U.S. patent application number 10/450255 was filed with the patent office on 2005-03-03 for transgenic animal expressing hla-a24 and utilization thereof.
Invention is credited to Gotoh, Masashi.
Application Number | 20050050580 10/450255 |
Document ID | / |
Family ID | 26605731 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050050580 |
Kind Code |
A1 |
Gotoh, Masashi |
March 3, 2005 |
Transgenic animal expressing hla-a24 and utilization thereof
Abstract
The present invention relates to a non-human transgenic mammal
which has had an HLA-A24 gene introduced and in which CTLs are
induced when stimulated by an HLA-A24-binding antigen, a method of
screening therapeutic or preventive agents for tumors or virus
infections comprising administering a test substance to said
transgenic non-human mammal and assaying and evaluating whether
CTLs specific for the test substance are induced, an
HLA-A24-binding tumor antigen peptide of PSA origin selected by
said screening method, a chimera DNA (DNA construct) useful in the
generation of said non-human transgenic mammal, a host cell
transformed by said chimera gene and use thereof, and the like.
Inventors: |
Gotoh, Masashi; (Osaka-fu,
JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
26605731 |
Appl. No.: |
10/450255 |
Filed: |
June 12, 2003 |
PCT Filed: |
December 12, 2001 |
PCT NO: |
PCT/JP01/10885 |
Current U.S.
Class: |
800/8 ;
800/18 |
Current CPC
Class: |
C12N 15/8509 20130101;
A01K 2267/03 20130101; A01K 2227/105 20130101; C07K 2319/00
20130101; A01K 2267/0393 20130101; A01K 67/0275 20130101; C07K
14/70539 20130101; A61P 31/12 20180101; A61P 35/00 20180101; A01K
2217/05 20130101; A61P 43/00 20180101 |
Class at
Publication: |
800/008 ;
800/018 |
International
Class: |
A01K 067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 13, 2000 |
JP |
2000-378556 |
Sep 6, 2001 |
JP |
2001-269746 |
Claims
1. A non-human transgenic mammal, which has had an HLA-A24 gene
introduced and in which CTLs are induced when stimulated by an
HLA-A24-binding antigen.
2. The non-human transgenic mammal according to claim 1, which
comprises the HLA-A24 gene homozygously.
3. The non-human transgenic mammal according to claim 1 or 2,
wherein the HLA-A24 gene is a chimera gene comprising the .alpha.1
and .alpha.2 domains of HLA-A24 gene and the .alpha.3 domain of
mouse MHC class I gene.
4. The non-human transgenic mammal according to any one of claims
1-3, wherein the HLA-A24 gene is HLA-A2402 gene.
5. The non-human transgenic mammal according to any one of claims
1-4, wherein the HLA-A24 gene comprises a nucleotide sequence
encoding the amino acid sequence shown in SEQ ID NO: 3.
6. The non-human transgenic mammal according to any one of claims
1-4, wherein the HLA-A24 gene comprises the nucleotide sequence
shown in SEQ ID NO: 2.
7. The non-human transgenic mammal according to any one of claims
1-4, wherein the HLA-A24 gene comprises a nucleotide sequence shown
in SEQ ID NO: 1.
8. The non-human transgenic mammal according to any one of claims
1-4, wherein the HLA-A24 gene is a DNA which hybridizes to the
complement of a nucleotide sequence set forth in any one of claims
5-7 under stringent conditions, and the expression product of said
DNA is capable of binding to HLA-A24-binding antigen peptide and
inducing CTLs.
9. The non-human transgenic mammal according to any one of claims
1-8, wherein the non-human mammal is mouse.
10. The non-human transgenic mammal according to claim 9 wherein
the mouse is of C57BL/6 mouse strain.
11. A method of screening an agent inducing antigen-specific CTLs,
which comprises administering a test substance to a transgenic
non-human mammal set forth in any one of claims 1-10, and assaying
and determining whether CTLs specific for the test substance are
induced.
12. A method of screening a therapeutic or preventive agent for
tumors or virus infections, which comprises administering a test
substance to a transgenic non-human mammal set forth in any one of
claims 1-10, and assaying and determining whether CTLs specific for
the test substance are induced.
13. The method of screening according to claim 11 or 12, which uses
transformants transformed by and expressing the HLA-A24 gene that
the transgenic non-human mammal set forth in any one of claims 1-10
contains as a target cell for assaying and determining whether CTLs
specific for the test substance are induced.
14. A transformant transformed by and expressing the HLA-A24 gene
that the transgenic non-human mammal set forth in any one of claims
3-10 contains.
15. A method of screening an antigen-specific CTL inducing agent,
which uses the transformant set forth in claim 14.
16. A method of screening a therapeutic or preventive agent for
tumors or virus infections, which uses the transformant set forth
in claim 14.
17. An isolated DNA comprising the nucleotide sequence shown in SEQ
ID NO: 1.
18. An isolated DNA comprising the nucleotide sequence shown in SEQ
ID NO: 2.
19. An isolated DNA comprising the nucleotide sequence shown in SEQ
ID NO: 3.
20. An isolated DNA, which hybridizes to the complement of a DNA
set forth in any one of claims 17-19 under stringent conditions,
and of which expression product is capable of binding to an
HLA-A24-binding antigen peptide and inducing CTLs.
21. The isolated DNA according to claim 20, which comprises
nucleotide sequences each encoding amino acid Nos. 25-114, 115-206
and 207-298 of the amino acid sequence shown in SEQ ID NO: 3.
22. The isolated DNA according to claim 20, which rises
polynucleotides each having the sequences corresponding to
nucleotide Nos. 72-339, 342-615 and 618-891 of the nucleotide
sequence shown in SEQ ID NO: 2.
23. An expression vector comprising a DNA set forth in any one of
claims 17-22.
24. A transformant transformed by the expression vector set forth
in claim 23.
25. A method of screening an antigen-specific CTL inducing agent,
which uses the transformant set fort in claim 24.
26. A method of screening a therapeutic or preventive agent for
tumors or virus infections, which uses the transformant set fort in
claim 24.
27. An HLA-A24-binding tumor antigen peptide of PSA origin, which
is obtainable by the screening method set fort in any one of claims
11-13, or a derivative thereof having functionally equivalent
characteristics.
28. The tumor antigen peptide according to claim 27, which
comprises the amino acid sequence shown in SEQ ID NO: 15, or a
derivative thereof having functionally equivalent
characteristics.
29. The tumor antigen peptide derivative according to claim 28,
which comprises the amino acid sequence shown in SEQ ID NO: 17.
30. A CTL inducing agent comprising as an active ingredient the
tumor antigen peptide or a derivative thereof set forth in any one
of claims 27-29.
31. A DNA encoding a tumor antigen peptide or a derivative thereof
set forth in any one of claims 27-29.
32. A recombinant DNA comprising the DNA set forth in claim 31.
33. A polypeptide obtainable by expressing the recombinant DNA set
forth in claim 32.
34. A CTL inducing agent comprising as an active ingredient the
recombinant DNA set forth in claim 32 or a polypeptide set forth in
claim 33.
35. An antigen presenting cell presenting a complex between an
HLA-A24 antigen and a tumor antigen peptide or a derivative thereof
set forth in any one of claims 27-29.
36. A CTL inducing agent comprising as an active ingredient the
antigen-presenting cell set forth in claim 35.
37. A CTL which recognizes a complex between an HLA-A24 antigen and
a tumor antigen peptide or a derivative thereof set forth in any
one of claims 27-29.
38. A therapeutic or preventive agent for tumors comprising as an
active ingredient the tumor antigen peptide or a derivative thereof
set forth in any one of claims 27-29, the recombinant DNA set forth
in claim 32, the polypeptide set forth in claim 33, the antigen
presenting cell set forth in claim 35 or the CTL set forth in claim
37.
39. A diagnosing agent comprising a tumor antigen peptide or a
derivative thereof set forth in any one of claims 27-29.
Description
TECHNICAL FIELD
[0001] The present invention relates to transgenic animals
expressing HLA-A24 and utilization thereof. More specifically, the
present invention relates to transgenic animals, into which an
HLA-A24 gene has been introduced and in which cytotoxic T
lymphocytes are induced when stimulated by an HLA-A24-binding
antigen (i.e., HLA-A24-restricted antigen), a method of screening
therapeutic or preventive agents for tumors or virus infections
using said transgenic animals, HLA-A24-binding tumor antigen
peptides of PSA origin selected by said method, recombinant DNA
constructs useful in the preparation of transgenic mice and the use
thereof, and the like.
BACKGROUD ART
[0002] The cellular immunity, particularly cytotoxic T lymphocyte
(hereinafter, referred to as "CTL"), plays an important role in the
removal of cancer cells or virus-affected cells from living body.
CTLs recognize a complex between a peptide fragment of antigen
proteins originated from cancer, virus etc. and an MHC class I
molecules which is referred to as "HLA" in the case of human, via
T-cell receptors, thereby specifically damaging cancer cells or
virus-affected cells or activating immune system through the
production of various cytokines. A peptide fragment which forms a
complex with an MHC class I molecule is referred to as an antigen
peptide and is generally about 8-11 amino acids in length. The
extracellular domain of an MHC class I molecule consists of
.alpha.1, .alpha.2 and .alpha.3 domains, wherein the .alpha.1 and
.alpha.2 domains participate in the formation of peptide binding
groove and the .alpha.3 domain in the binding with coreceptor CD8
molecule expressed on the surface of CTLs.
[0003] Typical examples of tumor antigen protein recognized by CTL
comprise those described in Table 1 of Immunity, vol. 10: 281,
1999. Specific examples include melanocyte tissue-specific proteins
such as gp100 (J. Exp. Med., 179:1005, 1994) and MART-1 (Proc.
Natl. Acad. Sci. USA, 91:3515, 1994), melanosome antigens such as
tyrosinase (J. Exp. Med., 178:489, 1993), and, as tumor antigen
proteins other than melanomas, tumor markers such as HER2/neu (J.
Exp. Med., 181:2109, 1995), CEA (J. Natl. Cancer. Inst., 87: 982,
1995) and PSA (J. Natl. Cancer. Inst., 89: 293, 1997), and SART-1
(J. Exp. Med., 187: 277, 1998) and cyclophilin B (Proc. Natl. Acad.
Sci. USA, 88: 1903, 1991) originated from squamous cancer, and the
like.
[0004] The so-called "cancer vaccine therapy" is considered to be
useful in the treatment or prevention of cancer or virus
infections, etc., which comprises administering to a subject any of
tumor antigen proteins or peptides, or DNAs encoding the same, or
virus-originated antigen proteins or peptides so as to enhance
specific T cells in vivo. The results of clinical studies conducted
with a tumor antigen peptide originated from MAGE-3, which is a
tumor antigen protein being overexpressed in melanoma, lung cancer
or head and neck cancer, showed significance of vaccine therapy in
the tumor rejection (Int. J. Cancer, 80: 219,1999).
[0005] In the course of development of agents for vaccine therapy,
evaluation/determination of a candidate agent for in vivo
usefulness in the vaccine therapy cannot be conducted using
pure-line mouse commonly used as experimental animals, and requires
transgenic animal expressing HLA (hereinafter, it may be referred
to as "animal model for human"). That is, human antigen peptide
usable in the vaccine therapy should be such peptide that can
induce specific immune response when presented to HLA which is a
human-specific MHC class I molecule. Accordingly, non-human
experimental animals lacking HLA are unavailable for in vivo
evaluation of agents for vaccine therapy directed to treatment of
human beings. As mentioned above, transgenic animals expressing HLA
(animal models for human) are essential for evaluation of
usefulness of agents for vaccine therapy.
[0006] Although the construction of transgenic animals is
technically understood from various basic textbooks, it is not easy
to obtain animals (animal models for human) having desired
function. There are many cases where intended animal models could
not be obtained; for example, a transgene was poorly or never
expressed, a transgenic animal lacked desirable functions as an
animal model even if a transgene was expressed, etc. Therefore, the
construction and/or establishment of animal models for human is
considered to be still an unpredictable technique.
[0007] As for animal models for HLA, it is considered to be
preferred that, when the animal is mouse, the mouse model for human
carries a chimera HLA molecule as a transgene, wherein the .alpha.3
domain of HLA gene is replaced by the corresponding domain of mouse
MHC class I molecule, and whereby CTLs of animal models can
effectively recognize a complex of HLA and antigen peptide.
However, there have been no successful examples regarding animal
models for human into which a chimera HLA molecule has been
introduced except for two reports, i.e., an animal model for
HLA-A2.1 (Eur. J. Immunol., 26: 97, 1996; and "HLA-A2.1/K.sup.b
transgenic mouse" in J. Exp. Med., 185: 2034, 1997) and an animal
model for HLA-A11 ("HLA-A11/K.sup.btransgenic mouse" in J.
Immunol., 159:4753, 1997). These publications showed that the
presence or absence of CTL induction in response to the
administration of an agent for vaccine therapy in transgenic mice
highly correlates with that in human, indicating that the
transgenic mice are useful as animal models for human.
[0008] The above-mentioned HLA-A2.1 and HLA-A11 are HLA haplotypes
dominant in Westerners. As a haplotype that is commonly shared by
many Asians including 60% of Japanese, HLA-A24 different from these
HLAs is well known. If a mouse model carrying a chimera HLA
transgene for HLA-A24 is established, it will become possible to
conduct in vivo evaluation of HLA-A24 restricted agent for vaccine
therapy widely applicable to Asians, which is expected to greatly
contribute to the development of pharmaceutical preparations in
this field. However, there have been no reports regarding animal
models for HLA-A24, and establishment thereof has been demanded.
Such animal models are useful in not only evaluation of vaccine
preparations but also screening thereof.
[0009] The tumor antigen protein PSA is a glycoprotein that is
specifically expressed even in normal prostatic epithelial cells
and has a blood half-life of 2-3 day. As the canceration
progresses, the expression level of PSA increases to give the tumor
antigen protein. Accordingly, PSA is used as a marker in diagnosis
of cancer, wherein the PSA level in serum of a patient is measured.
The prostatic cancer is placed 1st to 3rd place regarding both the
incidence and mortality rate among malignant male tumors in many
European countries and the United States, and tends to increase in
Japan in the late years. As the prostatic cancer is androgen
sensitive, treatment is conducted aiming at removal of androgen
(endocrine therapy). However, more than half of the cases of
prostatic carcinoma progress to recrudescence carcinoma (i.e.,
androgen refractory carcinoma) in 5 years, even if it responds to
and are controlled by endocrine therapy in the beginning of
treatment. Such decrease of androgen dependency greatly hampers
endocrine therapy of prostatic cancer. Accordingly, the development
of vaccine therapy with an antigen peptide of PSA origin is
demanded.
[0010] An antigenic peptide region of HLA-A2 type has recently been
identified in PSA (J. Natl. Cancer. Inst., 89: 293, 1997). However,
there have been no reports showing the existence of an HLA type A24
(HLA-A2402)-binding antigen peptide region. Incidentally, many
subtypes belonging to MHC class I molecule exist and it is known
that there are certain rules (binding motifs) in the amino acid
sequence of antigen peptide having a binding ability. In the
binding motif for HLA-A24 above, the second amino acid is tyrosine,
phenylalanine, methionine or tryptophan, and the C-terminal amino
acid is phenylalanine, leucine, isoleucine, tryptophan or
methionine. However, peptides identified on the basis of said motif
do not necessarily have the immunogenicity. That is, since an
antigen peptide is generated through the intracellular processing
of a tumor antigen protein, a peptide not having been produced by
in vivo processing cannot be an antigen peptide. Furthermore, even
if an amino acid region on a tumor antigen protein identified on
the basis of the binding motif is intracellularly generated as a
peptide, such a tumor antigen protein may be anergic by reason of,
for example, it per se exists in a living organism. As described
above, a simple prediction based on the binding motif for a given
HLA type is insufficient to identify an antigen peptide, and, from
this viewpoint, establishment of a mouse model for human which
enables to evaluate an HLA-A24-binding antigen peptides has been
demanded.
