytokine release reaction, in the individuals can be induced by
a simple method.
[0035] The RNA-introduced T lymphocytes of the present invention
can be prepared for example by introducing an RNA encoding an
antigen of interest into T lymphocytes derived from natural
origin.
[0036] The "T lymphocytes derived from natural origin" includes,
for example, CD3-positive cells derived from individuals. The "T
lymphocytes derived from natural origin" can be identified by
using, for example, a monoclonal antibody against CD3 and, if
necessary, a monoclonal antibody against a T cell antigen receptor.
Such CD3-positive cells may also be CD4-positive and/or
CD8-positive cells. The CD3-positive cells are preferably
CD4-positive.
[0037] The "antigen" includes, but not limited particularly to,
tumor-associated antigens, antigens of infectious microorganisms,
and the like.
[0038] The "tumor-associated antigens" include MAGE
(melanoma-associated antigen) family protein, SAGE (sarcoma
antigen), LAGE (L antigen), NY-ESO-1, WT-1, hTERT, and the
like.
[0039] The "antigens of infectious microorganisms" include antigens
of EB virus such as EBNA-3A, antigens of cytomegalovirus such as
CMVpp65, herpes virus antigen, influenza virus antigen, HIV
antigen, Salmonella antigen, Shigella antigen, Enterobacter
antigen, protozoa-derived antigen, fungus-derived antigen, and the
like.
[0040] The RNA may be an RNA prepared by usual genetic engineering
techniques, or an RNA prepared from cells. The RNA can be obtained,
for example, by extracting an RNA encoding an antigen of interest
from cells, reverse-transcribing the resulting RNA into cDNA,
amplifying the resulting cDNA by PCR, and carrying out
transcription using the amplified DNA as a template. Such RNA can
also be used for preparation of the RNA-introduced T lymphocytes of
the present invention.
[0041] The method of introducing the RNA into T lymphocytes
includes, for example, physical methods such as electroporation, a
particle gun method, a calcium phosphate method, a lipofection
method and a liposome method; biological techniques using viral
vectors such as a retrovirus vector, a lentivirus vector and an
adenovirus vector; and the like.
[0042] Specifically, the RNA-introduced T lymphocytes of the
present invention include, for example, CD4-positive cells
activated with phytohemagglutinin.
[0043] The second embodiment of the present invention relates to a
method of inducing T lymphocytes which recognize an antigen, which
comprises using, as antigen-presenting cells (also referred to
hereinafter as "APC"), RNA-introduced T lymphocytes into which an
RNA encoding the antigen of interest is introduced, to induce T
lymphocytes recognizing the antigen. Such induction method is based
on findings by the present inventors that an antigen-specific CTL
can be induced when autologous lymphocytes are stimulated with a
T-cell activator such as phytohemagglutinin and cells into which an
RNA encoding an antigen is introduced by electroporation are used
as antigen-presenting cells, and on surprising findings by the
present inventors that the RNA-introduced T lymphocytes exhibit an
ability to present the antigen.
[0044] The induction method of the present invention exhibits an
excellent effect that since the RNA-introduced T lymphocytes into
which an RNA encoding the antigen of interest is introduced are
used as antigen-presenting cells, T lymphocytes recognizing the
antigen of interest can be simply induced. The induction method of
the present invention is also advantageous in that the method is
excellent in handling (for example, collection, enlargement etc.)
of cells as compared with induction of a CTL with B cells,
macrophages or dendritic cells known as professional
antigen-presenting cells.
[0045] The "RNA encoding an antigen of interest" is exemplified by
the similar RNA as illustrated in the first embodiment.
Specifically, the RNA includes, for example, an RNA encoding an
antigen for which induction of cytotoxic T lymphocytes which
specifically recognize the antigen is desired (antigen of
interest). The antigen includes, for example, a tumor-associated
antigen or an antigen of an infectious microorganism and the like.
The antigen of infectious microorganisms includes EBNA3A, CMVpp65
and the like. The RNA includes, for example, an RNA prepared from
cancer tissues, and the like.
[0046] The method of artificially introducing an RNA encoding an
antigen of interest into the RNA-introduced T lymphocytes used as
antigen-presenting cells includes, but is not limited particularly
to, the physical methods and biological methods described above. By
such artificial introduction methods, the RNA can be introduced
into T lymphocytes, to express the RNA in the resulting
RNA-harboring T lymphocytes.
[0047] The T lymphocytes and RNA-introduced T lymphocytes can be
maintained, for example, in a medium such as RPMI, AIM-V and
X-VIVO10, physiologic saline, and buffer solutions such as
phosphate buffered physiologic saline.
[0048] In the induction method of the present invention, the
RNA-introduced T lymphocytes used as antigen-presenting cells may
be CD3-positive cells of an individual itself, for example, a
patient himself/herself (autologous T lymphocytes) or CD3-positive
cells derived from a donor having the same type of HLA as that of
an individual such as a patient. The T lymphocytes, similarly to
those in the first embodiment, may be CD4-positive or may be
CD8-positive. The RNA-introduced T lymphocytes are preferably
CD4-positive T lymphocytes. Here, when the individual is an
individual, such as a patient, who has underwent transplantation
such as allogeneic hematopoietic stem cell transplantation, the T
lymphocytes used can be T lymphocytes of a donor of the
transplant.
[0049] The RNA-introduced T lymphocytes may be activated preferably
with lectin such as phytohemagglutinin (PHA) and concanavalin
(ConA) or an anti-CD3 antibody in a medium containing IL-2, IL-7
and the like. Insofar as the activation conditions are usually used
conditions, the conditions are not limited particularly. For use as
antigen-presenting cells, the RNA-introduced T lymphocytes are
treated by gamma ray irradiation or with mitomycin.
[0050] Here, in the induction method of the present invention, the
RNA-introduced T lymphocytes are preferably CD4-positive cells
activated with phytohemagglutinin.
[0051] Specifically, the RNA-introduced T lymphocytes in the first
embodiment can be used as antigen-presenting cells to induce
antigen-recognizing T lymphocytes. The antigen-recognizing T
lymphocytes to be induced include CD8-positive cytotoxic T
lymphocytes (CTLs) and CD4-positive helper T lymphocytes. The
"antigen-recognizing T lymphocytes to be induced" vary depending on
a starting material used as a source of T lymphocytes derived from
natural origin. The "starting material" includes, for example,
peripheral blood mononuclear cells (hereinafter also referred to as
"PBMC") collected from blood, and CD8-positive lymphocytes
separated from the PBMC by a method using an anti-CD8 antibody and
magnetic beads. For example, when the PBMC is used as the "starting
material", lymphocytes usually in the form of a mixture of
antigen-recognizing CTLs and helper T lymphocytes are obtained. In
addition, for example, when CD8-positive cells are used as the
"starting material", lymphocytes containing a CTL are obtained.
[0052] When the CTL is to be induced in vitro, for example, the
RNA-introduced T lymphocytes and a sample excised extracorporeally
from a living body having the same type of HLA as in the
RNA-introduced T lymphocytes can be used to induce the CTL. The
"sample excised extracorporeally" in this specification means a
sample such as blood; lymph nodes, spleen and other various organs
excised by an operation, and the like. Particularly in the
induction method of the present invention, lymphocytes and
infiltrating lymphocytes occurring in these samples are preferably
used.
[0053] For example, when blood is used as the sample, the CTL of
interest can be induced by repetitive antigenic stimulation with
the T lymphocytes into which an RNA encoding the antigen of
interest is introduced, to lymphocytes obtained from PBMC prepared
from human blood having the corresponding type of HLA. By cloning,
the induced CTL can also be maintained as lymphocytes having
stabilized cytotoxicity. For example, the induced CTL can be
proliferated by stimulation with feeder cells, antigen, various
cytokines, or anti-CD3 antibody.
[0054] The activity of the induced "cytotoxic T lymphocytes which
recognize an antigen", i.e., the activity of the antigen-specific
CTL, can be evaluated in terms of cytotoxicity on target cells as
an indicator by measuring a radioactive substance released from the
target cells pulsed with a peptide from the CTL inducing antigen
and labeled with a radioactive substance and the like. In addition,
the activity of the antigen-specific CTL can be evaluated by
measuring the incorporation of radioactivity into the CTL in the
presence of the antigen-presenting cells pulsed with the antigen
peptide, whereby the proliferation reaction of the CTL against the
antigen-presenting cells pulsed with the antigen peptide can be
determined as an indicator of the activity of the antigen-specific
CTL. Each of the target cells or antigen-presenting cells which are
tranduced with a DNA encoding the antigen or into which an RNA
encoding the antigen is introduced can also be used for the above
purpose. The antigen-specific CTL can be detected by measuring the
amount of cytokines such as GM-CSF and IFN-.gamma. released in an
antigen-specific manner from the CTL or the target cells. The
antigen-specific CTL can also be confirmed directly by using an
antigen peptide/HLA complex labeled with a fluorescent dye and the
like. In this case, for example, the CTL can be contacted with a
first fluorescent marker coupled with a CTL-specific antibody, and
the resulting product can be contacted with an antigen peptide/MHC
complex coupled with a second fluorescent marker, to confirm the
antigen-specific CTL by detecting the presence of cells labeled
with both the first and second fluorescent markers by FACS
(fluorescence-activated cell sorting) analysis.
[0055] The third embodiment of the present invention relates to an
immune inducer comprising the RNA-introduced T lymphocytes in the
first embodiment. The immune inducer of the present invention
comprises the RNA-introduced T lymphocytes as an active ingredient,
and thus exhibits an excellent effect that the "cytotoxic T
lymphocytes which recognize an antigen" capable of acting
specifically on organisms causing an infectious disease or tumor
cells in individuals, especially Asian races, particularly
Japanese, exhibiting a therapeutic action on a tumor, and causing
specific cytolysis of target cells or cells presenting a specific
antigen, and cytokine release reaction, in the individuals, can be
induced. The immune inducer of the present invention can induce
immunity effective against cancer or an infectious disease in the
individuals.
[0056] The immune inducer of the present invention is provided in
the form of a suspension of the RNA-introduced T lymphocytes in the
first embodiment in a pharmaceutically acceptable diluent. Here,
the "diluent" means, for example, a medium suitable for storing the
RNA-introduced T lymphocytes in the first embodiment, or
physiologic saline or phosphate buffered physiologic saline. The
medium generally includes, but not limited particularly to, a
medium such as RPMI, AIM-V and X-VIVO10. These mediums are easily
commercially available.
[0057] For the purpose of stabilization and the like, a
pharmaceutically acceptable carrier may also be added to the immune
inducer of the present invention. Here, in this specification, the
carrier includes, for example, human serum albumin and the
like.
[0058] The content of the T lymphocytes in the first embodiment in
the immune inducer of the present invention is desirably
1.times.10.sup.4 cells/milliliter or more, preferably
5.times.10.sup.5 cells/milliliter or more, and 1.times.10.sup.8
cells/milliliter or less, preferably 5.times.10.sup.7
cells/milliliter or less, per type of T lymphocyte.
[0059] The immune inducer of the present invention is applied to
both administration into a donor of the T lymphocytes ("autologous
administration") and administration into another individual having
the same type of HLA ("allogenic administration").
[0060] When the immune inducer of the present invention is to be
administered into humans, the immune inducer of the present
invention can be administered subcutaneously, intracutaneously or
intravenously by a syringe. Although the amount of the immune
inducer of the present invention can be determined suitably
depending on body weight, disease state, and the like, it is
desired that the number of the RNA-introduced T lymphocytes
administered into an adult is usually from 10.sup.6 to 10.sup.10
cells per type of RNA-introduced T lymphocyte. The above range is a
standard and not restrictive. Administration of the immune inducer
of the present invention can be repeated until the desired effect
is obtained.
[0061] The RNA-introduced T lymphocytes contained in the immune
inducer of the present invention are T lymphocytes derived from an
individual (specifically human) as the subject of administration or
T lymphocytes having the same type of HLA as that of an individual
as the subject of administration. Therefore, toxicity of the
RNA-introduced T lymphocytes is not particularly recognized.
[0062] The immune inducer of the present invention exhibits an
excellent effect that a CTL recognizing an antigen encoded by an
RNA introduced into the RNA-introduced T lymphocytes contained in
the immune inducer is induced in an individuals (specifically
human) into which the immune inducer is administered.
[0063] Accordingly, the pharmacological evaluation of the immune
inducer of the present invention can be carried out, for example,
by measuring the activity of the CTL induced by the immune inducer
of the present invention in terms of cytotoxicity on the target
cells, proliferation reaction in the presence of antigen-presenting
cells, the amount of antigen-specific cytokines released, and the
like.
[0064] The T lymphocytes obtained by the induction method in the
second embodiment, for example, the CTL, can be used as a
therapeutic agent for cancer or an infectious disease. Accordingly,
a fourth embodiment of the present invention relates to a
therapeutic agent for cancer or an infectious disease, which
comprises, as an active ingredient, the T lymphocytes obtained by
the induction method in the second embodiment. The therapeutic
agent can be formulated pursuant to a therapeutic agent for cancer
in a sixth embodiment described below, to use in treatment.
[0065] In this specification, the therapeutic agent for cancer is
intended to encompass so-called carcinostatics.
[0066] Pharmacological evaluation of the therapeutic agent for
cancer or an infectious disease of the present invention, for
example, in the case of cancer, can be carried out where inhibition
of growth of cancer cells, death of cancer cells, induction of cell
death of cancer cells or shrinkage of cancer cells observed in
examination of the cytotoxicity of the therapeutic agent on cancer
cells, or shrinkage or disappearance of a cancer site, or
prevention of expansion of the cancer site, upon administration of
the therapeutic agent for cancer or an infectious disease into the
cancer site or therearound, is used as an indicator of the effect
of the therapeutic agent on cancer.
[0067] Pharmacological evaluation of the therapeutic agent for
cancer or an infectious disease of the present invention, for
example, in the case of an infectious disease, can be carried out
by using, as an indicator of the effect of the therapeutic agent on
an infectious disease, suppression of growth of an organism causing
an infectious disease or its cells or death of the organism or its
cells in examination of the cytotoxicity of the therapeutic agent
on the organism or its cells, or reduction or disappearance of
symptoms of an infectious disease, upon administration of the
therapeutic agent for cancer or an infectious disease in the
present invention into individuals (e.g. humans) with an infectious
disease.