DISCLOSURE OF INVENTION
[0011] One of purposes of the present invention is to provide a
novel animal model for HLA-A24 that enables in vivo evaluation of
antigenic proteins or peptides and to identify a novel
HLA-A24-binding antigen peptide using said animal model, and the
like. Specifically, the present invention is aimed at providing a
transgenic animal having had an HLA-A24 gene introduced, in which
CTLs are induced in response to stimulation with HLA-A24-binding
antigen, a method of screening a therapeutic or preventive agent
for tumors or virus infections using said transgenic animal, a
PSA-originated HLA-A24-binding tumor antigen peptide, etc. selected
by the said screening method, and the like.
[0012] The present inventors have intensively studied for
constructing transgenic animals (animal models for human) so that
in vivo evaluation of HLA-A24-binding antigenic proteins or
peptides can become possible.
[0013] When the present inventors attempted construction of a given
transgenic mouse, only one of two literatures above, which is
related to HLA-A2.1 (Eur. J. Immunol., 26:97 (1996)), seemed to
serve as a useful reference. However, the method described in this
literature was inappropriate for achieving the purpose of the
present invention. As mentioned above, it has been known a method
for generating animal models for HLA by introducing a gene of
chimera HLA molecule wherein the .alpha.3 domain of HLA is replaced
by the same domain of mouse MHC class I molecule (H-2 K.sup.b,
etc.). To obtain an appropriate chimera molecule, restriction sites
suited for the ligation with an H-2K.sup.b gene must exist on an
HLA molecule. In contrast to HLA-A2.1 gene, HLA-A24 gene does not
contain appropriate restriction sites suited for ligating the
.alpha.1 and .alpha.2 domains of said HLA-A24 gene to the .alpha.3
domain of mouse MHC (H-2K.sup.b) on the genome, and, therefore, an
appropriate artificial restriction site had to be constructed.
Furthermore, it was totally unpredictable whether or not a genomic
DNA of chimera HLA-A24 containing such an artificial sequence is
spliced normally to render the expression of the intended chimera
HLA-A24 molecule, when introduced into an individual. It was also
unclear whether or not the generated transgenic animals can express
normally the chimera HLA-A24 molecule and serve as animal models
for human having the desired CTL inducing ability. In addition,
most of the gene sequence of intron 3 region of mouse H-2K.sup.b,
which is responsible for the ligation with HLA-A24 gene, was
unknown and said gene was hardly available.
[0014] Under these circumstances, the present inventors succeeded
in the cloning of regions on both the HLA-A24 (HLA-A2402) and mouse
H-2K.sup.b genes necessary for the preparation of chimera molecule,
ligating these sequences through artificial restriction sites, and
constructing a chimera HLA gene (HLA-A2402/K.sup.b gene). The
present inventors then attempted to construct an HLA-A2402/K.sup.b
transgenic mouse by microinjecting said chimera gene into a
fertilized egg of C57BL/6 mouse strain. From 800 or more fertilized
eggs undergone microinjection, 8 lines of transgenic mouse were
obtained; however, only one line (04-1 line) was revealed to be a
transgenic mouse of homo-type that is homozygous for HLA-A24 gene
(chimera gene) of the present invention. When the mouse of 04-1
line was stimulated with a known HLA-A24-binding antigen peptide,
it showed a satisfactory CTL inducing activity and identified as
the objective HLA-A24 transgenic mouse of the present
invention.
[0015] The present inventors have also succeeded in identifying an
HLA-A24-binding tumor antigen peptide of PSA origin that has in
vivo antigenicity.
[0016] The present invention has been established on the basis of
these findings. Thus, the present invention can be summarized as
follows:
[0017] (1) A non-human transgenic mammal, which has had an HLA-A24
gene introduced and in which CTLs are induced when stimulated by an
HLA-A24-binding antigen;
[0018] (2) The non-human transgenic mammal according to (1), which
comprises the HLA-A24 gene homozygously;
[0019] (3) The non-human transgenic mammal according to (1) or (2),
wherein the HLA-A24 gene is a chimera gene comprising the .alpha.1
and .alpha.2 domains of HLA-A24 gene and the .alpha.3 domain of
mouse MHC class I gene;
[0020] (4) The non-human transgenic mammal according to any one of
(1)-(3), wherein the HLA-A24 gene is HLA-A2402 gene;
[0021] (5) The non-human transgenic mammal according to any one of
(1)-(4), wherein the HLA-A24 gene comprises a nucleotide sequence
encoding the amino acid sequence shown in SEQ ID NO: 3;
[0022] (6) The non-human transgenic mammal according to any one of
(1)-(4), wherein the HLA-A24 gene comprises the nucleotide sequence
shown in SEQ ID NO: 2;
[0023] (7) The non-human transgenic mammal according to any one of
(1)-(4), wherein the HLA-A24 gene comprises a nucleotide sequence
shown in SEQ ID NO: 1;
[0024] (8) The non-human transgenic mammal according to any one of
(1)-(4), wherein the HLA-A24 gene is a DNA which hybridizes to the
complement of a nucleotide sequence set forth in any one of (5)-(7)
above under stringent conditions, and the expression product of
said DNA is capable of binding to HLA-A24-binding antigen peptide
and inducing CTLs;
[0025] (9) The non-human transgenic mammal according to any one of
(1)-(8), wherein the non-human mammal is mouse;
[0026] (10) The non-human transgenic mammal according to (9)
wherein the mouse is of C57BL/6 mouse strain;
[0027] (11) A method of screening an agent inducing
antigen-specific CTLs, which comprises administering a test
substance to a transgenic non-human mammal set forth in any one of
(1)-(10) above, and assaying and determining whether CTLs specific
for the test substance are induced;
[0028] (12) A method of screening a therapeutic or preventive agent
for tumors or virus infections, which comprises administering a
test substance to a transgenic non-human mammal set forth in any
one of (1)-(10) above, and assaying and determining whether CTLs
specific for the test substance are induced;
[0029] (13) The method of screening according to (11) or (12),
which uses a transformant transformed by and expressing the HLA-A24
gene that the transgenic non-human mammal set forth in any one of
(1)-(10) contains as a target cell for assaying and determining
whether CTLs specific for the test substance are induced;
[0030] (14) A transformant transformed by and expressing the
HLA-A24 gene which the transgenic non-human mammal set forth in any
one of (3)-(10) contains;
[0031] (15) A method of screening an antigen-specific CTL inducing
agent, which uses the transformant set forth in (14);
[0032] (16) A method of screening a therapeutic or preventive agent
for tumors or virus infections, which uses the transformant set
forth in (14);
[0033] (17) An isolated DNA comprising the nucleotide sequence
shown in SEQ ID NO: 1;
[0034] (18) An isolated DNA comprising the nucleotide sequence
shown in SEQ ID NO: 2;
[0035] (19) An isolated DNA comprising the nucleotide sequence
shown in SEQ ID NO: 3;
[0036] (20) An isolated DNA, which hybridizes to the complement of
a DNA set forth in any one of (17)-(19) above under stringent
conditions and of which expression product is capable of inducing
CTLs when bound to an HLA-A24-binding antigen peptide;
[0037] (21) The isolated DNA according to (20), which comprises
nucleotide sequences each encoding amino acid Nos. 25-114, 115-206
and 207-298 of the amino acid sequence shown in SEQ ID NO: 3;
[0038] (22) The isolated DNA according to (20), which comprises
polynucleotides each having the sequences corresponding to
nucleotide Nos. 72-339, 342-615 and 618-891 of the nucleotide
sequence shown in SEQ ID NO: 2;
[0039] (23) An expression vector comprising a DNA set forth in any
one of (17)-(22) above;
[0040] (24) A transformant transformed by the expression vector set
forth in (23) above;
[0041] (25) A method of screening an antigen-specific CTL inducing
agent, which uses the transformant set fort in (24) above;
[0042] (26) A method of screening a therapeutic or preventive agent
for tumors or virus infections, which uses the transformant set
fort in (24) above;
[0043] (27) An HLA-A24-binding tumor antigen peptide of PSA origin,
which is obtainable by the screening method set fort in any one of
(11)-(13) above, or a derivative thereof having functionally
equivalent characteristics;
[0044] (28) The tumor antigen peptide according to (27), which
comprises the amino acid sequence shown in SEQ ID NO: 15, or a
derivative thereof having functionally equivalent
characteristics;
[0045] (29) The tumor antigen peptide according to (28), which
comprises the amino acid sequence shown in SEQ ID NO: 17;
[0046] (30) A CTL inducing agent comprising as an active ingredient
the tumor antigen peptide or a derivative thereof set forth in any
one of (27)-(29) above;
[0047] (31) A DNA encoding a tumor antigen peptide or a derivative
thereof set forth in any one of (27)-(29) above;
[0048] (32) A recombinant DNA comprising the DNA set forth in (31)
above;
[0049] (33) A polypeptide obtainable by expressing the recombinant
DNA set forth in (32) above;
[0050] (34) A CTL inducing agent comprising as an active ingredient
the recombinant DNA set forth in (32) above or a polypeptide set
forth in (33) above;
[0051] (35) An antigen presenting cell presenting a complex between
an HLA-A24 antigen and a tumor antigen peptide or a derivative
thereof set forth in any one of (27)-(29) above;
[0052] (36) A CTL inducing agent comprising as an active ingredient
the antigen presenting cell set forth in (35) above;
[0053] (37) A CTL which recognizes a complex between an HLA-A24
antigen and a tumor antigen peptide or a derivative thereof set
forth in any one of (27)-(29) above;
[0054] (38) A therapeutic or preventive agent for tumors comprising
as an active ingredient the tumor antigen peptide or a derivative
thereof set forth in any one of (27)-(29) above, the recombinant
DNA set forth in (32) above, the polypeptide set forth in (33)
above, the antigen presenting cell set forth in (35) above or the
CTL set forth in (37) above; and
[0055] (39) A diagnosing agent comprising a tumor antigen peptide
or a derivative thereof set forth in any one of (27)-(29)
above.
BRIEF DESCRIPTION OF DRAWINGS
[0056] FIG. 1 is a schematic diagram showing the process for
constructing an H-2K.sup.b genomic DNA used for constructing the
chimera gene (HLA-A2402/K.sup.b gene) of the present invention.
[0057] FIG. 2 is a schematic diagram showing the process for
constructing the HLA-A2402/K.sup.b gene of the present invention.
Plasmid H-2K.sup.b#20/26 containing H-2K.sup.b genomic DNA was
cleaved at BamHI site and the isolated BamHI fragment was ligated
into BglII-cleaved plasmid HLA-A2402#1 containing HLA-A24 genomic
DNA to construct the recombinant plasmid HLA-A2402/K.sup.b. Plasmid
non-A2402/K.sup.b was generated by ligating the BamHI fragment of
plasmid H-2K.sup.b#20/26 into plasmid HLA-A2402#1 cleaved at BglII
site in the opposite orientation.
[0058] FIG. 3 (A)-(P) is an alignment of HLA-A2402/K.sup.b genomic
sequence described in SEQ ID NO: 1 and HLA-A2402/K.sup.b cDNA
sequence described in SEQ ID NO: 2.
[0059] The Relationship between sequences in FIG. 3 is shown
below.
1 HLA-A2402/K.sup.b genomic HLA-A2402/K.sup.bcDNA sequence
(position) sequence (position) FIG. 3(A) No. 1-250 FIG. 3(B) No.
251-500 FIG. 3(C) No. 501-750 No. 1-98 FIG. 3(D) No. 751-1000 No.
99-343 FIG. 3(E) No. 1001-1250 No. 344-357 FIG. 3(F) No. 1251-1500
No. 358-607 FIG. 3(G) No. 1501-1750 No. 608-619 FIG. 3(H) No.
1751-2000 FIG. 3(I) No. 2001-2250 No. 620-816 FIG. 3(J) No.
2251-2500 No. 817-939 FIG. 3(K) No. 2501-2750 No. 940-1015 FIG.
3(L) No. 2751-3000 No. 1016-1087 FIG. 3(M) No. 3001-3250 No.
1088-1119 FIG. 3(N) No. 3251-3500 FIG. 3(O) No. 3501-3750 FIG. 3(P)
No. 3751-3857
[0060] FIG. 4 is a graph showing that specific CTLs were induced
when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with an HLA-24-binding antigen peptide
HER2/neu.sub.780-788 derived from human tumor antigen HER-2/neu.
The cytotoxic activity (% Specific Lysis) and the name of
respective transgenic mice are depicted in the vertical and
horizontal axes, respectively. In the figure, "pep+" refers to the
results obtained using target cells undergone peptide pulse and
"pep-" the results obtained using cells without peptide pulse. The
sample is a mouse splenocyte preparation prepared by pulsing
splenocytes, which have been isolated from an HLA-A24 expressing
transgenic mouse immunized with antigen peptide
HER2/neu.sub.780-788, with the same antigen peptide. As the target
cells, Jurkat-A2402/K.sup.b cells which are transformants carrying
the chimera gene HLA-A2402/K.sup.b and have been labeled with
.sup.51Cr and pulsed with the peptide above (pep.sup.+) were used.
As control cells, target cells that have not been pulsed with the
peptide (pep.sup.-) were used.
[0061] FIG. 5 is a graph showing that specific CTLs were induced
when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with an HLA-A24-binding antigen peptide
MAGE-3.sub.195-203 derived from cancer antigen MAGE-3. The
cytotoxic activity (% Specific Lysis) and the name of respective
transgenic mice are depicted in the vertical and horizontal axes,
respectively. In the figure, the open bar shows the results
obtained using target cells undergone peptide pulse and the solid
bar the results obtained using cells without peptide pulse. The
sample and the target cells were prepared in a manner similar to
that described in regard to FIG. 4 above.
[0062] FIG. 6 is a graph showing that specific CTLs were induced
when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with an HLA-A24-binding antigen peptide
CEA.sub.652-660 derived from cancer antigen CEA. The cytotoxic
activity (% Specific Lysis) and the name of respective transgenic
mice are depicted in the vertical and horizontal axes,
respectively. In the figure, the open bar shows the results
obtained using target cells undergone peptide pulse and the solid
bar the results obtained using cells without peptide pulse. The
sample and the target cells were prepared in a manner similar to
that described in regard to FIG. 4 above.
[0063] FIG. 7 is a graph showing that specific CTLs were induced
when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with an HLA-A24-binding antigen peptide
CEA.sub.268-277 derived from cancer antigen CEA. The cytotoxic
activity (% Specific Lysis) and the name of respective transgenic
mice are depicted in the vertical and horizontal axes,
respectively. In the figure, the open bar shows the results
obtained using target cells undergone peptide pulse and the and
solid bar the results obtained using cells without peptide pulse.
The sample and the target cells were prepared in a manner similar
to that described in regard to FIG. 4 above.
[0064] FIG. 8 is a graph showing that specific CTLs were induced
when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with a PSA-origin antigen peptide
PSA.sub.152-160 prepared from human cancer antigen PSA protein on
the basis of information about human HLA-A24-binding motifs. The
cytotoxic activity (% Specific Lysis) and the name of respective
transgenic mice are depicted in the vertical and horizontal axes,
respectively. In the figure, "pep+" refers to the results obtained
using target cells undergone peptide pulse and "pep-" the results
obtained using cells without peptide pulse. The sample and the
target cells were prepared in a manner similar to that described in
regard to FIG. 4 above.