[0068] A fifth embodiment of the present invention relates to
cytotoxic T lymphocytes which recognize cells presenting a complex
of a human major histo-compatibility antigen (HLA)-A24-restricted
antigen peptide represented by SEQ ID NO: 1 or 2 and an HLA-A24
molecule, or a complex of a functional derivative of the antigen
peptide and an HLA-A24 molecule on the surfaces of the cells and
which are positive to CD8.
[0069] The peptide of an amino acid sequence represented by SEQ ID
NO: 1 is an HLA-A24-restricted antigen peptide derived from
MAGE-A4. The peptide of an amino acid sequence represented by SEQ
ID NO: 2 is an HLA-A24-restricted antigen peptide derived from
SAGE. Accordingly, the CTL of the present invention exhibits an
excellent property of specifically causing cytolysis or cytokine
release reaction of the target cells or antigen-presenting
cells.
[0070] In this specification, the "functional derivative of the
HLA-A24-restricted antigen peptide" means a substance having an
ability to form a complex with an HLA-A24 molecule, and the formed
complex is recognized by a CTL recognizing a complex of an antigen
peptide represented by SEQ ID NO: 1 or 2 and an HLA-A24 molecule.
The "functional derivative of the HLA-A24-restricted antigen
peptide" is, for example, a peptide having an ability to form a
complex with an HLA-A24 molecule, the formed complex being
recognized by the CTL of the present invention, wherein the amino
acid sequence of the functional derivative is different from the
amino acid sequence represented by SEQ ID NO: 1 or 2 by: [0071] 1)
deletion, [0072] 2) substitution with other amino acid residues or
amino acid analogues, [0073] 3) addition of one or more amino acid
residues or amino acid analogues, or [0074] 4) a combination
thereof of one or several amino acid residues. The length of the
amino acid sequence of the functional derivative is preferably from
9 to 10 residues, but is not limited thereto particularly. The
"amino acid analogues" in this specification mean N-acylated amino
acids, O-acylated amino acids, esterified amino acids, amide amino
acids, alkylated amino acids, and the like.
[0075] Insofar as the complex of the HLA-A24-restricted antigen
peptide or the functional derivative thereof and the HLA-A24
molecule is recognized by the CTL of the present invention, a
formyl group, an acetyl group, a t-butoxycarbonyl (t-Boc) group or
the like may be bound to the N-terminal amino group of the antigen
peptide or the functional derivative thereof, or to a free amino
group of a side chain of an amino acid residue. In addition,
insofar as the complex of the HLA-A24-restricted antigen peptide or
the functional derivative thereof and the HLA-A24 molecule is
recognized by the CTL, a methyl group, an ethyl group, a t-butyl
group, a benzyl group or the like may be bound to the C-terminal of
the antigen peptide or the functional derivative thereof, or to a
free carboxyl group in a side chain of an amino acid residue
thereof.
[0076] The functional derivative can be identified by using the CTL
recognizing a complex of the antigen peptide represented by SEQ ID
NO: 1 or 2 and an HLA-A24 molecule. The method of identifying the
functional derivative includes, for example, the following
methods.
[0077] The first method is a method wherein a candidate substance
as the functional derivative is mixed with HLA-A24-expressing
cells, and the candidate substance not bound to HLA-A24 molecules
is washed away, to react the resulting product with the CTL. When
candidate-specific cytotoxicity, cytokine release or proliferation
reaction is recognized in this method, the candidate substance can
be judged to be the functional derivative.
[0078] The second method is a method wherein a candidate substance
is mixed with cells having an ability to present the antigen, and
incubated for an appropriate time, for example, for a time required
for the antigen to be incorporated and processed and for the
antigen peptide and HLA molecule complex to be presented to the
surface of the cell, to react the resulting product with the CTL.
When candidate-specific cytokine release or proliferation reaction
is recognized in this method, the candidate substance can be judged
to be the functional derivative.
[0079] A third method is a method wherein a nucleic acid encoding
an amino acid sequence of the candidate substance is bound to an
expression vector capable of presenting a peptide on HLA-A24
molecules on cells having an ability to present the antigen
described below, and wherein suitable cells are transformed by the
resulting recombinant vector, thereby reacting the resulting cells
having an ability to present the antigen with the CTL. In such
method, when candidate substance-specific cytokine release or
proliferation reaction is recognized, the candidate substance can
be judged to be the functional derivative.
[0080] The functional derivative includes, for example, peptides
having an ability to bind to HLA-A24 molecules to give a complex of
the peptide and an HLA-A24 molecule recognized by a CTL recognizing
a complex of a peptide represented by SEQ ID NO: 1 or 2 and an
HLA-A24 molecule, out of peptides having the amino acid sequence of
SEQ ID NO: 1 or 2, wherein: [0081] 1) a second amino acid residue
from the N-terminal is substituted with an amino acid selected from
the group consisting of a Tyr residue, a Phe residue, a Met residue
and a Trp residue typical of the peptide binding to HLA-A24
molecules and/or [0082] 2) the C-terminal amino acid is substituted
with an amino acid selected from the group consisting of a Leu
residue, an Ile residue, a Trp residue and a Phe residue typical of
the peptide binding to HLA-A24 molecules, in order to enhance
binding to HLA-A24 molecules. More specifically, the functional
derivative is, for example, a peptide wherein the C-terminal amino
acid Phe residue is substituted with a Leu residue in the amino
acid sequence of SEQ ID NO: 2.
[0083] Those peptides which have an ability to bind to HLA-A24
molecules to give a complex of the peptide and an HLA-A24 molecule
recognized by the CTL of the present invention, out of those
peptides wherein one or several amino acid residues (amino acid
residues to be substituted) are substituted with amino acid
residues or amino acid analogues similar in side chain to the amino
acid residues to be substituted in the amino acid sequence of SEQ
ID NO: 1 or 2 are also included.
[0084] The "amino acid residues similar in side chain to the amino
acid residues to be substituted" refer to other amino acid
residue(s) belonging to the same group as an amino acid residue in
any one of the following groups 1 to 7: [0085] 1. glycine (Gly)
residue and alanine (Ala) residue; [0086] 2. valine (Val) residue,
isoleucine (Ile) residue, leucine (Leu) residue and methionine
(Met) residue; [0087] 3. asparagine (Asn) residue and glutamine
(Gln) residue; [0088] 4. aspartic acid (Asp) residue and glutamic
acid (Glu) residue; [0089] 5. serine (Ser) residue and threonine
(Thr) residue; [0090] 6. lysine (Lys) residue and arginine (Arg)
residue; and [0091] 7. phenylalanine (Phe) residue and tyrosine
(Tyr) residue.
[0092] A sixth embodiment of the present invention relates to a
therapeutic agent for cancer, which comprises the CTL in the fifth
embodiment as an active ingredient. Since the therapeutic agent for
cancer of the present invention comprises the CTL of the present
invention, the therapeutic agent for cancer exhibits an excellent
effect that the agent can act specifically on tumor cells of
individuals, especially Asian races, particularly Japanese.
[0093] Particularly, the therapeutic agent of the present invention
is useful for treatment of cancer wherein expression of the antigen
recognized by the CTL in the fifth embodiment, i.e., MAGE-4 or
SAGE, is recognized.
[0094] The therapeutic agent for cancer according to the present
invention is provided in the form of a suspension of the CTL in a
pharmaceutically acceptable diluent.
[0095] In this specification, the "diluent" refers to, for example,
a medium suitable for storage of the CTL, or physiologic saline,
phosphate buffered physiologic saline, and the like.
[0096] The medium includes, but is not limited to, for example,
RPMI, AIM-V, X-VIVO10 and the like.
[0097] For the purpose of stabilization, a pharmaceutically
acceptable carrier may also be added to the therapeutic agent for
cancer according to the present invention. Here, the carrier
includes, for example, human serum albumin and the like.
[0098] The content of the CTL in the therapeutic agent for cancer
according to the present invention is 1.times.10.sup.4
cells/milliliter or more, preferably 5.times.10.sup.5
cells/milliliter or more, and 1.times.10.sup.8 cells/milliliter or
less, preferably 5.times.10.sup.7 cells/milliliter or less, per
kind of CTL.
[0099] When the therapeutic agent for cancer according to the
present invention is to be administered to human, the agent can be
administered, for example, with a syringe. The dose of the
therapeutic agent for cancer according to the present invention can
be determined suitably depending on body weight, disease state, and
the like of the individual, and the number of CTLs administered
into an adult is desirably set to be 1.times.10.sup.6 to
1.times.10.sup.10 cells per kind of CTL. Here, the above range is a
standard and not restrictive. The active ingredient CTL is a CTL
derived from a human as the subject of administration or a CTL
having the same type of HLA as in the human, and thus the toxicity
of the therapeutic agent for cancer according to the present
invention is not particularly recognized.
[0100] Pharmacological evaluation of the therapeutic agent for
cancer according to the present invention can be carried out in the
same manner as described above.
[0101] A seventh embodiment of the present invention relates to an
inducer of the cytotoxic T lymphocytes (CTLs) in the fifth
embodiment, which comprises a human major histo-compatibility
antigen (HLA)-A24-restricted antigen peptide represented by SEQ ID
NO: 1 or 2, or its functional derivative, as an active ingredient.
The present invention is based on the present inventors' finding
that the peptide consisting of an amino acid sequence represented
by SEQ ID NO: 1 or 2, identified by the present inventors as the
HLA-A24-restricted antigen peptide recognized by an
HLA-A24-restricted CTL induced against tumor antigen MAGE-A4 or
SAGE, is useful for inducing a CTL from human peripheral blood
lymphocytes.
[0102] Since the CTL inducer of the present invention comprises the
HLA-A24-restricted antigen peptide or the functional derivative
thereof as an active ingredient, the CTL inducer of the present
invention exhibits an excellent effect that the CTL in the fifth
embodiment can be induced by a simple technique. Further, the CTL
inducer of the present invention exhibits an excellent effect that
an action can be exerted specifically on organisms causing an
infectious disease or tumor cells in individuals, especially Asian
races, particularly Japanese.
[0103] The CTL inducer of the present invention is provided in the
form of a suspension of the HLA-A24-restricted antigen peptide or
the functional derivative thereof alone or in a mixture thereof
with other molecules (helper T cell antigen peptide and/or
adjuvant) in physiologic saline or phosphate buffered physiologic
saline, or in such a form that the peptide or the functional
derivative thereof alone, or in the mixture, can be suspended at
use.
[0104] The HLA-A24-restricted antigen peptide or the functional
derivative thereof used in the CTL inducer of the present invention
may be bound covalently to a higher fatty acid or a helper T cell
antigen peptide or may be formed into a complex with an HLA-A24
molecule. Desirably, the content of the HLA-A24-restricted antigen
peptide or the functional derivative thereof in the CTL inducer of
the present invention is 0.01% by weight or more, preferably 0.1%
by weight or more and 100% by weight or less, preferably 95% by
weight or less, per kind of HLA-A24-restricted antigen peptide or
the functional derivative thereof.
[0105] The CTL inducer of the present invention can be utilized as
an additive for a medium for in vitro proliferation of the CTL of
the present invention; in diagnosis of an immune-sensitized state
with T lymphocyte proliferation activity, delayed skin reaction or
the like as an indicator; and the like.
[0106] When the CTL inducer of the present invention is used, for
example, as an additive in a medium, it is desirable that the
amount of the CTL inducer of the invention used is, in terms of
peptide concentration in the medium, 1 ng/milliliter or more,
preferably 100 ng/milliliter or more and 100 .mu.g/milliliter or
less, preferably 1 .mu.g/milliliter or less, per type of the
HLA-A24-restricted antigen peptide or the functional derivative
thereof. The medium includes mediums such as serum-containing RPMI
or AIM-V.
[0107] The effect of the CTL inducer of the present invention on
induction of a CTL can be evaluated, for example, by measuring the
activity of the CTL induced with the inducer, in terms of
cytotoxicity on the target cells, proliferation reaction in the
presence of antigen-presenting cells, the amount of
antigen-specific cytokines released, or the like.
[0108] An eighth embodiment of the present invention relates to a
therapeutic agent for cancer, which comprises an HLA-A24-restricted
antigen peptide represented by SEQ ID NO: 1 or 2 or the functional
derivative thereof as an active ingredient. Since the therapeutic
agent for cancer according to the present invention comprises the
HLA-A24-restricted antigen peptide or the functional derivative
thereof as an active ingredient, the therapeutic agent for cancer
according to the present invention exhibits an excellent effect
that the agent can act specifically on tumor cells in individuals,
especially Asian races, particularly Japanese.
[0109] The therapeutic agent for cancer according to the present
invention is provided in the form of: [0110] 1) the antigen peptide
alone, [0111] 2) a mixture of the antigen peptide and a
pharmaceutically acceptable carrier and/or diluent, or [0112] 3)
the above-mentioned 1) or 2) to which a subsidiary agent was added
if necessary.
[0113] Here, the carrier includes, for example, human serum albumin
and the like.
[0114] The diluent includes, for example, a phosphate buffer,
distilled water, physiologic saline, and the like.
[0115] Furthermore, the subsidiary agent includes pharmaceutically
acceptable adjuvants and the like. The adjuvants include, but are
not limited to, for example, (a) Freund complete adjuvant, (b)
Freund incomplete adjuvant, (c) inorganic gel such as aluminum
hydroxide, alum, (d) surfactants such as lysolecithin, dimethyl
octadecyl ammonium bromide, (e) polyanions such as dextran sulfate,
poly IC, (f) peptides such as muramyl peptide, tuftsin, and (g)
monophosphoryl lipid (MPL) A manufactured by Ribi Corporation or
functional equivalents thereof.
[0116] When the therapeutic agent for cancer according to the
present invention is administered into humans, the therapeutic
agent for cancer according to the present invention may be
administered, for example, subcutaneously, intracutaneously or
intravenously with a syringe, or may be administered by transdermal
absorption through a mucosa by a method such as spraying.
[0117] The content of the HLA-A24-restricted antigen peptide or the
functional derivative thereof in the therapeutic agent for cancer
according to the present invention is desirably 0.01% by weight or
more, preferably 0.1% by weight or more, and 100% by weight or
less, preferably 95% by weight, per type of HLA-A24-restricted
antigen peptide or the functional derivative thereof.