[0065] FIG. 9 is a graph showing that specific CTLs were not
induced when an HLA-A24 expressing transgenic mouse of the present
invention was immunized with a PSA-origin antigen peptide
PSA.sub.248-257 obtained from human cancer antigen PSA protein on
the basis of information about human HLA-A24-binding motifs. The
cytotoxic activity (% Specific Lysis) and the name of respective
transgenic mice are depicted in the vertical and horizontal axes,
respectively. In the figure, "pep+" refers to the results obtained
using target cells undergone peptide pulse and "pep-" the results
obtained using cells without peptide pulse. The sample and the
target cells were prepared in a manner similar to that described in
regard to FIG. 4 above.
BEST MODE FOR CARRYING OUT THE INVENTION
[0066] The present invention for the first time provides a
transgenic animal expressing HLA-A24. Specifically, the present
invention provides a non-human transgenic animal, into which an
HLA-A24 gene has been introduced and in which CTLs are induced when
stimulated with an HLA-A24-binding antigen (HLA-A24-restricted
antigen peptide).
[0067] The present invention also relates to use (utilization) of
the above-mentioned transgenic non-human mammal.
[0068] The present invention also relates to a DNA construct (e.g.,
chimera gene) useful in the construction of the above-mentioned
non-human transgenic mammal, a host cell transformed by said
chimera gene and use thereof.
[0069] Examples of an HLA-A24 gene to be introduced when
constructing the non-human transgenic mammal of the present
invention include subspecies such as HLA-A2402 gene, HLA-A2403
gene, etc. (J. Immunother., 23:282, 2000). However, considering
that most of HLA-A24 carriers also contain HLA-A2402 gene, it would
be preferred to use HLA-A2402 gene from the aspect of construction
of a transgenic animal enabling the in vivo evaluation of an agent
for vaccine therapy that is adaptable to many patients. HLA-A2402
and HLA-A2403 genes are registered at GenBank under Accession Nos.
M64740 and M64741, respectively. A desired HLA-A24 gene may be
cloned by PCR using appropriate primers prepared on the basis of
these sequences and, as a template, a genomic DNA or mRNA derived
from human tumor cells, etc.
[0070] As a transgene, a naturally occurring HLA-A24 gene (genome
or cDNA) may be used; however, it is preferred to use a chimera
gene wherein the .alpha.3 region of said HLA-A24 gene is replaced
by the corresponding .alpha.3 region of the animal into which it is
transferred.
[0071] The extracellular domain of an MHC class I molecule consists
of .alpha.1, .alpha.2 and .alpha.3 domains, wherein the .alpha.1
and .alpha.2 domains participate in the formation of peptide
binding groove and the .alpha.3 domain in the binding with a
coreceptor: CD8 molecule expressed on the surface of CTL cells.
Besides, it has been suggested to be preferred that an HLA animal
model expresses a chimera HLA molecule wherein the .alpha.3 domain
of HLA gene is replaced by the corresponding domain of MHC of the
subject animal so that CTLs of the resultant HLA animal model
effectively recognize a complex of HLA and antigen peptide (Eur. J.
Immunol., 26: 97, 1996, J. Immunol., 159:4753,1997). Accordingly,
as mentioned above, it is preferred that the .alpha.3 domain of HLA
gene is replaced by the corresponding .alpha.3 domain of the
subject animal into which it should be introduced
[0072] Such a chimera HLA-A24 gene is also falls within the scope
of the "HLA-A24 gene" of the present invention as far as it can be
expressed and retain the function as HLA-A24.
[0073] For the purposes of the present invention, it is necessary
that the .alpha.1 and .alpha.2 domains of the chimera HLA-A24 gene,
which domains participate in the formation of peptide binding
groove, are at least derived from HLA-A24. On the other hand, it is
preferred that the .alpha.3 domain is replaced by the corresponding
domain of MHC of the subject animal into which it should be
introduced, and it is more preferred that the region including the
.alpha.3 domain and the succeeding domain thereof is replaced by
the corresponding region of MHC of the subject animal into which it
should be introduced.
[0074] For example, when the subject into which the gene is
introduced is a mouse, mouse MHC corresponding to human HLA is H-2
of MHC class I molecule (H-2K.sup.b, etc., Immunogenetics., 41:178,
1995). Therefore, preferred examples of HLA-A24 gene to be
introduced into a mouse include a chimera HLA-A2402 gene comprising
the .alpha.1 and .alpha.2 domains of HLA-A2402 gene and the
.alpha.3 domain of mouse H-2K.sup.b gene.
[0075] Thus, the term "HLA-A24 gene" herein used in relation to the
present invention includes various subspecies belonging to HLA-A24
gene. Examples thereof include not only a naturally occurring
HLA-A24 gene comprising all of the .alpha.1, .alpha.2 and .alpha.3
domains composing said HLA-A24 gene but also DNA constructs
comprising at least .alpha.1 and .alpha.2 domains of these three
domains. Examples of the DNA construct include a chimera gene
consisting of the .alpha.1 and .alpha.2 domains of HLA-A24 origin
and the .alpha.3 domain originated from any mammal such as mouse,
and the like.
[0076] The specific examples of chimera HLA-A2402 gene useful for
the present invention include the followings:
[0077] (1) A DNA comprising a nucleotide sequence encoding the
amino acid sequence shown in SEQ ID NO:3;
[0078] (2) A DNA comprising a nucleotide sequence for cDNA shown in
SEQ ID NO:2;
[0079] (3) A DNA comprising a nucleotide sequence for genomic DNA
shown in SEQ ID NO:1;
[0080] (4) A DNA which hybridizes to the complement of any one of
DNAs set forth in (1)-(3) above under stringent conditions, and of
which expression product is capable of binding to HLA-A24-binding
antigen peptide and inducing CTLs (i.e., having CTL-inducing
activity).
[0081] The DNA set forth in (4) refers to a DNA having a similar
structure to those set forth in (1) to (3) above (e.g., DNAs
comprising modifications of one to several amino acids). Specific
examples of such DNA include those hybridizing to the complement of
a DNA set forth in any one of (1) to (3) above under stringent
conditions and having the above-mentioned activities. In this
context, "stringent conditions" refer to, for example, such
conditions wherein hybridization is conducted under a condition
(formamide concentration: 45% (v/v), salt concentration:
5.times.SSPE, temperature: 42.degree. C.) and washing is conducted
under a condition (salt concentration: 2.times.SSPE, temperature:
42.degree. C.). The assay for the activity will hereinafter be
described. Another example includes a DNA encoding an amino acid
sequence, which has substitution, deletion or insertion of one or
more amino acid residues (preferably, 1-30 amino acid residues,
more preferably 1-20 amino acid residues, still more preferably one
to several amino acid residues) in an amino acid sequence encoded
by a DNA set forth in (1)-(3) above and which has the
above-mentioned activity. Still another example includes a DNA
encoding an amino acid sequence which is homologous to an amino
acid encoded by a DNA set forth in (1)-(3) above at about 50% or
more, preferably at about 70% or more, more preferably at about 80%
or more, yet preferably at about 90% or more, and most preferably
at about 95% or more homology and which has the above-mentioned
activity.
[0082] More specifically, examples include a DNA which comprises
nucleotide sequences each encoding amino acid Nos. 25-114 (.alpha.1
domain), Nos. 115-206 (.alpha.2 domain), and Nos., 207-298
(.alpha.3 domain) of the amino acid sequence shown in SEQ ID NO: 3
and which has the above-mentioned activity. Further examples
include a DNA which comprises nucleotide sequences each
corresponding to the nucleotide Nos. 72-339 (.alpha.1 domain), Nos.
342-615 (.alpha.2 domain), and Nos. 618-891 (.alpha.3 domain) of
the nucleotide sequence shown in SEQ ID NO: 2.
[0083] The process for preparing said chimera HLA-A2402 gene is
described below by way of illustration for the preparation of
HLA-A24 gene of the present invention, which, however, should not
be construed as limiting the scope of the present invention.
[0084] A region including promoter, exons 1-3 and introns 1-3,
which comprises .alpha.1 and .alpha.2 domains needed for the
construction of a chimera HLA-A2402 gene is cloned on the basis of
sequence information about human HLA-A2402 genomic DNA (GenBank
Acc. No. L47206). In this connection, it is desirable that an
appropriate restriction site(s) is constructed in the intron 3 so
as to facilitate the ligation with mouse H-2K.sup.b gene. Examples
of preferred restriction site is BglII site. Said BglII site can be
constructed by modifying the downstream primer for PCR reaction so
as to generate a BglII site, and conducting PCR reaction using said
downstream primer. Specifically, such a modification can be
effected using the primer shown in SEQ ID NO: 5.
[0085] The aforementioned cloning can be carried out by conducting
PCR reaction using, for example, as a template a genomic DNA
originated from human tumor cell line and an appropriate primer as
mentioned above. Said PCR can be carried out easily according to
the teaching in a textbook such as Molecular Cloning 2nd Ed., Cold
Spring Harbor Laboratory Press (1989), etc. Also, There are a
number of commercially availabe PCR kits at present.
[0086] On the other hand, the region downstream from .alpha.3
domain (exons 4-8, and introns 3-7) is cloned by PCR using as a
template, for example, a genome DNA originated from mouse tumor
cell line on the basis of sequence information about mouse
H-2K.sup.b genome DNA (GenBank Acc. No. v00746, v00747). The
sequence registered at GenBank was incomplete, that is, most of
intron 3 region needed for the ligation with HLA-A2402 gene was not
registered, and, therefore, the unregistered region had to be
cloned and sequenced first of all. Since there exist homologous
pseudogenes or highly homologous genes of H-2K.sup.b gene, adequate
primers were required to clone appropriate H-2K.sup.b gene.
Examples of preferred primer include, for example, various primers
described in Example 2 (SEQ ID NO: 6-9).
[0087] The thus obtained HLA-A2402 genome fragment and mouse
H-2K.sup.b genome fragment are ligated to construct the intended
chimera gene (HLA-A2402/K.sup.b gene). When a BglII site is
constructed in the intron 3 of HLA-A2402 gene, as mentioned above,
the both genes are preferably ligated at said BglII site and the
BamHI site in the intron 3 of mouse H-2K.sup.b gene. In this
manner, the chimera HLA-A2402 gene (HLA-A2402/K.sup.b gene) of the
present invention can be prepared.
[0088] The technique for making 1 to several amino acid
modifications to the resultant chimera HLA-A2402 gene is also well
known to one of ordinary skill in the art and can be conducted
according to the method described in a literature such as Molecular
Cloning 2nd Edt., Cold Spring Harbor Laboratory Press (1989).
[0089] The processes for introducing HLA-A24 gene into fertilized
eggs and construction of transgenic animals are hereinafter
described.
[0090] A region corresponding to HLA-A24 gene is excised from the
HLA-A24-containing plasmid constructed above and the resultant
fragment is purified in a conventional manner to such a degree that
it can be introduced into fertilized eggs. When the
HLA-A2402/K.sup.b gene possesses the native promoter region, the
excised HLA-A24 gene fragment can be introduced into fertilized
eggs as it is; however, when it does not, a foreign promoter
necessary for expression should be added. Examples of such a
promoter include .beta.-actin promoter, methalothionine promoter,
etc. Besides, an enhancer may be added to increase the expression
amount and examples of such an enhancer include CMV enhancer, SV40
enhancer, immunoglobulin enhancer, etc.
[0091] The purified HLA-A24 gene is then introduced into a
fertilized egg of the subject animal. The subject animal may be
specifically, for example, mouse, rat, rabbit, etc.; however,
rodents such as mouse, rat, etc. are preferred from the viewpoint
of feasibility of generation, fosterage and application, and mouse
is especially preferred. Above all, a mouse of strain C57BL/6 is
more preferred. C57BL/6 mouse strain has an advantage because said
mouse expresses H-2K.sup.b as the class I molecule and not
H-2K.sup.d having similar binding motif as HLA-A24. That is, when
an HLA-A24-binding antigen is administered, there is no danger of
presentation of an antigen on the cell surface or
cross-reaction.
[0092] The construction of transgenic mammal will hereinafter be
described using a mouse as an example.
[0093] The method for introducing a gene into a mouse fertilized
egg includes microinjection, electroporation, etc., without
limitation. After introduction, the resultant egg cells are
incubated and transferred to oviduct of a foster parent mouse, and
the recipient animal is raised. Among the offspring born, those
containing the HLA-A24 gene of the present invention are selected
to generate the transgenic mouse. The identification of said
transgenic mouse can be carried out by, for example, PCR using
primers specific for the introduced HLA-A24 gene and a DNA
preparation derived from mouse tail as a template.
[0094] The so generated transgenic mouse line can be examined
whether the introduced HLA-A24 gene is expressed on the cell
surface by, for example, recovering splenocytes from spleen
isolated from the transgenic mouse and analyzing said splenocytes
by flow cytometry. The resultant transgenic mouse of the present
invention preferably carries the HLA-A24 transgene
homozygously.
[0095] The above-mentioned transgenic animals can be generated by
making reference to "Handbook of New Gengenetic Engineering
Technique, p269-276, Yodo-sha (1996), "Operating Manual for Mouse
Embryo", Kindai-syuppan (1989) and the like. For example, with the
aim of integrating a DNA construct, as a chimera HLA-A24 gene, in
the HLA-A24 gene locus of mouse genome, said construct is
introduced into mouse embryonic stem cells and the resultant
recombinant stem cells are then selected and introduced into mouse
blastocysts to obtain chimeric embryos. The chimeric embryos are
then transferred to pseudo pregnant mice to obtain offspring, which
are then screened for the presence of alleles of chimeric transgene
to identify the heterozygous mice. The resultant mice are then
subjected to crossbreeding to yield homozygous transgenic mice
having a phenotype characterized in that CTLs are induced when
stimulated with an HLA-A24-binding antigen.
[0096] The transgenic mice of the present invention can be examined
whether or not they are usable as animal models for human, that is,
whether or not specific CTLs are induced in response to stimulation
with an HLA-A24-binding antigen by any methods known to one of
ordinary skill in the art. Typical example of such methods is
provided below.
[0097] A known HLA-A24-binding antigen peptide is mixed with
Freund's incomplete adjuvant in a conventional manner to prepare
water-in-oil emulsion. The emulsion is then used to immunize a
transgenic mouse. The inoculation is preferably conducted via mouse
tail, subcutaneous of back, peritoneal cavity, plantar, etc.
Several days later, the spleen is removed. The splenocytes are then
recovered and prepared in a conventional manner. Splenocytes are
used herein because they contain antigen-presenting cells such as
dendritic cells, but the present invention is not limited to the
use of splenocytes.
[0098] The splenocytes are then subjected to hemolytic treatment
and X-ray irradiation, and then pulsed with the above-mentioned
antigen peptide. In so doing, non-irradiated, non-peptide-pulsed
splenocytes are also added and re-stimulated at 37.degree. C.
Stimulation is continued for several days to obtain a test sample
for evaluation.
[0099] Target cells for the assay of the test sample above are
prepared separately. Any cells expressing HLA-A24 on the cell
surface can be used as the target cells, including both of cells
naturally expressing HLA-A24 and transformed prepared by
introducing HLA-A24 gene.
[0100] Examples of cells naturally expressing HLA-A24 include
isolated and purified lymphocytes originated from the transgenic
mouse of the present invention, RERF-LC-AI cells (Riken Cell Bank,
RCB0444) expressing HLA-A24 naturally, etc.