[0118] Desirably, the dose of the therapeutic agent for cancer
according to the present invention per adult is, in terms of
peptide concentration, 0.1 .mu.g/kg or more, preferably 1 .mu.g/kg
or more, and 10 mg/kg or less, preferably 1 mg/kg or less, more
preferably 100 .mu.g/kg or less, per type of HLA-A24-restricted
antigen peptide or the functional derivative thereof. Here, upon
administration into humans, toxicity of the therapeutic agent for
cancer according to the present invention is not particularly
recognized.
[0119] A ninth embodiment of the present invention relates to a
tetramer for detecting a T cell receptor possessed by the CTL in
the fifth embodiment, which comprises an HLA-A24-restricted antigen
peptide represented by SEQ ID NO: 1 or 2 or a functional derivative
thereof. Such tetramer binds to TCR possessed by the CTL. The
tetramer of the present invention comprises the HLA-A24-restricted
antigen peptide or the functional derivative thereof, and thus
exhibits an excellent effect that the CTL can be monitored in
individuals, especially Asian races, particularly Japanese. The
present invention is based on the present inventor's finding that a
MAGE-A4- or SAGE-specific HLA-A24-restricted CTL can be detected by
a tetramer formed by, with biotin-streptavidin, tetramerizing
MHC/antigen peptide complexes prepared from the antigen peptide of
SEQ ID NO: 1 or 2.
[0120] A CTL is activated to cause various immune reactions, upon
recognition of, along with the MHC molecule, the antigen peptide
binding to the MHC molecule on the cell surface of an
antigen-presenting cell or a target cell by a complex of a T cell
receptor (TCR) and a CD3 molecule on the cell surface of the
CTL.
[0121] According to the tetramer of the present invention, the MHC
tetramer can be utilized for a different TCR, i.e., an
HLA-A24-restricted antigen peptide from MAGE-A4 or SAGE, and is
useful in analysis on behavior or functions of a CTL, particularly
for a method of specific measurement of a CTL having the TCR of
interest.
[0122] The tetramer of the present invention can be obtained, for
example, by tetramerizing complexes formed from the
HLA-A24-restricted antigen peptide of SEQ ID NO: 1 or 2 or the
functional derivative thereof, .beta.2 microglobulin and an HLA-A24
molecule by a biotin-streptavidin method.
[0123] The tetramer of the present invention binds to a T cell
receptor possessed by a CTL, and is thus useful, for example, in
that the tetramer can be utilized in a method for measuring a CTL
which can be carried out in a short time and is simple, by using
the formation of complex of the tetramer and a CTL as an indicator
in place of measurement of the cytotoxicity of a CTL. The tetramer
of the present invention is useful particularly in detection or
separation of a CTL in a sample such as PBMC containing only a
small amount of CTL.
[0124] The tetramer of the present invention can also be used in
ELISPOT (enzyme-linked immunospot) used for monitoring T
lymphocytes in the body of a patient, or for target cells in a
cytotoxicity test.
[0125] The tetramer of the present invention is also useful in
monitoring a CTL.
[0126] Hereinafter, the present invention will be described in more
detail by reference to the Examples, but the present invention is
not limited to the Examples.
EXAMPLE 1
Identification of HLA-A2402-Restricted CTL Epitope (Antigen
Peptide) Using HLA-A2402 Transgenic Mice
(1) Materials and Method
HHDA2402.+-..beta.2m-/- mice
[0127] A DNA construct (HHDA2402) containing an HLA-A2402 leader
sequence, human .beta.2 microglobulin, HLA-A2402 .alpha.1 and
.alpha.2 domains, an H-2D.sup.b .alpha.3 transmembrane domain, and
a cytoplasm domain was constructed. The resulting construct was
cloned into an expression vector pcDNA 3.1 (manufactured by
Invitrogen Corporation). The HHDA2402 construct (4 kb SalI-NotI
fragment) was injected into fertilized eggs of C57BL/6 mice, to
give HHDA2402-expressing mice. Then, the HHDA2404-expressing mice
were bred with .beta.2m-/- mice (manufactured by The Jackson
Laboratory). The resulting HHDA2402.+-..beta.2m.+-. were bred with
.beta.32m-/- mice to give HHDA 2402.+-..beta.2m-/- mice (referred
to hereinafter as "HHDA2402 mice").
(2) Cell Strain
[0128] TAP transporter-deficient strain T2 (J. Immunol., 167,
p.2529-2537 (2001)) was transfected with an HLA-A2402 cDNA to
prepare T2A24 strain. The following cell strains were also used in
preparation of CTL target cells: breast cancer cell strain R27
(A2402-negative), esophagus cancer strain KE-4 (A2402-positive),
esophagus cancer strain TE-10 (A2402-positive), chronic myelocytic
leukemia strain K562 (A2402-negative), lung cancer strain 11-18
(A2402-positive) and embryonic renal cell 293 (A2402-negative). The
above-mentioned R27, K562 and 293 were transfected with an
HLA-A2402 cDNA to prepare R27A24, K562A24 and 293A24. Human
B-lymphoblast (LCL) was prepared in a usual manner from
HLA-A2402-positive or negative cells derived from volunteers by
using EBV.
(3) Plasmid
[0129] cDNA of the full-length MAGE-A4, SAGE or EBNA-3A was cloned
into pcDNA 3.1.
(4) Immunization with a Gene Gun
[0130] Using Helios Gene Gun System (trade name, manufactured by
Bio-Rad Laboratories, Inc.), gold particles coated with plasmid DNA
were administered endoabdominally at a helium pressure of from 350
to 400 psi into HHDA2402 mice (6- to 8-week-old, female). The gold
particles were prepared according to a manufacturer's manual. After
2 weeks, booster administration was carried out. After 1 week,
spleen cells were collected.
(5) Preparation of Mouse CD8-Positive T Cells
[0131] One week after the final immunization, MACS system (trade
name, manufactured by Mitlenyi Biotec) using CD8 alpha (Lyt 2)
microbeads was used, to select CD8-positive T cells. The purity of
the resulting T cell fraction analyzed by flow cytometry was 95% or
more.
(6) ELISPOT Assay (Mice)
[0132] A 96-well nitrocellulose ELISPOT plate (trade name: MAHA
S4510, manufactured by Millipore Corporation) was incubated
overnight with 2 .mu.g/milliliter anti-mouse IFN -gamma mAb (trade
name: clone R4-6A2, manufactured by PharMingen Company) at
4.degree. C., to coat the plate. Each well was washed with
phosphate buffered physiologic saline (PBS). The well after washing
was blocked by incubation at 37.degree. C. for 2 hours in
FCS-containing RPMI1640 medium.
[0133] Fresh CD8-positive cells (1.times.10.sup.5 cells/well)
derived from the immunized mice and CD8-negative cells
(1.times.10.sup.6 cells/well) pulsed with various peptides were
seeded to each well and regulated so as to have a final volume of
200 .mu.l. Then, the cells were cultured at 37.degree. C. for 22
hours. Thereafter, the cells were washed sufficiently with PBS
containing 0.05 wt % Tween.TM. 20 (referred to as "PBS-Tween"). Two
hundreds microliters of 1.25 .mu.g/milliliter biotinylated
anti-mouse IFN-gamma mAb (trade name, manufactured by PharMingen
Company) was added to the cells after washing, and the cells were
then cultured overnight at 4.degree. C. The cells thus obtained
were washed with PBS-Tween. One hundred microliters of 1
.mu.g/milliliter streptavidin-alkaline phosphatase conjugate (trade
name, manufactured by Mabtech Ltd.) was added to the cells after
washing. Thereafter, the resulting mixture was incubated at room
temperature for 90 minutes, to carry out reaction. The resulting
product was washed with PBS-Tween and then stained with an alkaline
phosphatase conjugate substrate kit (trade name, manufactured by
Bio-Rad Laboratories, Inc.). Thereafter, the product after staining
was washed with distilled water to terminate the reaction. Then,
the plate was dried and spots were counted.
(7) Estimation of Epitope Peptides
[0134] Nine-residue peptide which might have an ability to bind to
HLA-A2402 were searched for by using HLA Peptide Binding
Predictions running on a web site of BioInformatics & Molecular
Analysis Section (BIMAS).
[0135] Each of five peptides derived from SAGE and ten peptides
derived from MAGE-A4 shown in Table 1 below were chemically
synthesized by a peptide synthesizer. TABLE-US-00001 TABLE 1
Antigen name Amino acid position Amino acid sequence SAGE 841-848
NYERIFILL (SEQ ID NO: 3) 776-784 LYKPDSNEF (SEQ ID NO: 4) 715-723
LYATVIHDI (SEQ ID NO: 2) 621-629 QYAAVTHNI (SEQ ID NO: 5) 250-258
TYNVPEEKM (SEQ ID NO: 6) MAGE-A4 239-247 VYGEPRKLL (SEQ ID NO: 7)
143-151 NYKRCFFVI (SEQ ID NO: 1) 271-279 EFLWGPRAL (SEQ ID NO: 8)
151-159 IFGKASESL (SEQ ID NO: 9) 113-121 KVDELAHFL (SEQ ID NO: 10)
283-291 SYVKVLEHV (SEQ ID NO: 11) 124-132 KYRAKELVT (SEQ ID NO: 12)
199-207 KTGLLIIVL (SEQ ID NO: 13) 301-309 AYPSLREAA (SEQ ID NO: 14)
43-51 SSPLVPGTL (SEQ ID NO: 15)
[0136] Here, the "amino acid position" in the table indicates the
position of each peptide in an amino acid sequence of SAGE or
MAGE-A4.
[0137] Furthermore, similarly, EB virus-derived EBNA 3A.sub.246-254
(SEQ ID NO: 16, RYSIFFDYM) and HER2-derived HER2.sub.63-71 (SEQ ID
NO: 17, TYLPTNASL) were synthesized. The purity of the peptides
used was 90% or more.
[0138] HHDA2402 mice were immunized with an expression plasmid
carrying EB virus-derived EBNA 3A gene by a gene gun. After
immunized twice, spleen-derived CD8+T cells were prepared.
CD8-negative cells pulsed with the EBNA 3A.sub.246-254 peptide were
used as target cells, to carry out ELISPOT assay. The results are
shown in FIG. 1A.
[0139] As a result, EBNA 3A.sub.246-254 peptide-specific CTLs were
detected as shown in FIG. 1A.
[0140] A tumor antigen MAGE-A4-derived candidate peptide and a
testis antigen SAGE-derived candidate peptide (Table 1 above) were
then subjected to ELISPOT assay in the same manner. The results are
shown in FIGS. 1B and 1C.
[0141] As a result, two peptides (MAGE-A4.sub.239-247 and
MAGE-A4.sub.143-151) were positive in MAGE-A4, as shown in FIG. 1B.
Similarly, as shown in FIG. 1C, two peptides (SAGE.sub.776-784 and
SAGE.sub.715-723) were positive in SAGE.
[0142] Wild-type C57BL6 mice were then used, to conduct the same
experiment as above. As a result, only MAGE-A4.sub.239-247 and
SAGE.sub.776-784 were positive.
[0143] Accordingly, MAGE-A4.sub.143-151 and SAGE.sub.715-723 were
identified as antigen peptides presented by HLA-A2402.
EXAMPLE 2
Preparation of HLA-2402 Antigen Peptide-Specific Human Cytotoxic T
Lymphocytes (CTLs) Using CD4-Positive PHA Blast Cells into which
mRNA was Introduced
(1) Preparation of mRNA
[0144] MAGE-A4 plasmid and SAGE plasmid were linearized. The
resulting products and T7 polymerase (trade name: mMESSAGE mMACHINE
T7 Kit, manufactured by Ambion, Inc.) were used, to conduct in
vitro transfer according to a manufacturer's manual. Thereafter,
the resulting product was polyadenylated with poly A polymerase
(trade name: Poly(A) Tailing Kit, manufactured by Ambion, Inc.)
according to a manufacture's manual. The resulting RNA was stored
at -80.degree. C. until use.
(2) Preparation of CD4-Positive Phytohemagglutinin (PHA) Blast
Cells
[0145] By using positive selection using MACS CD4 microbeads
(manufactured by Miltenyi Biotec), fresh CD4-positive cells were
separated from PBMC. The resulting CD4-positive cells were seeded
on a 24-well plate (manufactured by Coming Incorporated) at a cell
density of from 1 to 2.times.10.sup.6 cells/milliliter RPMI-1640
medium (composition: containing 25 mM Hepes, 10 wt % inactivated
human AB serum, 2 mM L-glutamine, 100 U/milliliter penicillin, 100
.mu.g/milliliter streptomycin) per well.
[0146] On the 0th day, PHA (trade name: HA15, manufactured by Murex
S.A.) was added to the medium in each well of the plate at a final
concentration of 10 .mu.g/milliliter. On the 3rd day, half amount
of the medium was exchanged with the above medium containing IL-2
(20 U/milliliter) and IL-7 (40 ng/milliliter). Exchange of the
medium was repeated every 3 days, whereby activated CD4-positive
cells were obtained. The mRNA was introduced by electroporation
into the activated CD4-positive cells at 14 to 28 days after
culture was initiated. The resulting cells were used below as
antigen-presenting cells.
(3) In Vitro Induction of Human CTLs Using mRNA-Introduced
CD4-Positive Blast Cells
[0147] MACS CD8 Microbeads (trade name, manufactured by Miltenyi
Biotec) were used, to separate CD8+ T cells from PBMC. The number
5.times.10.sup.5 cells of CD8+ T cells thus obtained were
stimulated with radiation (30 Gy)-irradiated 1.times.10.sup.5
mRNA-introduced CD4+ PHA blast cells in a 96-well round plate
(manufactured by Nunc Corporation). After 7 days, the CD8+ T cells
were stimulated again with radiation (30 Gy)-irradiated
1.times.10.sup.5 mRNA-introduced CD4+ PHA blast cells, and then
cultured in an IL-2 (20 IU/milliliter)-containing RPMI1640 medium
for 7 days.