[0101] Examples of transformed cells prepared by introducing an
HLA-A24 gene include the followings:
[0102] The HLA-A24 gene to be transformed into cells may be an
HLA-A24 gene of natural-type or a chimera HLA-A24 gene as mentioned
above. However, it is preferred to transform a cell by the same
HLA-A24 gene as that the transgenic animal contains so as to
facilitate the recognition by CTLs of the transgenic animal. That
is, when a transgenic animal contains a chimera HLA-A2402 gene
(HLA-A2402/K.sup.b gene), it is preferable that an
HLA-A2402/K.sup.b gene is transformed into the target cells to be
used for the evaluation of said transgenic animal.
[0103] The said HLA-A24 gene for transformation may be in the form
of cDNA or genomic DNA. In addition, the HLA-A24 may be isolated
from the transgenic animal itself or other cells.
[0104] For example, when an HLA-A24 cDNA should be introduced, the
intended cDNA can be obtained by PCR reaction using HLA-A24
specific primers and mRNA isolated from the transgenic animal as a
template. When a genomic DNA should be introduced, the intended
genomic DNA can be obtained by PCR reaction using HLA-A24 specific
primers and genomic DNA isolated from RERF-LC-AI cells (Riken Cell
Bank, RCB0444) expressing HLA-A24 naturally as a template. In
addition, the HLA-A24 gene (chimera HLA-A24 gene, etc.) prepared
for introducing into transgenic animals above can be used.
[0105] Specific examples of cDNA or genomic DNAs to be introduced
include DNAs described in (1) to (4) above, that is, a cDNA
comprising a nucleotide sequence encoding the amino acid sequence
shown in the SEQ ID NO: 3, a cDNA comprising the nucleotide
sequence shown in the SEQ ID NO: 2, a genomic DNA comprising the
nucleotide sequence shown in the SEQ ID NO: 1, and the like.
[0106] The desired recombinant expression vector expressing HLA-A24
can be constructed by inserting the resultant cDNA or genomic DNA
into a commercially available expression vector such as pcDNA3.1,
pcDNA3.1 derivative, pRc/RSV, pRc/CMV, pEF derivative, pREP9
derivative (Invitrogen), pIRESneo (Chlontech), or the like.
[0107] The host cells include human lymphocytes such as Jurkat cell
(ATCC T1B-152) and T2 cell (ATCC CRL-1992), and mouse lymphocytes
such as EL4 cell (ATCC T1B-39), RMA-S cell (Nature.,
346:476-80(1990)), K562 cell (ATCC CCL-243), C1R cell (ATCC
CRL-2369), and the like. Examples of method for introducing an
expression vector containing HLA-A24 gene into host cells include a
method utilizing a gene-transfer device, calcium phosphate method
(J. Virol., 52,456-467(1973)), a method utilizing LT-1 (PanVera
Corp.), a method utilizing a lipid for gene transfer
(Lipofectamine, Lipofectin; Gibco-BR, Inc.), and the like. When
host cells surviving in a selective medium are cultivated, cells
expressing a gene introduced stably are established.
[0108] The thus established target cells can be used for
evaluating/estimating the above-mentioned transgenic animal (i.e.,
evaluation of test samples derived from the transgenic animal) in a
manner well known to a person skilled in the art. Specifically, it
can be carried out by a technique wherein the amount of various
cytokines (e.g., IFN-.gamma.) produced by CTLs in response to the
target cells is measured by ELISA or the like or wherein the
cytotoxicity of CTL against target cells is measured (.sup.51Cr
release assay, Int. J. Cancer, 58:317, (1994)), which CTLs are
induced by stimulation with antigen.
[0109] The ".sup.51Cr release assay" is a technique comprising
labeling a target cells with .sup.51Cr, pulsing the labeled cells
with the subject antigen peptide followed by addition of a test
sample (splenocytes) of the transgenic animal, and measuring the
amount of .sup.51Cr released from target cells damaged by CTLs. If
induction of antigen specific CTLs is recognized by such an assay,
the said transgenic animals are estimated to be model animals for
human that are capable of evaluating HLA-A24-binding tumor antigen
protein or tumor antigen peptide.
[0110] The so constructed transgenic animals of the present
invention are those wherein CTLs are induced when stimulated by
HLA-A24-binding antigen. Accordingly, said transgenic animals are
usable for determining whether or not a test substance has an
activity of inducing antigen-specific CTLs in vivo. That is, the
present invention provides a method of screening a CTL inducing
agent specific for an antigen, which comprises administering a test
substance to a transgenic animal of the present invention, and
assaying and determining whether or not CTLs specific for the test
substance are induced.
[0111] Examples of the test substance used in the screening method
include antigen proteins, which have proved to have activity of
inducing specific CTLs by in vitro test, antigen peptides derived
therefrom and plasmid DNAs encoding the same, without limitation.
Also included are proteins or peptides of which activity is unknown
or plasmid DNAs encoding the same.
[0112] Specific examples include tumor antigen proteins such as
MAGE (Science, 254:1643, 1991), gp100 (J. Exp. Med., 179: 1005,
1994), MART-1 (Proc. Natl. Acad. Sci. USA, 91: 3515, 1994),
tyrosinase (J. Exp. Med., 178: 489, 1993), HER2/neu (J. Exp. Med.,
181: 2109, 1995), CEA (J. Natl. Cancer. Inst., 87: 982, 1995), PSA
(J. Natl. Cancer. Inst., 89: 293, 1997), SART-1 (J. Exp. Med., vol.
187, p277-288, 1998, WO97/46676), Cyclophilin B (Proc. Natl. Acad.
Sci. U.S.A., 88:1903, 1991), SART-3 (WO00/12701), ART-1
(WO00/32770), and the like, partial peptides thereof, and plasmid
DNAs encoding the same. Additional examples include viruses such as
HIV, HCV, HBV, influenza virus, HPV, HTLV, EBV etc., antigen
proteins derived from viruses and antigen peptides thereof, antigen
proteins derived from bacteria such as tubercle bacillus and
antigen peptides thereof.
[0113] The determination and/or evaluation whether HLA-A24
restricted CTLs specific for a test substance have been induced by
administration of said test substance can be carried out in a
manner similar to the evaluation method for transgenic animals
described above.
[0114] For example, the transgenic animal of the present invention
is immunized with a test substance, and spleen is extracted and
splenocytes are recovered. The splenocytes are subjected to
hemolytic treatment and X-ray irradiation, followed by pulsing with
a test substance. In so doing, non-irradiated, non-peptide-pulsed
splenocytes are also added and re-stimulated at 37.degree. C.
Stimulation is continued for several days to yield a test sample
for evaluation.
[0115] On the other hand, target cells for assay as described above
are labeled with .sup.51Cr, pulsed with antigen peptide of a test
substance, and the test sample (splenocytes) above is added
thereto. When the induction of antigen specific CTLs in response to
the addition of a test sample is observed, said test substance
proves to be one having ability to induce antigen specific CTLs in
vivo. The CTL inducing agents thus selected can be used as a
therapeutic and/or preventive agent for tumors or virus
infections.
[0116] The target cells described above are used for evaluating not
only transgenic animals or test substances administered to
transgenic animals, but also any substances which should be
evaluated whether they induce HLA-A24 antigen specific CTLs or
not.
[0117] The present invention also encompasses an HLA-A24-binding
tumor antigen peptide of PSA origin, which is obtainable according
to the screening method using the transgenic animal of the present
invention as described above, and a derivative thereof having
characteristics functionally equivalent to that of said tumor
antigen peptide. In this regard, the term "PSA" refers to a tumor
antigen comprising the amino acid sequence disclosed in Biochem.
Biophys. Res. Commun. 160(2), 903-910(1989) and GenBank Acc No.
M26663.
[0118] It has been known that there is a rule (motif) for the
sequence of antigen peptides that are presented after binding to
HLA antigen, and in the case of HLA-A24 antigen, the amino acid at
the second position from the N-terminus of 8 to 11 amino acid
peptide is phenylalanine, tyrosine, methionine or tryptophan, and
the C-terminal amino acid is phenylalanine, leucine, isoleucine,
tryptophan or methionine (Immunogenetics, 41: 178, 1995, J.
Immunol., 152: p3913,1994, J. Immunol., 155: 4307,1994). An
HLA-A24-binding tumor antigen peptide expressing activity in vivo
can be identified and obtained by selecting a partial peptide
region on the amino acid sequence of PSA, which region has the
binding motif, and applying the selected peptide to the transgenic
animal of the present invention.
[0119] Specific examples of said tumor antigen peptide include a
tumor antigen peptide comprising the amino acid sequence shown in
SEQ ID NO: 15.
[0120] The tumor antigen peptide of the present invention can be
synthesized according to a method generally used in the peptide
chemistry. The synthetic method includes those described in
literatures (Peptide Synthesis, Interscience, New York,1966; The
Proteins, Vol. 2, Academic Press Inc., New York, 1976; Peptide
Gosei (Peptide Synthesis), Maruzen, Co. Ltd., 1975; Peptide Gosei
no Kiso to Jikken (Fundamentals and Experiments for Peptide
Synthesis), Maruzen, Co. Ltd., 1985; Iyakuhin no Kaihatsu
(Developments of Pharmaceuticals, Sequel Vol. 14, Peptide Gosei
(Peptide Synethesis), Hirokawa-shoten, 1991), and the like.
[0121] In this regard, "a derivative thereof having characteristics
functionally equivalent to that of said tumor antigen peptide",
which hereinafter may be referred to as "tumor antigen peptide
derivative" is a variant that is derived from the tumor antigen
peptide of the present invention by modifying one to several amino
acid residues in the amino acid sequence of the same and yet has
characteristics as a tumor antigen peptide, i.e., being recognized
by CTLs when bound to HLA-A24 antigen.
[0122] In this regard, the term "modification" of amino acid
residue(s) means substitution, deletion and/or addition of amino
acid residue(s), including addition of amino acid residue(s) to the
N-- and/or C-terminus of peptide, and preferably substitution of
amino acid residue(s). When the modification is related to amino
acid substitution, the number and the position of amino acid
residue to be replaced can be selected optionally, as long as an
activity as tumor antigen peptide is maintained.
[0123] As mentioned above, it has been known that there is a rule
(motif) for the sequence of antigen peptide that is presented after
binding to HLA antigen, and in the case of HLA-A24 antigen, the
amino acid at the second position from the N-terminus of 8 to 11
amino acid peptide is phenylalanine, tyrosine, methionine or
tryptophan, and the C-terminal amino acid is phenylalanine,
leucine, isoleucine, tryptophan or methionine (Immunogenetics,
41:178, 1995 J. Immunol., 152:3913,1994, J. Immunol., 155:4307,
1994). More preferably, in the motif, the second amino acid from
the N-terminus is phenylalanine, tyrosine or tryptophan, and the
C-terminal amino acid is phenylalanine, leucine, isoleucine or
tryptophan. Besides, an amino acid residue(s) having similar
properties to amino acids available in the motif may possibly be
accepted.
[0124] Accordingly, the specific examples of the tumor antigen
peptide derivative of the present invention includes a tumor
antigen peptide derivative that has been derived by replacing the
amino acid at the second and/or C-terminal position of the motif
above by an amino acid exchangeable in the motif, and that has an
activity as an tumor antigen peptide. The length of the peptide is
preferably from about 8 to 11 amino acids considering that it
bounds to an HLA-A24 antigen and presented.
[0125] Specific examples include a peptide comprising an amino acid
sequence wherein the amino acid at the second position of the amino
acid sequence shown in SEQ ID NO: 15 is replaced by any one
selected from phenylalanine, tyrosine, methionine and tryptophan,
and/or the amino acid at the C terminus is replaced by any one
selected from phenylalanine, leucine, isoleucine, tryptophan and
methionine, as shown in SEQ ID NO: 17, and having an activity as a
tumor antigen peptide.
[0126] Similar to the tumor antigen peptide of the present
invention, the tumor antigen peptide derivative of the present
invention can be identified whether or not it has characteristics
functionally equivalent to the tumor antigen peptide by
synthesizing a candidate peptide according to a method for peptide
synthesis, subjecting the resulting peptide to the transgenic
animal of the present invention and examining the same for the
activity of inducing HLA-A24 restricted CTLs as mentioned
above.
[0127] The tumor antigen peptide or its derivative of the present
invention has, as shown in the Examples below, an activity of
inducing CTLs, which CTLs have cytotoxic activity or generate
lymphokines and thereby are able to exert anti-tumor effects.
Accordingly, the tumor antigen peptide or its derivative of the
present invention can be used as an active ingredient of a
therapeutic or preventive agent for tumors. The present invention
provides a therapeutic or preventive agent for tumors comprising as
an active ingredient a tumor antigen peptide or a derivative
thereof. When the therapeutic or preventive agent for tumors of the
present invention is administered to a patient who is
HLA-A24,positive and PSA-positive, the tumor antigen peptide or a
derivative thereof is effectively presented to an HLA-A24 antigen
of the antigen-presenting cells, and CTLs specific for the complex
of HLA-A24 antigen presented then proliferate and destroy the tumor
cells. In this manner, treatment or prevention of tumors would be
achieved. Since PSA is expressed in prostate cancer with high
frequency, the therapeutic or preventive agent for tumors of the
present invention can be used effectively for prostate cancer.
[0128] The therapeutic or preventive agent for tumors of the
present invention may be administered along with an adjuvant, or
may be administered in a particulate dosage form in order to
effectively establish the cellular immunity. Examples of available
adjuvant include those described in a literature (Clin. Microbiol.
Rev., 7:277-289, 1994). In addition, liposomal preparations,
particulate preparations in which the peptides are bound to beads
having a diameter of several .mu.m, or preparations in which the
peptides are attached to lipids, are also usable. Administration
may be achieved, for example, intradermally, hypodermically, by
intravenous injection, or the like. Although the dose of a tumor
antigen peptide or a derivative thereof of the present invention in
the preparation may be adjusted as appropriate depending on, for
example, the disease to be treated, the age and the body weight of
a particular patient, it would be usually from 0.0001 mg to 1000
mg, preferably from 0.001 mg to 1000 mg, more preferably from 0.1
mg to 10 mg of the peptide every several days to every several
months.
[0129] The present invention also provides a DNA encoding the tumor
antigen peptide or a derivative thereof of the present invention.
The DNA of the present invention can be synthesized by one ordinary
skilled in the art in ease on the basis of the amino acid sequence
of the tumor antigen peptide or a derivative thereof of the present
invention. Preferred examples of the present DNA include a DNA
encoding the amino acid sequence of SEQ ID NO: 15.
[0130] The DNA of the present invention can be used effectively in
the production of the tumor antigen peptide or a derivative thereof
of the present invention. In addition, the DNA of the present
invention can also be used effectively for inducing CTLs, i.e., for
treating or preventing tumors.
[0131] Thus, there has recently been developed a vaccination method
which uses a DNA encoding "polytope" wherein plural of CTL-epitopes
are ligated as a DNA vaccine. See, for example, Journal of
Immunology, 160, p1717, 1998 etc. Accordingly, a DNA prepared by
ligating one or more DNAs encoding a tumor antigen peptide or a
derivative thereof of the present invention and, if desired, a
DNA(s) encoding other tumor antigen peptide is inserted into an
appropriate expression vector to obtain an active ingredient of a
CTL-inducing agent (i.e., therapeutic or preventive agent for
tumors). Said DNA resulted from ligation is referred to as
"recombinant DNA". In addition to the above-mentioned use of
recombinant DNA as a therapeutic or preventive agent, a polypeptide
as an expression product of the recombinant DNA in host cells is
also useful as an active ingredient of a therapeutic or preventive
agent for tumors.