(4) In Vitro Amplification of CTLs
[0148] To amplify the sensitized CD8+ T cell group containing the
antigen-specific CTLs, autologous LCLs (5.times.10.sup.6 cells)
into which the target antigen mRNA was introduced, autologous PBMCs
(2.5.times.10.sup.7 cells), and IL-2 (20 IU/milliliter) were added
to the sensitized CD8+ T cell group. The resulting mixture was
cultured in the absence of anti-CD3 antibody in a 25-milliliter
flask (manufactured by Corning Incorporated).
(5) Limiting Dilution
[0149] The CD8+ T cells were diluted to a density of 0.3 cell/well
in a 96-well round plate (manufactured by Nunc Corporation).
Furthermore, autologous PBMCs (5.times.10.sup.4 cells/well),
radiation-irradiated LCLs (1.times.10.sup.4 cells/well), IL-2 (20
IU/milliliter), and anti-CD3 mAb (30 ng/milliliter) were added to
the resulting dilution. The resulting mixture was cultured. As a
result, CD8+ T cells lyzing the antigen-presenting cells as the
target were proliferated in the presence of radiation-irradiated
PBMC, radiation-irradiated LCL, and anti-CD3 mAb.
(6) ELISPOT Assay (Human)
[0150] A 96-well nitrocellulose ELISPOT plate (trade name: MAHA
S4510, manufactured by Millipore Corporation) was coated by
overnight incubation at 4.degree. C. with 2 .mu.g/milliliter
antihuman IFN-.gamma. mAb (trade name: 1-D1K). Each well was washed
with PBS, and then blocked by incubation at 37.degree. C. for 2
hours with RPMI1640 medium containing 10 wt % human AB serum.
Effecter cells (2.times.10.sup.4 cells/well) and peptide-pulsed
T2A24 cells (5.times.10.sup.4 cells/well) were seeded to each well.
The cells in the well were cultured at 37.degree. C. for 18 hours.
The resulting cells were washed sufficiently with PBS-Tween. After
washing, the cells were incubated overnight with 1.25
.mu.g/milliliter biotinylated anti-mouse IFN-gamma mAb
(manufactured by PharMingen Company) at 4.degree. C. The resulting
product was washed with PBS-Tween. One hundred microliters of 1
.mu.g/milliliter streptavidin-alkaline phosphatase conjugate
(manufactured by Mabtech Ltd.) was added to the resulting product
and reacted by incubation at room temperature for 60 minutes. The
resulting product was washed with PBS-Tween and then stained with
an alkaline phosphatase conjugate substrate kit (trade name,
manufactured by Bio-Rad Laboratories, Inc.). The resulting product
was then washed with distilled water to terminate the reaction.
Then, the plate was dried and spots were counted.
(7) .sup.51Cr Release Cytotoxicity Assay
[0151] The cytotoxicity was evaluated in a usual manner as follows.
The target cells were labeled with 100 .mu.Ci (3.7.times.10.sup.6
Bq) .sup.51Cr. Then, 1.times.10.sup.4 target cells were reacted in
a 96-well V-bottomed plate (manufactured by Nunc Corporation) with
effecter cells at various density at 37.degree. C. After 5 hours,
100 .mu.l supernatant was collected and measured for radioactivity.
This measurement was conducted in triplicate, and the average of
specific lysis % in 3 wells was calculated on the basis of the
following formula: Specific cytotoxic activity (%)=[(measurement of
each well-minimum release level)/(maximum release level-minimum
release level)].times.100
[0152] In the above formula, the "minimum release level" is the
.sup.51Cr level of the well containing only the target cells and
K562 cells, and indicates the natural release level of .sup.51Cr
from the target cells. The "maximum release level" is indicative of
the .sup.51Cr release level upon disruption of the target cells
with surfactant Triton.TM. X-100 added to the cells.
[0153] According to the above-mentioned items (1) to (7), the
following evaluation was conducted.
[0154] CD8+ cells prepared from healthy normal humans were
sensitized in vitro with autologous CD4+ PHA blast cells into which
mRNA was introduced. The CD8+ cells were stimulated twice with the
autologous CD4+ PHA blast cells into which MAGE-A4 mRNA was
introduced. The results of ELISPOT assay are shown in FIG. 2.
[0155] As a result, MAGE-A4-specific bulk CTLs were obtained as
shown in panel A in FIG. 2. As shown in panel B in FIG. 2, cells
obtained by amplifying the MAGE-A4-specific bulk CTLs with
mRNA-introduced autologous LCL, autologous PBMC and IL-2 showed
reaction specific to T2A24 cells pulsed with MAGE-A4.sub.143-151
peptide.
[0156] Furthermore, MAGE-A4.sub.143-151-specific #2-28 cells
obtained by limiting dilution were analyzed by flow cytometry using
MAGE-A4.sub.143-151 tetramer and anti-CD8 antibody, and the results
are shown in panels A and B in FIG. 3. The cytotoxicity by
MAGE-A4.sub.143-151-specific #2-28 cells was examined, and the
results are shown in panels C and D in FIG. 3.
[0157] As a result, the #2-28 cells obtained by limiting dilution
were stained positively with the MAGE-A4.sub.143-151 tetramer, but
not stained with a tetramer used as a control, as shown in panels A
and B in FIG. 3. As shown in panel C in FIG. 3, the #2-28 cells
showed HLA-A2402-restricted cytotoxicity on the target cells pulsed
with the MAGE-A4.sub.143-151-peptide. Furthermore, as shown in
panel D in FIG. 3, the #2-28 cells showed cytotoxicity on the tumor
cell strain expressing both MAGE-A4 and HLA-A2402. This indicates
that the MAGE-A4.sub.143-151 peptide is presented not only against
the mRNA-introduced CD4+ PHA blast cells but also against the
HLA-2402-positive tumor cells by intracellular processing of
MAGE-A4 antigen.
[0158] Similarly, human bulk CTL cells were obtained by
sensitization twice with SAGE mRNA-introduced autologous CD4+PHA
blast cells. IFN-.gamma. ELISPOT assay was conducted using the
resulting human bulk CTL cells. As the target cells, T2A24 cells
pulsed with SAGE.sub.715-723 peptide or T2A24 cells pulsed with the
control peptide were used. The results are shown in FIG. 4A.
[0159] As a result, SAGE.sub.715-723-specific bulk CTLs were
inducted by stimulation twice with CD4+ blast cells into which
truncated SAGE mRNA was introduced, as shown in FIG. 4A.
[0160] The bulk CTLs were amplified in vitro. The resulting CTL was
analyzed by flow cytometry using SAGE.sub.715-723 tetramer and
anti-CD8 antibody. The results are shown in FIG. 4B.
[0161] As a result, the resulting bulk CTL was positive in staining
with SAGE.sub.715-723 A24 tetramer, as shown in FIG. 4B.
Accordingly, it was revealed that bulk CTLs contained CD8+ cells
positive to SAGE.sub.715-723 HLA-A24 tetramer.
[0162] T2A24 cells were then pulsed with SAGE.sub.715-723 peptide.
As the control, the cells were pulsed with HER2.sub.63-71 peptide.
Thereafter, the pulsed cells were subjected to ELISPOT assay. The
results are shown in FIG. 4C.
[0163] As a result, IFN-.gamma. was released only when T2A24 pulsed
with SAGE.sub.715-723 peptide was used as the target cell, as shown
in FIG. 4C.