[0132] The term "recombinant DNA" can be easily prepared by a
method of DNA synthesis or ordinary genetic engineering technique
according to the teaching of Molecular Cloning 2nd. Edit., Cold
Spring Harbor Laboratory Press (1989), etc. Furthermore, insertion
of the recombinant DNA into an expression vector can also be
conducted in accordance with the teaching of the aforementioned
textbook and the like.
[0133] The evaluation whether or not the resultant recombinant DNA
of the present invention gives a tumor antigen peptide that can be
recognized by CTL after binding to HLA-A24 antigen is also
conducted using the transgenic animal of the present invention.
[0134] When applying the recombinant DNA of the present invention
to the therapeutic or prophylactic agent for tumors, the following
methods are usable.
[0135] Thus, examples of a method of introducing the recombinant
DNA of the present invention into cells include a method which
employs viral vectors and those described in literatures
(Nikkei-Science, April, 1994, p20-45; Gekkan-Yakuji, 36(1), 23-48
(1994); Jikken-Igaku-Zokan, 12(15), 1994, and references cited
therein), and any one of such methods may be applied to the present
invention.
[0136] Examples of methods which use viral vectors include those
wherein the DNA of the present invention is incorporated into DNA
or RNA virus such as retrovirus, adenovirus, adeno-associated
virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, or
Sindbis virus, and then introduced into cells. Among them, the
methods using retrovirus, adenovirus, adeno-associated virus, or
vaccinia virus are particularly preferred.
[0137] Examples of another method include a method wherein
expression plasmids are directly injected intramuscularly (DNA
vaccination), the liposome method, Lipofectin method,
microinjection, the calcium phosphate method, and electroporation.
Among them, DNA vaccination and the liposome method are
particularly preferred.
[0138] In order to make the recombinant DNA of the present
invention act as pharmaceutical in practice, one can use either of
two methods: in vivo method in which DNA is directly introduced
into the body, or ex vivo method in which certain kinds of cells
are removed from human, and after introducing DNA into said cells
outside of the body, reintroduced into the body (Nikkei-Science,
April, 1994, p20-45; Gekkan-Yakuji, 36(1), 23-48 (1994);
Jikkenn-Igaku-Zokan, 12(15), 1994; and references cited therein).
In vivo method is more preferable.
[0139] In the case of in vivo methods, a DNA of the present
invention may be administered via any appropriate route depending
on the diseases and symptoms to be treated, and other factors. For
example, it may be administered via intravenous, intraarterial,
subcutaneous, intracutaneous, or intramuscular routes. In the case
of in vivo methods, such pharmaceuticals may be administered in
various dosage forms such as solution, and they are typically
formulated into injections containing a recombinant DNA of the
present invention as an active ingredient, which may also include,
as needed, conventional carriers. When a recombinant DNA of the
present invention is included in liposomes or membrane-fused
liposomes (such as Sendai virus (HVJ)-liposomes), such medicines
may be in the form of suspension, frozen drug,
centrifugally-concentrated frozen drug or the like.
[0140] Although the amount of a recombinant DNA of the present
invention in such formulations varies depending on, for example,
the disease to be treated, the age and body weight of a particular
patient, it is usually preferred to administer 0.0001-100 mg, more
preferably 0.001-10 mg, of a recombinant DNA of the present
invention at every several days to every several months.
[0141] When a recombinant DNA of the present invention as described
above is administered to a patient, polypeptides corresponding to
the said recombinant DNA are expressed to a high extent. Individual
tumor antigen peptide resulted from intracellular processing forms
a complex with an HLA antigen and presented on the cell surface of
antigen presenting cells at high density, and CTLs specific for the
complex proliferate efficiently in vivo and destroy tumor cells.
The treatment or prevention of tumor would be achieved in this
manner. The recombinant DNA of the present invention is especially
useful for treatment or prevention of prostate cancer.
[0142] In addition, the "polypeptide" as an expression product of
the above-mentioned recombinant DNA can be expressed and produced
by transforming a host cell with an expression vector constructed
by inserting the above-mentioned recombinant DNA into an
appropriate expression vector (e.g., pSV-SPORT1 etc.), and
culturing the resultant transformant in an appropriate medium.
Examples of host cell include prokaryotes such as Escherichia coli,
unicellular eukaryotes such as yeast, and cells derived from
multicellular eukaryotes such as insects or animals. Transformation
of host cells with an expression plasmid can be carried out by a
known method such as the calcium phosphate method, DEAE-dextran
method, or the electric pulse method. The polypeptide thus obtained
can be isolated and purified according to standard biochemical
procedures. It can be determined whether the said polypeptide gives
a tumor antigen peptide(s) capable of being recognized by CTLs when
bound to HLA-A24 antigen using the transgenic animal of the present
invention.
[0143] When using the polypeptide of the present invention as a
therapeutic or preventive agent for tumors, the dosage form,
administration method and dose are the same as that mentioned above
in connection with the tumor antigen peptide or a derivative
thereof of the present invention. When the polypeptide of the
present invention is administered to a tumor patient, it is uptaken
by antigen presenting cells. Individual tumor antigen peptide
produced by intracellular processing then forms a complex with an
HLA antigen and presented on the cell surface of antigen presenting
cells at high density, and CTLs specific for said complex
efficiently proliferate in vivo and destroy tumor cells. In this
manner, the treatment and prophylaxis of tumors would be achieved.
The polypeptide of the present invention is useful especially in
the prevention or treatment of prostate cancer.
[0144] The present invention also provides an antigen presenting
cell on which a complex of an HLA-A24 antigen and a tumor antigen
peptide or a derivative thereof of the present invention is
presented.
[0145] As shown in the working Examples below, a potent cytotoxic
activity was observed after stimulation with the peptide of the
present invention, which very shows that antigen presenting cells
presenting a complex between the peptide of the present invention
and an HLA-A24 antigen exist in the lymph node of a transgenic
animal, and that CTLs specifically recognize said antigen
presenting cells are induced. Such antigen presenting cells on
which a complex between an HLA-A24 antigen and the tumor antigen
peptide of the present invention is presented can be effectively
used in the cell therapy (DC therapy) as described below.
[0146] Antigen presenting cells used for cell therapy are prepared
as follows. Cells having an antigen-presenting ability are isolated
from a tumor patient and pulsed ex vivo with the tumor antigen
peptide or its derivative of the present invention, thereby
allowing the cells to present a complex between an HLA-A24 antigen
and the peptide or its derivative of the present invention. In this
regard, the "cell having an antigen-presenting ability" is not
restricted to particular one and includes cells on which an HLA-A24
antigen capable of presenting the tumor antigen peptide or a
derivative thereof of the present invention is expressed; however,
dendritic cells known to have antigen presenting ability are
preferred.
[0147] In addition, the substance used for pulsing the
above-mentioned antigen presenting cells may be in the form of
peptide, as mentioned above, and also in the form of recombinant
DNA or RNA, or a polypeptide of the present invention.
[0148] The antigen presenting cells of the present invention can be
prepared by, for example, isolating cells having an
antigen-presenting ability from a tumor patient, pulsing ex vivo
with the tumor antigen peptide or its derivative of the present
invention to form a complex between an HLA-A24 antigen and the
peptide above or its derivative (Cancer Immunol. Immunother.,
46:82, 1998; J. Immunol., 158: p1796, 1997; Cancer Res., 59: p1184,
1999). In a case where dendritic cells are used, the antigen
presenting cells of the present invention can be prepared as
follows. Lymphocytes are isolated from peripheral blood of a tumor
patient by Ficoll method; adherent cells are separated from
non-adherent cells; the adherent cells are then cultured in the
presence of GM-CSF and IL-4 to induce dendritic cells; and the
dendritic cells are pulsed by culturing with a tumor antigen
peptide or a polypeptide of the present invention to yield the
antigen presenting cells of the present invention.
[0149] When the antigen presenting cells of the present invention
is prepared by a process comprising introducing a recombinant DNA
of the present invention into the above-mentioned cells having
antigen-presenting ability, said process can be carried out in
accordance with the teaching in Cancer Res., 56: p5672, 1996 or J.
Immunol., 161: p5607, 1998, and the like. Furthermore, RNA, as well
as DNA, is usable for the preparation of antigen presenting cells
in accordance with the teaching of J. Exp. Med., 184: p465, 1996,
and the like.
[0150] The present invention also provides a CTL inducing agent
(therapeutic agent for tumors) containing as an active ingredient
the antigen presenting cell above. Said therapeutic agent
preferably contains physiological saline, phosphate buffered saline
(PBS), culture medium, or the like in order to stably maintain the
antigen presenting cell. Administration may be achieved, for
example, intravenously, hypodermically, or intradermally. The
dosage can be, for example, the same as that described in the
afore-mentioned literatures.
[0151] By returning the above therapeutic agent into the patient's
body, specific CTLs are efficiently induced in the patient who is
positive for both HLA-A24 and PSA, and thereby tumor can be
treated. The antigen presenting cells of the present invention are
especially useful for treating prostate cancer.
[0152] The present invention also provides CTLs that recognize a
complex between an HLA-A24 antigen and the tumor antigen peptide or
a derivative thereof of the present invention. The CTLs of the
present invention may be used effectively in the following adoptive
immunotherapy.
[0153] In the case of melanoma, it has been observed that an
adoptive immunotherapy wherein intratumoral T cell infiltrate taken
from the patient himself/herself are cultured ex vivo in large
quantities, and then returned into the patient achieves a
therapeutic gain (J. Natl. Cancer. Inst., 86:1159, 1994).
Furthermore, in mouse melanoma, suppression of metastasis has been
observed by stimulating splenocytes in vitro with a tumor antigen
peptide TRP-2, thereby proliferating CTLs specific for the tumor
antigen peptide, and then administering said CTLs into a mouse
carrying grafted melanoma (J. Exp. Med., 185:453, 1997). This
resulted from in vitro proliferation of CTLs that specifically
recognize the complex between an HLA antigen of antigen presenting
cell and the tumor antigen peptide. Accordingly, a method for
treating tumors which comprises stimulating in vitro peripheral
blood lymphocytes of a patient with a tumor antigen peptide, a
derivative thereof, a recombinant DNA or a polypeptide of the
present invention to make tumor-specific CTLs proliferate, and
returning the CTLs into the patient is believed to be useful.
[0154] Furthermore, the present invention also provides a
therapeutic agent for tumors containing as an active ingredient the
CTLs of the present invention. It is preferred that the therapeutic
agent contains physiological saline, phosphate buffered saline
(PBS), culture medium, or the like in order to stably maintain
CTLs. Administration may be achieved, for example, intravenously,
hypodermically, or intradermally. The dosage can be, for example,
the same as that described in the afore-mentioned literatures.
[0155] By returning the above therapeutic agent into the patient's
body, the toxicity of CTLs on tumor cells is enhanced in the
patient who is positive for both HLA-A24 and PSA efficiently and
destroy the tumor cells, and thereby achieving the treatment of
tumor. The CTLs of the present invention are especially useful in
the treatment of prostatic cancer.
[0156] Besides, the tumor antigen peptide or a derivative thereof,
or a polypeptide of the present invention can be used as an
ingredient of an agent for diagnosis of tumors. That is, the tumor
antigen peptide or its derivative of the present invention can
serve as a diagnostic agent which is useful in the detection of an
antibody in a sample (such as blood, a tumor tissue, or the like)
obtained from a patient suspected to have a tumor. In this manner,
one can detect tumors in early-stage, or diagnose recurrent or
metastatic tumors. Furthermore, it may be used for screening of
tumor patients adaptable to pharmaceuticals containing tumor
antigen peptides or the like of the present invention as an active
ingredient. Specifically, the diagnosis can be effected using
immunoblotting, RIA, ELISA, or fluorescent or luminescent assay.
The diagnostic agent of the present invention is especially useful
in the diagnosis of prostatic cancer.
[0157] Furthermore, there have recently been established a new
method of detecting antigen-specific CTLs that uses a complex
between an antigen peptide and an HLA antigen (Science, 274: p94,
1996). A complex of a tumor antigen peptide or its derivative of
the present invention and an HLA antigen may be applied to the said
detection method to detect CTLs specific for tumor antigen whereby
one can detect tumors in early-phase, or diagnose recurrence or
metastasis of tumor. It can also be used for selecting patients
adaptable to the pharmaceuticals of the present invention or for
evaluating therapeutic effects thereof, which pharmaceuticals
contain as an active ingredient a tumor antigen peptide or the like
of the present invention. Thus, the present invention provides a
diagnostic agent for tumors comprising a tumor antigen peptide or
its derivative of the present invention.
[0158] Specifically, the diagnosis above can be carried out as
follows: a tetramer of a complex between tumor antigen peptide and
fluorescence-labeled HLA antigen obtained by a method described in
Science, 274: p94, 1996 is prepared and subjected to the
flowcytometry and the amount of CTLs specific for antigen peptide
among peripheral blood lymphocytes derived from a patient suspected
to have a tumor is determined.
[0159] The following Examples are provided to further illustrate
the present invention and are not to be construed as limiting the
scope thereof.
EXAMPLE 1
Cloning of HLA-A2402 Genomic DNA Fragment
[0160] (1) Cloning of HLA-A2402 Genomic DNA Fragment
[0161] For the purpose of cloning human HLA-A2404 genomic DNA by
PCR, a human tumor cell line, RERF-LC-AI cells (Riken Cell Bank
RCB0444) were cultured and human genomic DNA was purified using
Genomic Prep Cells and Tissue DNA Isolation Kit (Amersham) as per
attached protocol. GenBank database was then searched for HLA-A2402
genomic DNA needed for the construction of chimeric HLA gene, which
revealed that one registered under Accession No. Z72422 was
relevant, but a 270 bp promoter region was not registered. The
construction of the objective transgenic mouse requires promoter,
exons 1-3 and introns 1-3. To clone HLA-A2402 genomic DNA
containing promoter, PCR was conducted using the upstream primer
HLA26-1F:
[0162] 5'-CCC AAG CTT ACT CTC TGG CAC CAA ACT CCA TGG GAT-3' (36
mer, SEQ ID NO: 4)
[0163] which was designed making reference to the nucleotide
sequence of the promoter of HLA-A2601 (Accession No. AB005048)
frequently found in Japanese; and the downstream primer A24-BglII
30:
[0164] 5'-CGG GAG ATC TAC AGG CGA TCA GGT AGG CGC-3' (30 mer, SEQ
ID NO:5)
[0165] which comprises a modification in the nucleotide sequence in
intron 3, specifically, the nucleotide at 1282 position from the 5'
terminus of Accession No. Z72422 is changed from G to A. Said
modification of nucleotide was needed for the following reasons.
The present invention is aimed at obtaining an transgenic mouse
expressing a chimeric HLA consisting of exons 1-3 of HLA-A2402 and
exons 4-8 of H-2K.sup.b, which chimeric HLA can be constructed by
ligating the region upstream from the BamHI restriction site in
intron 3 of HLA-A2402 genomic DNA and the region downstream from
intron 3 of H-2K.sup.b genomic DNA and, for this end, it was
necessary to construct an artificial BglII restriction site in the
intron 3 of HLA-A2402.
[0166] PCR cloning of HLA-A2402 genomic DNA fragment was then
conducted using Native Pfu DNA Polymerase (Stratagene) having high
3'.fwdarw.5' exonuclease activity as per attached protocol, and the
pair of primers above. The PCR comprised heat treatment at
95.degree. C. for 45 seconds, 35 cycles of reaction at 95.degree.