[0164] SAGE.sub.715-723-specific HLA-A24 CTL cells #22 (#22 cells)
were prepared by limiting dilution. The resulting #22 cells were
analyzed by flow cytometry using SAGE.sub.715-723 HLA-A24 tetramer
and anti-CD8 antibody. The results are shown in FIG. 5A. The
cytotoxicity by the #22 cells was also examined. The results are
shown in FIG. 5C.
[0165] As a result, the #22 cells were positive to SAGE.sub.715-723
HLA-A24 tetramer, as shown in FIG. 5A. When the #22 cells were
co-cultured with 293-A2402 into which a plasmid carrying the full
length of SAGE gene was introduced, the #22 cells secreted
IFN-.gamma.. The #22 cells (1.times.10.sup.4 cells), and 293A24
cells (1.times.10.sup.4 cells) transformed with SAGE cDNA, were
cultured in a 96-well round plate for 18 hours. As a result,
IFN-.gamma. was released as shown in FIG. 5B. As shown in panels A
and B in FIG. 5C, the #22 cells showed cytotoxicity to both K562A24
and R27A24. Here, both the K562A24 and R27A24 expressed HLA-A2402
and SAGE. Furthermore, as shown in panel C in FIG. 5C, there was
specific cytotoxicity on A2402+LCL cells into which mRNA of SAGE
gene was introduced.
EXAMPLE 3
SAGE.sub.715-723-Specific CD8+ T Cells are Induced from
A2402-Positive Healthy Normal Humans with High Probability
In Vitro Induction of Human CTLs Using CD8- PBMC Pulsed with a
Peptide
[0166] The number 1.times.10.sup.7 of CD8-negative PBMC were pulsed
with 10 .mu.M peptide by incubation at room temperature for 1 hour
and at 37.degree. C. in 5% CO.sub.2 for 1 hour, in 200 .mu.l
RPMI1640 medium containing 25 mM Hepes, 10 wt % inactivated human
AB-positive serum, 2 mM L-glutamine, 100 U/milliliter penicillin,
and 100 .mu.g/milliliter streptomycin. The resulting cells were
used as antigen-presenting cells. The number 5.times.10.sup.5 of
CD8+ T cells that were separated were then stimulated by incubation
for from 10 to 12 days with 1.times.10.sup.6 cells of the
peptide-pulsed CD8- PBMC. On Days 1, 4 and 7, half amount of the
medium was exchanged with fresh one, and human IL-2 (20
IU/milliliter) and IL-7 (50 ng/milliliter) were added thereto.
Culturing for induction was conducted in 200 .mu.l RPMI1640 medium
(containing 25 mM Hepes, 10 wt % inactivated human AB serum, 2 mM
L-glutamine, 100 U/milliliter penicillin, 100 .mu.g/milliliter
streptomycin).
[0167] It was examined whether an HLA-A2402-restricted
SAGE.sub.715-723-specific CTL was induced in vitro by once
stimulating CD8+ cells from HLA-A2402-positive healthy normal
volunteers, with CD8-negative PBMC pulsed with SAGE.sub.715-723
peptide.
[0168] As a result, SAGE.sub.715-723 HLA-A24 tetramer-positive T
cells were detected in three out of six HLA-A2402+ healthy normal
humans at 10 days after mixed lymphocyte reaction in vitro. FIGS.
6A to 6D each show one example of the analysis results.
EXAMPLE 4
Preparation of a Tetramer and Flow Cytometry Analysis
[0169] HLA-A2402 heavy chain and .beta.2-microglobulin were
expressed as an insoluble polymer in Escherichia coli. Here, in
order to add a sequence serving as a substrate for biotinylating
enzyme BirA to the C-terminal of the HLA-A2402 heavy chain, a
corresponding polynucleotide was added to the nucleic acid encoding
the HLA-A2402 heavy chain. A monomer
HLA/.beta.2-microglobulin/peptide complex was prepared in vitro by
folding the insoluble polymer in the presence of
MAGE-A4.sub.143-151 peptide or SAGE.sub.715-723. The resulting
product was biotinylated with a recombinant BirA enzyme
(manufactured by Avidity) and tetramerized with
phycoerythrin-labelled streptavidin (manufactured by Molecular
Probes Inc.) to give an MHC/peptide tetramer.
[0170] In staining, sensitized CD8+ T cells were reacted with 20
.mu.g/milliliter tetramer at 37.degree. C. for 30 minutes, and then
reacted with a tricolor anti-CD8 monoclonal antibody (trade name,
Caltag Laboratories, Burlingame, Calif., USA) on ice for 15
minutes. After washing, the stained cells were analyzed by flow
cytometry (trade name: FACSCalibur, manufactured by Becton
Dickinson, and Company). As a result, it was confirmed that the
prepared tetramer was bound to the sensitized CD8+ T cells.
EQUIVALENTS
[0171] The present invention may be embodied in other specific
forms without departing from the spirit or essential
characteristics thereof. The present invention is therefore to be
considered in all respects as illustrative and not restrictive. The
scope of the invention is indicated by the appended claims rather
than by the foregoing description. Furthermore, all changes which
come within the meaning and range of equivalency of the claims are
therefore intended to be embraced therein.
Sequence CWU 1
1
17 1 9 PRT Homo sapiens 1 Asn Tyr Lys Arg Cys Phe Pro Val Ile 1 5 2
9 PRT Homo sapiens 2 Leu Tyr Ala Thr Val Ile His Asp Ile 1 5 3 9
PRT Homo sapiens 3 Asn Tyr Glu Arg Ile Phe Ile Leu Leu 1 5 4 9 PRT
Homo sapiens 4 Leu Tyr Lys Pro Asp Ser Asn Glu Phe 1 5 5 9 PRT Homo
sapiens 5 Gln Tyr Ala Ala Val Thr His Asn Ile 1 5 6 9 PRT Homo
sapiens 6 Thr Tyr Asn Val Pro Glu Glu Lys Met 1 5 7 9 PRT Homo
sapiens 7 Val Tyr Gly Glu Pro Arg Lys Leu Leu 1 5 8 9 PRT Homo
sapiens 8 Glu Phe Leu Trp Gly Pro Arg Ala Leu 1 5 9 9 PRT Homo
sapiens 9 Ile Phe Gly Lys Ala Ser Glu Ser Leu 1 5 10 9 PRT Homo
sapiens 10 Lys Val Asp Glu Leu Ala His Phe Leu 1 5 11 9 PRT Homo
sapiens 11 Ser Tyr Val Lys Val Leu Glu His Val 1 5 12 9 PRT Homo
sapiens 12 Lys Tyr Arg Ala Lys Glu Leu Val Thr 1 5 13 9 PRT Homo
sapiens 13 Lys Thr Glu Leu Leu Ile Ile Val Leu 1 5 14 9 PRT Homo
sapiens 14 Ala Tyr Pro Ser Leu Arg Glu Ala Ala 1 5 15 9 PRT Homo
sapiens 15 Ser Ser Pro Leu Val Pro Gly Thr Leu 1 5 16 9 PRT
Epstein-Barr virus 16 Arg Tyr Ser Ile Phe Phe Asp Tyr Met 1 5 17 9
PRT Homo sapiens 17 Thr Tyr Leu Pro Thr Asn Ala Ser Leu 1 5
* * * * *