C. for 45 seconds, 66.degree. C. for 1 minute and 72.degree. C. for
4 minutes, and reaction at 72.degree. C. for 10 minutes, followed
by cooling to 4.degree. C. The amplified gene fragment was ligated
into HindIII and BamHI restriction sites of phagemid vector
pBluescript to obtain a recombinant plasmid. The recombinant
plasmid was introduced into E. coli JM109 (Toyobo) by heat shock
method at 42.degree. C., and white colonies of E. coli to which the
recombinant plasmid has been introduced were selected on ampicillin
(50 .mu.g/ml)-containing LB agar medium (1% bacto-tryptone, 0.5%
yeast extract, 1% NaCl, 2% agar) coated with X-Gal and IPTG to
obtain the transformants.
[0167] (2) Determination of Nucleotide Sequence of HLA-A2402
Promoter Region
[0168] Four transformants obtained in the above were incubated
overnight in LB medium containing ampicillin (3 ml), followed by
purification of plasmid clone contained in each transformant by
alkaline lysis method (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
edited by F. M. Ausubel, et al., John Wiley & Sons, Inc.). The
nucleotide sequence was then determined by means of ABI PRISM.TM.
377 DNA Sequencing System (PE Biosystems). Samples for sequencing
were subjected to ABI PRISM.TM. Dye Terminator Cycle Sequencing
Ready Reaction kit (PE Biosystems) to sequence each clone as per
attached protocol. When the promoters of respective clones were
compared, it was revealed that they are totally the same. Thus, the
nucleotide sequence of promoter region of HLA-A2402 was determined,
which sequence had not been registered at GenBank database. The
nucleotide sequence registered under the Accession No. Z72422 was
compared with that of respective clones, which revealed that there
is one normal clone free of PCR mutation.
EXAMPLE 2
Cloning of H-2K.sup.b Genomic DNA Fragment
[0169] (1) Cloning of H-2K.sup.b Genomic DNA Fragment
[0170] Mouse tumor cell line EL4 (ATCC T1B-39) was cultured, and
mouse genomic DNA was purified and used in the PCR cloning.
Purification of DNA was carried out using TaKaRa LA Taq.TM. (Takara
Shuzo) suited for the amplification of long-chain DNA as per the
attached protocol. The GenBank database was then searched for
H-2K.sup.b gene needed for the construction of chimeric HLA gene,
which revealed that said gene was divided in two segments
registered under the Accession Nos. v00746 and v00747. As v00746,
the upstream 1594 bp region of H-2K.sup.b down to midstream of
intron 3 was registered and, as v00747, the downstream 1837 bp
region of H-2K.sup.b down to midstream of intron 7 was registered.
Because there was no BamHI restriction site in intron 3, which is
divided and registered as v00746 and v00747, the H-2K.sup.b gene
registered at the database was thought to have incomplete
length.
[0171] There are homologous pseudogenes or highly homologous genes
of H-2K.sup.b gene (Cell., 25:683, 1981). PCR was conducted with
TaKaRa LA Taq.TM. (Takara Shuzo) as per attached protocol using the
upstream primer H-2KB F3:
[0172] 5'-CGC AGG CTC TCA CAC TAT TCA GGT GAT CTC-3' (30 mer, SEQ
ID NO:6)
[0173] which has low homology with said complementary gene and is
coded by exon 3 of v00746, and the downstream primer H-2KB 3R:
[0174] 5'-CGG AAT TCC GAG TCT CTG ATC TTT AGC CCT GGG GGC TC-3' (38
mer, SEQ ID NO:7)
[0175] which corresponds to v00747 having EcoRI restriction site
added at the terminus, and, as a template, the purified mouse
genomic DNA above. The PCR comprised 35 cycles of reaction at
98.degree. C. for 10 seconds and 66.degree. C. for 4 minutes, and
reaction at 68.degree. C. for 10 minutes, followed by cooling to
4.degree. C.
[0176] The amplified gene fragment was ligated into KpnI and EcoRI
restriction sites of phagemid vector pBluescript to obtain a
recombinant plasmid. The recombinant plasmid was introduced into E.
coli JM109 (Toyobo) by heat shock method at 42.degree. C., and
white colonies of E. coli to which the recombinant plasmid has been
introduced were selected on ampicillin-containing LB agar medium
coated with X-Gal and IPTG to obtain the transformants. Three
transformants were incubated overnight in LB medium containing
ampicillin (3 ml). The plasmid clone contained in each transformant
was purified and subjected to analysis of nucleotide sequence in a
similar manner to the above. The nucleotide sequence of respective
clones and that of v00747 were compared, which revealed that there
was one PCR mutation independently in two clones and three PCR
mutations in one clone. There were five nucleotides commonly found
in these three clones, which were different from those of v00747.
These nucleotides were found in regions corresponding to intron 6
and 3' non-coding region. Furthermore, the unregistered intron 3
region contained a nucleotide resulted from PCR mutation that is
different among 3 clones. The determination of nucleotide sequence
was therefore partly impossible concerning the unregistered region,
which could be achieved after re-cloning the unregistered intron 3
region using a polymerase with high 3'.fwdarw.5' exonuclease
activity.
[0177] (2) Determination of Nucleotide Sequence of H-2K.sup.b
Intron 3
[0178] To determine the nucleotide sequence of the unregistered
region, a region containing the unregistered intron 3 region was
cloned by PCR with Native Pfu DNA Polymerase (Stratagene) as per
attached protocol using the purified mouse genomic DNA as a
template. The PCR was carried out using an upstream primer H-2kb
F5:
[0179] 5'-AGG ACT TGG ACT CTG AGA GGC AGG GTC TT-3' (29 mer, SEQ ID
NO:8),
[0180] which is registered as v00746, and the downstream primer
H-2kb 5R:
[0181] 5'-CAT AGT CCC CTC CTT TTC CAC CTG TGA GAA-3' (30 mer, SEQ
ID NO:9),
[0182] which is registered as v00747. The PCR comprised heat
treatment at 95.degree. C. for 45 seconds, 25 cycles of reaction at
95.degree. C. for 45 seconds, 68.degree. C. for 1 minute and
72.degree. C. for 4 minutes, and reaction at 72.degree. C. for 10
minutes, followed by cooling to 4.degree. C. The amplified gene
fragment was ligated into BamHI and BglII restriction sites of
phagemid vector pBluescript to obtain a recombinant plasmid. The
recombinant plasmid was introduced into E. coli JM109 (Toyobo) by
heat shock method at 42.degree. C., and white colonies of E. coli
to which the recombinant plasmid has been introduced were selected
on ampicillin (50 .mu.g/ml)-containing LB agar medium (1%
bacto-tryptone, 0.5% yeast extract, 1% NaCl, 2% agar) coated with
X-Gal and IPTG to obtain the transformants. Five transformants were
incubated overnight in LB medium containing ampicillin (3 ml) and
the plasmid clone contained in each transformant was purified and
subjected to analysis of nucleotide sequence in a similar manner to
the above. The intron 3 regions of respective clones analyzed were
compared, which revealed that the sequences agreed completely. The
nucleotide sequence of intron 3 region was thus determined. In
addition, the region spanning from the BamHI site in the
unregistered region to v00747 revealed to be 463 bp.
[0183] (3) Construction of H-2K.sup.b Genomic DNA
[0184] As a result of determination of nucleotide sequence of the
unregistered region in (2) above, the entire nucleotide sequence of
H-2K.sup.b genomic DNA necessary for the construction of the
objective chimeric HLA gene was determined. It became clear that
the objective H-2K.sup.b genomic DNA can be constructed by
combining two clones obtained in the above, i.e., H-2K.sup.b#20
free of PCR mutation and H-2K.sup.b#26 free of PCR mutation, in 5'-
and 3'-regions, respectively. Accordingly, these clones were
cleaved by a restriction enzyme and respective regions having no
PCR mutations were combined to construct the H-2K.sup.b genomic DNA
free of PCR mutations. The schematic diagram for construction is
shown in FIG. 1.
[0185] The both clones were cleaved at the BglII and EcoRI
restriction sites and ligated to obtain recombinant plasmid. The
recombinant plasmid was introduced into E. coli JM109 (Toyobo) by
heat shock method at 42.degree. C., and white colonies of E. coli
to which the recombinant plasmid has been introduced were selected
on ampicillin-containing LB agar medium coated with X-Gal and IPTG
to obtain the transformants. Three transformants were incubated
overnight in LB medium containing ampicillin (3 ml). The plasmid
clone contained in each transformant was purified by alkaline lysis
method and subjected to sequence analysis in a similar manner to
the above. As a result, it was revealed that all the transformants
contained a plasmid encoding H-2K.sup.b genomic DNA free of PCT
mutation.
[0186] The nucleotide sequence of H-2K.sup.b genomic DNA herein
obtained corresponds to the nucleotide sequence downstream from the
nucleotide at position 1551 of SEQ ID NO: 1 inclusive, which is
described below.
EXAMPLE 3
Construction of Chimera Genomic DNA (HLA-A2402/K.sup.b DNA)
[0187] The Plasmid HLA-A2402#1 containing HLA-A2402 genomic DNA
obtained in Example 1 was cleaved at BglII restriction site and the
plasmid H-2K.sup.b#20/26 containing H-2K.sup.b genomic DNA obtained
in Example 2 was cleaved at BamHI restriction site, and the
resultant fragments were ligated to give a recombinant plasmid. The
schematic construction is shown in FIG. 2. The recombinant plasmid
was introduced into E. coli JM109 (Toyobo) by heat shock method at
42.degree. C., and white colonies of E. coli to which the
recombinant plasmid has been introduced were selected on
ampicillin-containing LB agar medium coated with X-Gal and IPTG to
obtain the transformants. Ten transformants were incubated
overnight in LB medium containing ampicillin (3 ml). The plasmid
clone contained in each transformant was purified and subjected to
sequence analysis in a similar manner to the above. As a result, it
was revealed that three transformants contained a plasmid carrying
the intended chimeric gene HLA-A2402/K.sup.b DNA, which may be
referred to as simply "A2402/K.sup.b DNA". The genomic sequence of
the constructed HLA-A2402/K.sup.b is shown in SEQ ID NO: 1.
EXAMPLE 4
Splicing Analysis of Chimera Genomic DNA
[0188] Mouse tumor cell line EL4 was transfected with the
constructed chimeric HLA gene (HLA-A2402/K.sup.b gene) with Electro
Gene Transfer GTE-10 (Shimadzu) as per the attached protocol. Two
days later, total RNA was purified from transfected EL4 cells and
un-transfected EL4 cells (control) by using ISOGEN (Nippon Gene) as
per the attached protocol. Reverse transcription was performed
using SuperScript Choice System (GIBCO BRL) as per the attached
protocol using Oligo(dT).sub.12-18 and a part of said RNA as a
template to synthesize cDNA. In addition, chimera gene was
specifically amplified by PCR using Native Pfu DNA Polymerase
(Stratagene) and a part of said cDNA as a template.
[0189] PCR was conducted using an upstream primer Chimera-F2:
[0190] 5'-CGA ACC CTC GTC CTG CTA CTC TC-3' (23 mer, SEQ ID NO:10),
which is encoded in exon 1 of HLA-A2402 gene and has low homology
with H-2K.sup.b gene, and a downstream primer Chimera-R2:
[0191] 5'-AGC ATA GTC CCC TCC TTT TCC AC-3' (23 mer, SEQ ID NO:11),
which is encoded in exon 8 of H-2K.sup.b gene and has low homology
with HLA-A2402 gene, under the conditions of heat treatment at
95.degree. C. for 45 seconds, 40 cycles of reaction at 95.degree.
C. for 45 seconds, 53.degree. C. for 1 minute and 72.degree. C. for
2 minutes, and reaction at 72.degree. C. for 10 minutes, followed
by cooling to 4.degree. C.
[0192] As a result, about 1.1 kbp gene fragments were specifically
amplified only in transfected EL4 cells. Based on this result, it
was estimated that the transferred chimera genomic DNA was
transcribed in mouse cells, that is, HLA promoter functioned and
mRNA spliced at the predicted position was expressed. The amplified
fragment by PCR above was sequenced, and whereby the nucleotide
sequence of cDNA encoding HLA-A2402/K.sup.b was determined as
expected. The nucleotide sequence of cDNA encoding said
HLA-A2402/K.sup.b is shown in SEQ ID NO:2 and the amino acid
sequence thereof in SEQ ID NO:3. Furthermore, FIG. 3(A) to 3(P)
show the relationship between the genome sequence of
HLA-A2402/K.sup.b (SEQ ID NO: 1) and the cDNA sequence (SEQ ID
NO:2) aligned in parallel. In the figure, the upper line is
HLA-A2402/K.sup.b genome sequence shown in SEQ ID NO: 1 and the
lower line is HLA-A2402/K.sup.b cDNA sequence shown in SEQ ID NO:
2.
EXAMPLE 5
Preparation of DNA Solution for Microinjection
[0193] Plasmid (11 .mu.g) encoding the constructed chimeric HLA
gene was digested with restriction enzymes HindIII and EcoRI, and
also restriction enzyme DraI that cleaves only vector. After gel
electrophoresis (1% SeaKem GTG, Nippon Gene), gel fragment
containing chimera DNA was recovered. A DNA solution for
microinjection was prepared by purifying the transgene with
Prep-A-Gene purification kit (BioRad) as per the attached protocol
and dissolving in {fraction (1/10)} TE buffer (10 mM Tris (pH 8),
0.1 mM EDTA (pH 8)).
EXAMPLE 6
Introduction into Mouse Fertilized Egg and Identification of
Transgenic Mouse
[0194] The injection of chimera gene construct was performed using
fertilized eggs derived from a C57BL/6 mouse strain.
[0195] The fertilized eggs of C57BL/6 mouse strain were used
because C57BL/6 mice express as the class I molecule H-2b not
H-2K.sup.d having similar binding motifs to HLA-A2402. Accordingly,
a transgenic mouse of said C57BL/6 line can advantageously avoid
cross reaction when an HLA-A24-binding tumor antigen peptide is
administered, because the endogenous mouse class I cannot present
said peptide on the cell surface.
[0196] In the first injection, the chimera construct was injected
into 81 fertilized eggs, and the eggs were transferred to 4
recipient mice, which resulted in no delivery.
[0197] In the second injection, the chimera construct was injected
into 50 fertilized eggs, and the eggs were transferred to 2
recipient mice, which resulted in delivery of 4 offspring, but all
of them died before weaning.
[0198] In the third injection, the chimera construct was injected
into 101 fertilized eggs, and the eggs were transferred to 4
recipient mice, which resulted in delivery of 11 offspring, but all
of them died before weaning.
[0199] In the fourth injection, the chimera construct was injected
into 168 fertilized eggs, and the eggs were transferred to 6
recipient mice, which resulted in delivery of 22 offspring, and 19
of them were weaned from the breast. Four of them, i.e., 01-4,
04-2, 05-1 and 05-6 were identified as a transgenic mouse; however,
01-4 mouse was unable to mate due to malformation and 05-6 mouse
died shortly after weaning.
[0200] In the fifth injection, the chimera construct was injected
into 221 fertilized eggs, and the eggs were transferred to 8
recipient mice, which resulted in delivery of 14 offspring, and 6
of them were weaned from the breast. Three of them, i.e., 04-1,
04-5 and 04-6 were identified as a transgenic mouse.
[0201] In the sixth injection, the chimera construct was injected
into 225 fertilized eggs, and the eggs were transferred to 8
recipient mice, which resulted in delivery of 13 offspring, and 9
of them were weaned from the breast. Three of them, i.e., 10-5,
14-1 and 15-2 were identified as a transgenic mouse.
[0202] The transgenic mouse was identified by carrying out PCR with
TaKaRa LA Taq.TM. (Takara Shuzo) as per the attached protocol using
the same primers as those used for cloning of HLA-A2402 gene
(HLA26-1F, SEQ ID NO: 4; and A24-BglII30, SEQ ID NO: 5) and a tail
DNA preparation as a template, applying to 1% agarose gel
electrophoresis, and selecting a mouse on the basis of the
existence of 1.5 kbp DNA band.
EXAMPLE 7
Expression of Transgene Product in Transgenic Mouse
[0203] Splenocytes were recovered from spleens isolated from mice
of 8 transgenic lines 04-2, 05-1, 04-1, 04-5, 04-6, 10-5, 14-1 and
15-2 constructed in Example 6, according to CURRENT PROTOCOLS IN
IMMUNOLOGY, edited by J. E. Coliganl et al., John Wiley & Sons,
Inc. Expression of HLA-A2402/K.sup.b, which is a protein derived
from transgene, on the cell surface of transgenic mouse splenocytes
was analyzed by flow cytometry. As control, splenocytes prepared
from C57BL/6 strain were used. Specifically, 5.times.10.sup.6
splenocytes were stained by FITC-labeled anti-HLA antibody B9.12.1
(Immunotech). Endogenous mouse class I was stained by FITC-labeled
anti-H-2K.sup.b monoclonal antibody AF6-88.5 (Pharmingen).
[0204] As a result, 5 lines, i.e., 04-1, 04-5, 10-5, 14-1 and 15-2
showed expression specific for HLA class I. Among them, only 04-1
line revealed to have ability of reproduction. On the other hand,
the other 3 lines, i.e., 04-6, 04-2 and 05-1, showed no expression
specific for HLA class I. Thus, 8 transgenic mouse lines were
constructed but, among them, only 04-1 line showed class I
expression manner and achieved homozygosity.
EXAMPLE 8
Establishment of Transformed Cells Expressing HLA-A2402
[0205] A transformed cell Jurkat-A2402/K.sup.b which stably
expresses HLA-2402/K.sup.b was established in order to evaluate the
CTL inducing ability of the transgenic mouse prepared in the
above.
[0206] (1) Construction of Expression Vector
[0207] Spleen was removed from a Tg mouse and splenocytes were
prepared. Total RNA was prepared with ISOGEN (Nippon Gene) as per
the attached protocol. Reverse transcription was then performed
with SuperScript Choice System (GIBCO BRL) as per the attached
protocol using Oligo(dT).sub.12-18 and, as a template, a part of
said RNA to synthesize cDNA. PCR was then conducted by LA-PCR kit
(Takara Shuzo) as per the attached protocol using a part of said
cDNA as a template, and the upstream primer chi.PF1:
[0208] 5'-CCC AAG CTT CGC CGA GGA TGG CCG TCA TGG CGC CCC GAA-3'
(SEQ ID NO: 12); and the downstream primer chi.PR1:
[0209] 5'-CCG GAA TTC TGT CTT CAC GCT AGA GAA TGA GGG TCA TGA
AC-3', SEQ ID NO: 13). PCR comprised heat treatment at 95.degree.
C. for 45 seconds, 25 cycles of reaction at 95.degree. C. for 45
seconds, 60.degree. C. for 1 minute and 68.degree. C. for 2
minutes, and reaction at 72.degree. C. for 10 minutes, followed by
cooling to 4.degree. C. The PCR amplified gene was introduced into
an expression vector pcDNA3.1(+) (Invitrogen) to construct an
expression vector encoding HLA-A2402/K.sup.b.
[0210] (2) Introduction into Jurkat Cells
[0211] The vector above (10 .mu.g) was linealized by digesting with
PvuI restriction enzyme. Jurkat cells (ATCC T1B-152)
5.times.10.sup.6 were transfected with the constructed chimeric HLA
gene by means of a gene-transfer device (GIBCO BRL) as per the
attached protocol. Cells were seeded into 96-well plate at 0.5
cells/well and cultured in a medium containing Geneticin (0.6
mg/ml). As a result, cell proliferation was observed in 6 wells (6
clones, A-2, A-4, A-6, A-9, A-10 and A-11). Among them, A-10 showed
the highest expression of transgene and said clone was established
as Jurkat-A2402/K.sup.b cell.
EXAMPLE 9
Test for CTL Inducing Ability of Transgenic Mouse
[0212] Human tumor antigen HER-2/neu is known to be overexpressed
in breast, ovarian and lung cancers, and is shown by in vitro
experiment that a peptide derived therefrom has an activity of
inducing specific CTLs in peripheral blood of HLA-A24 positive
healthy subjects (Int. J. Cancer., 87:553, 2000).
[0213] The transgenic mouse was immunized with HLA-A24-binding
peptide HER-2/neu.sub.780-788 (SEQ ID NO: 14) derived from said
human tumor antigen and MHC Class II I-A.sup.b-restricted helper
peptide originated from tetanus toxin (FNNFTVSFWLRVPKVSASHLE), and
examined whether specific CTLs are induced as is the case with
human. That is, HER-2/neu.sub.780-788 and helper peptide were
adjusted to 40 mg/ml and 20 mg/ml, respectively, in DMSO and
diluted with physiological saline to 2 mg/ml and 1 mg/ml,
respectively. They were mixed with equal amount of Freund's
incomplete adjuvant (Wako Pure Chemical Industries, Ltd.) using
glass syringe to prepare water-in-oil emulsion. The resultant
preparation (200 .mu.l) was injected into a transgenic mouse (04-1
line) subcutaneously in the base of the tail for immunization.
Seven days after initiation of experiment, spleen was removed and
grounded on the frosted part of glass slide, and splenocytes were
recovered and prepared. A portion of splenocytes undergone
hemolysis treatment with ACK buffer (0.15 M NH.sub.4Cl, 10 mM
KHCO.sub.3, 0.1 mM EDTA, pH 7.2-7.4) was exposed to X ray radiation
(2,000 rad), pulsed with the above-mentioned peptide (100 .mu.g/ml)
for 1 hour, and seeded into 24-well plate at
0.7.times.10.sup.6/well. Non-radiated, non-peptide-pulsed
splenocytes (7.times.10.sup.6/well) were added together and
stimulated again at 37.degree. C. (final concentration of peptide,
1 .mu.g/ml). In vitro stimulation was carried out for 6 days in 10
ml of a culture solution (CTM culture solution) containing 10% FCS,
10 mM HEPES, 20 mM L-glutamine, 1 mM sodium pyruvate, 1 mM MEM
nonessential amino acid, 1% MEM vitamin and 55 .mu.M
2-mercaptoethanol in RPMI1640 medium.
[0214] On the other hand, Jurkat-A2402/K.sup.b cells prepared in
Example 8 were labeled with .sup.51Cr (3.7 MBq/10.sup.6 cells) and
pulsed with the peptide above at 100 .mu.g/ml for one hour. The
labeling was carried out over 2 hours, and 1 hour after initiation
of labeling, peptide was added to make the final concentration 100
.mu.g/ml. Cells that were not pulsed with peptide were prepared as
control target cells.
[0215] CTL-inducing activity was determined by .sup.51Cr release
assay (J. Immunol., 159:4753, 1997), wherein the previously
prepared transgenic mouse splenocyte preparation was added to said
Jurkat-A2402/K.sup.b cells as the target cells. The results are
shown in FIG. 4. As a result, induction of specific CTLs by
stimulation with HER-2/neu.sub.780-788 was observed.
[0216] Furthermore, the CTL inducing ability was tested in the same
manner using MAGE-3.sub.195-203 (SEQ ID NO: 18), CEA652-660 (SEQ ID
NO: 19) and CEA.sub.268-277 (SEQ ID NO: 20), which are also known
to be HLA-A24-binding tumor antigen peptide like
HER-2/neu.sub.780-788. The results are shown in FIG. 5 to FIG. 7.
As a result, induction of specific CTLs by stimulation with these
known HLA-A24-binding tumor antigen peptides was observed.
[0217] From these results, the HLA-A24 transgenic mouse of the
present invention were revealed to be an animal model for human
that can be used for evaluation of HLA-A24-binding tumor antigen
protein or tumor antigen peptide.
EXAMPLE 10
CTL Induction by Tumor Antigen PSA-Originated Peptide
[0218] Since it was found that the HLA-A24 transgenic mouse of the
present invention makes it possible to evaluate HLA-A24-binding
tumor antigen protein or tumor antigen peptide in vivo, evaluation
of peptides that have not been identified as a tumor antigen
peptide yet was conducted using said mouse. In the Experiment,
peptides originated from human tumor antigen PSA were used. There
have not been any reports regarding HLA-A24-binding tumor antigen
peptide PSA originated from regions so far.
[0219] The amino acid sequence of PSA protein was searched for the
sequence corresponding to human HLA-A24-binding motifs, that is, a
motif wherein the 2nd amino acid is tyrosine, phenylalanine,
methionine or tryptophan and the C-terminal amino acid is
phenylalanine, tryptophan, leucine, isoleucine or methionine, and
two kinds of sequences consisting of 9 amino acids or 10 amino
acids (PSA.sub.152-160: SEQ ID NO:15 and PSA.sub.248-257: SEQ ID
NO:16) were identified. Said PSA.sub.152-160 and PSA.sub.248-257
correspond to partial sequences of amino acid Nos. 152-160 and
248-257, respectively, of the amino acid sequence of PSA.
[0220] Tumor antigenicity of PSA.sub.152-160 or PSA.sub.248-257 was
analyzed using HLA-A2402/K.sup.b transgenic mouse of 04-1 line
constructed in Example 7. A transgenic mouse was immunized with
PSA.sub.152-160 or PSA.sub.248-257 in association with tetanus
toxin-derived mouse MHC class II I-A.sup.b-restricted helper
peptide (FNNFTVSFWLRVPKVSASHLE), and the CTL-inducing activity was
determined in a manner similar to that described in Example 9.
FIGS. 8 and 9 show the results of determination of CTL induction by
PSA.sub.152-160, and PSA.sub.248-257, respectively. PSA.sub.152-160
caused specific CTL induction but PSA.sub.248-257 did not.
Accordingly, it became evident that PSA.sub.152-160 has tumor
antigenicity in vivo, in other words, it is an HLA-A24-binding
tumor antigen peptide. The results above also demonstrate that
peptides having the binding motif of HLA-A24 type do not
necessarily have antigenicity in vivo and that the present method
is useful for identifying active peptides.
Sequence Listing Free Text
[0221] In the nucleotide sequence shown in SEQ ID NO: 1, the region
between No. 1 and No. 1550 is of human origin, and the region
between No. 1551 and No. 3587 is of mouse origin.
[0222] In the nucleotide sequence shown in SEQ ID NO: 2, the region
between No. 1 and No. 618 is of human origin and the region between
No. 619 and No. 1119 is of mouse origin.
[0223] In the amino acid sequence shown in SEQ ID NO: 3, the region
between No. 1 and No. 206 is of human origin and the region between
No. 207 and No. 372 is of mouse origin.
[0224] The nucleotide sequence shown in SEQ ID NO: 4 is a PCR
primer.
[0225] The nucleotide sequence shown in SEQ ID NO: 5 is a PCR
primer.
[0226] The nucleotide sequence shown in SEQ ID NO: 6 is a PCR
primer.
[0227] The nucleotide sequence shown in SEQ ID NO: 7 is a PCR
primer.
[0228] The nucleotide sequence shown in SEQ ID NO: 8 is a PCR
primer.
[0229] The nucleotide sequence shown in SEQ ID NO: 9 is a PCR
primer.
[0230] The nucleotide sequence shown in SEQ ID NO: 10 is a PCR
primer.
[0231] The nucleotide sequence shown in SEQ ID NO: 11 is a PCR
primer.
[0232] The nucleotide sequence shown in SEQ ID NO: 12 is a PCR
primer.
[0233] The nucleotide sequence shown in SEQ ID NO: 13 is a PCR
primer.
[0234] In the amino acid sequence of SEQ ID NO: 17, the second
amino acid is phenylalanine, tyrosine, methionine or tryptophan,
and the ninth amino acid is phenylalanine, leucine, isoleucine,
tryptophan or methionine.
INDUSTRIAL APPLICABILITY
[0235] According to the present invention, it is provided
transgenic mice which have had an HLA-A24 gene introduced and in
which CTL is induced in response to stimulation with an
HLA-A24-binding antigen, a method of screening a therapeutic or
preventive agent for tumors or virus infections using said
transgenic mice, and HLA-A24-binding tumor antigen peptides of PSA
origin selected by the screening method.
Sequence CWU 1
1
21 1 3857 DNA Artificial Sequence Description of Artificial
Sequence The DNA region from position 1 to position 1550 is derived
from human, and the DNA region from position 1551 to position 3857
is derived from mouse. 1 aagcttactc tctggcacca aactccatgg
gatgattttt cttctagaag agtccaggtg 60 gacaggtaag gagtgggagt
cagggagtcc agttcaggga cagagattac gggatgaaaa 120 gtgaaaggag
agggacgggg cccatgccga gggtttctcc cttgtttctc agacagctct 180
tgggccaaga ttcagggaga cattgagaca gagcgcttgg cacagaagca gaggggtcag
240 ggcgaagtcc cagggcccca ggcgtggctc tcagggtctc aggccccgaa
ggcggtgtat 300 ggattgggga gtcccagcct tggggattcc ccaactccgc
agtttctttt ctccctctcc 360 caacctatgt agggtccttc ttcctggata
ctcacgacgc ggacccagtt ctcactccca 420 ttgggtgtcg ggtttccaga
gaagccaatc agtgtcgtcg cggtcgctgt tctaaagtcc 480 gcacgcaccc
accgggactc agattctccc cagacgccga ggatggccgt catggcgccc 540
cgaaccctcg tcctgctact ctcgggggcc ctggccctga cccagacctg ggcaggtgag
600 tgcggggtcg ggagggaaac ggcctctgcg gggagaagca aggggcccgc
ctggcggggg 660 cgcaagaccc gggaagccgc gccgggagga gggtcgggcg
ggtctcagcc actcctcgtc 720 cccaggctcc cactccatga ggtatttctc
cacatccgtg tcccggcccg gccgcgggga 780 gccccgcttc atcgccgtgg
gctacgtgga cgacacgcag ttcgtgcggt tcgacagcga 840 cgccgcgagc
cagaggatgg agccgcgggc gccgtggata gagcaggagg ggccggagta 900
ttgggacgag gagacaggga aagtgaaggc ccactcacag actgaccgag agaacctgcg
960 gatcgcgctc cgctactaca accagagcga ggccggtgag tgaccccggc
ccggggcgca 1020 ggtcacgacc cctcatcccc cacggacggg ccgggtcgcc
cacagtctcc gggtccgaga 1080 tccaccccga agccgcggga ccccgagacc
cttgccccgg gagaggccca ggcgccttaa 1140 cccggtttca ttttcagttt
aggccaaaaa tccccccggg ttggtcgggg ccgggcgggg 1200 ctcgggggac
tgggctgacc gcggggtcgg ggccaggttc tcacaccctc cagatgatgt 1260
ttggctgcga cgtggggtcg gacgggcgct tcctccgcgg gtaccaccag tacgcctacg
1320 acggcaagga ttacatcgcc ctgaaagagg acctgcgctc ttggaccgcg
gcggacatgg 1380 cggctcagat caccaagcgc aagtgggagg cggcccatgt
ggcggagcag cagagagcct 1440 acctggaggg cacgtgcgtg gacgggctcc
gcagatacct ggagaacggg aaggagacgc 1500 tgcagcgcac gggtaccagg
ggccacgggg cgcctacctg atcgcctgta gatcctgtgt 1560 gacacacctg
taccttgtcc cccagagtca ggggctggga gtcattttct ctggctacac 1620
acttagtgat ggctgttcac ttggactgac agttaatgtt ggtcagcaag gtgactacaa
1680 tggttgagtc tcaatggtgt caccttccag gatcatacag ccctaatttt
aatatgaact 1740 caaacacata ttaaattagt tattttccat tccctcctcc
attctttgac tacctctctc 1800 atgctattga acatcacata aggatggcca
tgtttaccca atggctcatg tggattccct 1860 cttagcttct gagtcccaaa
agaaaatgtg cagtcctgtg ctgaggggac cagctctgct 1920 tttggtcact
agtgcgatga cagttgaagt gtcaaacaga cacatagttc actgtcatca 1980
ttgatttaac tgagtcttgg gtagatttca gtttgtcttg ttaattgtgt gatttcttaa
2040 atcttccaca cagattcccc aaaggcccat gtgacccatc acagcagacc
tgaagataaa 2100 gtcaccctga ggtgctgggc cctgggcttc taccctgctg
acatcaccct gacctggcag 2160 ttgaatgggg aggagctgat ccaggacatg
gagcttgtgg agaccaggcc tgcaggggat 2220 ggaaccttcc agaagtgggc
atctgtggtg gtgcctcttg ggaaggagca gtattacaca 2280 tgccatgtgt
accatcaggg gctgcctgag cccctcaccc tgagatgggg taaggagagt 2340
gtgggtgcag agctggggtc agggaaagct ggagctttct gcagaccctg agctgctcag
2400 ggctgagagc tggggtcatg accctcacct tcatttcttg tacctgtcct
tcccagagcc 2460 tcctccatcc actgtctcca acatggcgac cgttgctgtt
ctggttgtcc ttggagctgc 2520 aatagtcact ggagctgtgg tggcttttgt
gatgaagatg agaaggagaa acacaggtag 2580 gaaagggcag agtctgagtt
ttctctcagc ctcctttaga gtgtgctctg ctcatcaatg 2640 gggaacacag
gcacacccca cattgctact gtctctaact gggtctgctg tcagttctgg 2700
gaacttccta gtgtcaagat cttcctggaa ctctcacagc ttttcttctc acaggtggaa
2760 aaggagggga ctatgctctg gctccaggtt agtgtgggga cagagttgtc
ctggggacat 2820 tggagtgaag ttggagatga tgggagctct gggaatccat
aatagctcct ccagagaaat 2880 cttctaggtg cctgagttgt gccatgaaat
gaatatgtac atgtacatat gcatatacat 2940 ttgttttgtt ttaccctagg
ctcccagacc tctgatctgt ctctcccaga ttgtaaaggt 3000 gacactctag
ggtctgattg gggaggggca atgtggacat gattgggttt caggaactcc 3060
cagaatcccc tgtgagtgag tgatgggttg ttcgaatgtt gtcttcacag tgatggttca
3120 tgaccctcat tctctagcgt gaagacagct gcctggagtg gacttggtga
cagacaatgt 3180 cttctcatat ctcctgtgac atccagagcc ctcagttctc
tttagtcaag tgtctgatgt 3240 tccctgtgag cctatggact caatgtgaag
aactgtggag cccagtccac ccctctacac 3300 caggaccctg tccctgcact
gctctgtctt cccttccaca gccaaccttg ctggttcagc 3360 caaacactga
gggacatctg tagcctgtca gctccatgct accctgacct gcaactcctc 3420
acttccacac tgagaataat aatttgaatg taaccttgat tgttatcatc ttgacctagg
3480 gctgatttct tgttaatttc atggattgag aatgcttaga ggttttgttt
gtttgtttga 3540 ttgatttgtt tttttgaaga aataaatgat agatgaataa
acttccagaa tctgggtcac 3600 tatgctgtgt gtatctgttg ggacaggatg
agactgtagc agctgagtgt gaacagggct 3660 gtgccgaggt gggctcagtt
tgctttgatc tgtgatgggg ccacacctcc actgtgtcac 3720 ctctgggctc
tgttccctct atcactatga ggcacatgct gagagtttgt ggtcacaaag 3780
acacagggaa ggcctgagcc ttgccctgtc cccaggatta tgagccccca gggctaaaga
3840 tcagagactc ggaattc 3857 2 1119 DNA Artificial Sequence
Description of Artificial Sequence The DNA region from position 1
to position 618 is derived from human, and the DNA region from
position 619 to position 1119 is derived from mouse. 2 atg gcc gtc
atg gcg ccc cga acc ctc gtc ctg cta ctc tcg ggg gcc 48 Met Ala Val
Met Ala Pro Arg Thr Leu Val Leu Leu Leu Ser Gly Ala 5 10 15 ctg gcc
ctg acc cag acc tgg gca ggc tcc cac tcc atg agg tat ttc 96 Leu Ala
Leu Thr Gln Thr Trp Ala Gly Ser His Ser Met Arg Tyr Phe 20 25 30
tcc aca tcc gtg tcc cgg ccc ggc cgc ggg gag ccc cgc ttc atc gcc 144
Ser Thr Ser Val Ser Arg Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala 35
40 45 gtg ggc tac gtg gac gac acg cag ttc gtg cgg ttc gac agc gac
gcc 192 Val Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp
Ala 50 55 60 gcg agc cag agg atg gag ccg cgg gcg ccg tgg ata gag
cag gag ggg 240 Ala Ser Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu
Gln Glu Gly 65 70 75 80 ccg gag tat tgg gac gag gag aca ggg aaa gtg
aag gcc cac tca cag 288 Pro Glu Tyr Trp Asp Glu Glu Thr Gly Lys Val
Lys Ala His Ser Gln 85 90 95 act gac cga gag aac ctg cgg atc gcg
ctc cgc tac tac aac cag agc 336 Thr Asp Arg Glu Asn Leu Arg Ile Ala
Leu Arg Tyr Tyr Asn Gln Ser 100 105 110 gag gcc ggt tct cac acc ctc
cag atg atg ttt ggc tgc gac gtg ggg 384 Glu Ala Gly Ser His Thr Leu
Gln Met Met Phe Gly Cys Asp Val Gly 115 120 125 tcg gac ggg cgc ttc
ctc cgc ggg tac cac cag tac gcc tac gac ggc 432 Ser Asp Gly Arg Phe
Leu Arg Gly Tyr His Gln Tyr Ala Tyr Asp Gly 130 135 140 aag gat tac
atc gcc ctg aaa gag gac ctg cgc tct tgg acc gcg gcg 480 Lys Asp Tyr
Ile Ala Leu Lys Glu Asp Leu Arg Ser Trp Thr Ala Ala 145 150 155 160
gac atg gcg gct cag atc acc aag cgc aag tgg gag gcg gcc cat gtg 528
Asp Met Ala Ala Gln Ile Thr Lys Arg Lys Trp Glu Ala Ala His Val 165
170 175 gcg gag cag cag aga gcc tac ctg gag ggc acg tgc gtg gac ggg
ctc 576 Ala Glu Gln Gln Arg Ala Tyr Leu Glu Gly Thr Cys Val Asp Gly
Leu 180 185 190 cgc aga tac ctg gag aac ggg aag gag acg ctg cag cgc
acg gat tcc 624 Arg Arg Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg
Thr Asp Ser 195 200 205 cca aag gcc cat gtg acc cat cac agc aga cct
gaa gat aaa gtc acc 672 Pro Lys Ala His Val Thr His His Ser Arg Pro
Glu Asp Lys Val Thr 210 215 220 ctg agg tgc tgg gcc ctg ggc ttc tac
cct gct gac atc acc ctg acc 720 Leu Arg Cys Trp Ala Leu Gly Phe Tyr
Pro Ala Asp Ile Thr Leu Thr 225 230 235 240 tgg cag ttg aat ggg gag
gag ctg atc cag gac atg gag ctt gtg gag 768 Trp Gln Leu Asn Gly Glu
Glu Leu Ile Gln Asp Met Glu Leu Val Glu 245 250 255 acc agg cct gca
ggg gat gga acc ttc cag aag tgg gca tct gtg gtg 816 Thr Arg Pro Ala
Gly Asp Gly Thr Phe Gln Lys Trp Ala Ser Val Val 260 265 270 gtg cct
ctt ggg aag gag cag tat tac aca tgc cat gtg tac cat cag 864 Val Pro
Leu Gly Lys Glu Gln Tyr Tyr Thr Cys His Val Tyr His Gln 275 280 285
ggg ctg cct gag ccc ctc acc ctg aga tgg gag cct cct cca tcc act 912
Gly Leu Pro Glu Pro Leu Thr Leu Arg Trp Glu Pro Pro Pro Ser Thr 290
295 300 gtc tcc aac atg gcg acc gtt gct gtt ctg gtt gtc ctt gga gct
gca 960 Val Ser Asn Met Ala Thr Val Ala Val Leu Val Val Leu Gly Ala
Ala 305 310 315 320 ata gtc act gga gct gtg gtg gct ttt gtg atg aag
atg aga agg aga 1008 Ile Val Thr Gly Ala Val Val Ala Phe Val Met
Lys Met Arg Arg Arg 325 330 335 aac aca ggt gga aaa gga ggg gac tat
gct ctg gct cca ggc tcc cag 1056 Asn Thr Gly Gly Lys Gly Gly Asp
Tyr Ala Leu Ala Pro Gly Ser Gln 340 345 350 acc tct gat ctg tct ctc
cca gat tgt aaa gtg atg gtt cat gac cct 1104 Thr Ser Asp Leu Ser
Leu Pro Asp Cys Lys Val Met Val His Asp Pro 355 360 365 cat tct cta
gcg tga 1119 His Ser Leu Ala 370 3 372 PRT Artificial Sequence
Description of Artificial Sequence The polypeptide region from
position 1 to position 206 is derived from human, 207 to position
372 is derived from mouse. 3 Met Ala Val Met Ala Pro Arg Thr Leu
Val Leu Leu Leu Ser Gly Ala 5 10 15 Leu Ala Leu Thr Gln Thr Trp Ala
Gly Ser His Ser Met Arg Tyr Phe 20 25 30 Ser Thr Ser Val Ser Arg
Pro Gly Arg Gly Glu Pro Arg Phe Ile Ala 35 40 45 Val Gly Tyr Val
Asp Asp Thr Gln Phe Val Arg Phe Asp Ser Asp Ala 50 55 60 Ala Ser
Gln Arg Met Glu Pro Arg Ala Pro Trp Ile Glu Gln Glu Gly 65 70 75 80
Pro Glu Tyr Trp Asp Glu Glu Thr Gly Lys Val Lys Ala His Ser Gln 85
90 95 Thr Asp Arg Glu Asn Leu Arg Ile Ala Leu Arg Tyr Tyr Asn Gln
Ser 100 105 110 Glu Ala Gly Ser His Thr Leu Gln Met Met Phe Gly Cys
Asp Val Gly 115 120 125 Ser Asp Gly Arg Phe Leu Arg Gly Tyr His Gln
Tyr Ala Tyr Asp Gly 130 135 140 Lys Asp Tyr Ile Ala Leu Lys Glu Asp
Leu Arg Ser Trp Thr Ala Ala 145 150 155 160 Asp Met Ala Ala Gln Ile
Thr Lys Arg Lys Trp Glu Ala Ala His Val 165 170 175 Ala Glu Gln Gln
Arg Ala Tyr Leu Glu Gly Thr Cys Val Asp Gly Leu 180 185 190 Arg Arg
Tyr Leu Glu Asn Gly Lys Glu Thr Leu Gln Arg Thr Asp Ser 195 200 205
Pro Lys Ala His Val Thr His His Ser Arg Pro Glu Asp Lys Val Thr 210
215 220 Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Asp Ile Thr Leu
Thr 225 230 235 240 Trp Gln Leu Asn Gly Glu Glu Leu Ile Gln Asp Met
Glu Leu Val Glu 245 250 255 Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln
Lys Trp Ala Ser Val Val 260 265 270 Val Pro Leu Gly Lys Glu Gln Tyr
Tyr Thr Cys His Val Tyr His Gln 275 280 285 Gly Leu Pro Glu Pro Leu
Thr Leu Arg Trp Glu Pro Pro Pro Ser Thr 290 295 300 Val Ser Asn Met
Ala Thr Val Ala Val Leu Val Val Leu Gly Ala Ala 305 310 315 320 Ile
Val Thr Gly Ala Val Val Ala Phe Val Met Lys Met Arg Arg Arg 325 330
335 Asn Thr Gly Gly Lys Gly Gly Asp Tyr Ala Leu Ala Pro Gly Ser Gln
340 345 350 Thr Ser Asp Leu Ser Leu Pro Asp Cys Lys Val Met Val His
Asp Pro 355 360 365 His Ser Leu Ala 370 4 36 DNA Artificial
Sequence Description of Artificial Sequence PCR primer 4 cccaagctta
ctctctggca ccaaactcca tgggat 36 5 30 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 5 cgggagatct
acaggcgatc aggtaggcgc 30 6 30 DNA Artificial Sequence Description
of Artificial Sequence PCR primer 6 cgcaggctct cacactattc
aggtgatctc 30 7 38 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 7 cggaattccg agtctctgat ctttagccct
gggggctc 38 8 29 DNA Artificial Sequence Description of Artificial
Sequence PCR primer 8 aggacttgga ctctgagagg cagggtctt 29 9 30 DNA
Artificial Sequence Description of Artificial Sequence PCR primer 9
catagtcccc tccttttcca cctgtgagaa 30 10 23 DNA Artificial Sequence
Description of Artificial Sequence PCR primer 10 cgaaccctcg
tcctgctact ctc 23 11 23 DNA Artificial Sequence Description of
Artificial Sequence PCR primer 11 agcatagtcc cctccttttc cac 23 12
39 DNA Artificial Sequence Description of Artificial Sequence PCR
primer 12 cccaagcttc gccgaggatg gccgtcatgg cgccccgaa 39 13 41 DNA
Artificial Sequence Description of Artificial Sequence PCR primer
13 ccggaattct gtcttcacgc tagagaatga gggtcatgaa c 41 14 9 PRT Homo
sapiens 14 Pro Tyr Val Ser Arg Leu Leu Gly Ile 5 15 9 PRT Homo
sapiens 15 Cys Tyr Ala Ser Gly Trp Gly Ser Ile 5 16 10 PRT Homo
sapiens 16 His Tyr Arg Lys Trp Ile Lys Asp Thr Ile 5 10 17 9 PRT
Artificial Sequence VARIANT 2 Xaa is Phe, Tyr, Met or Trp. 17 Cys
Xaa Ala Ser Gly Trp Gly Ser Xaa 5 18 9 PRT Homo sapiens 18 Ile Met
Pro Lys Ala Gly Leu Leu Ile 5 19 9 PRT Homo sapiens 19 Thr Tyr Ala
Cys Phe Val Ser Asn Leu 5 20 10 PRT Homo sapiens 20 Gln Tyr Ser Trp
Phe Val Asn Gly Thr Phe 5 10 21 21 PRT Unknown MHC Class II
I-Ab-restricted helper peptide originating from tetanus toxin 21
Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5
10 15 Ala Ser His Leu Glu 20
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