U.S. patent application number 13/059617 was filed with the patent office on 2011-11-17 for inhbb epitope peptides and vaccines containing the same.
This patent application is currently assigned to Oncotherapy Science, Inc.. Invention is credited to Ryuji Ohsawa, Takuya Tsunoda, Sachiko Yoshimura.
Application Number | 20110280898 13/059617 |
Document ID | / |
Family ID | 41707002 |
Filed Date | 2011-11-17 |
United States Patent
Application |
20110280898 |
Kind Code |
A1 |
Tsunoda; Takuya ; et
al. |
November 17, 2011 |
INHBB EPITOPE PEPTIDES AND VACCINES CONTAINING THE SAME
Abstract
Peptide vaccines against cancer are described herein. In
particular, the present invention describes epitope peptides
derived from INHBB that elicit CTLs. The present invention also
provides established CTLs that specifically recognize HLA-A02
positive target cells pulsed with the peptides. Antigen-presenting
cells and exosomes that present any of the peptides, as well as
methods for inducing antigen-presenting cells are also provided.
The present invention further provides pharmaceutical compositions
containing the INHBB polypeptides or polynucleotides encoding
thereof, as well as exosomes and antigen-presenting cells as active
ingredients. Furthermore, the present invention provides methods
for the treatment and/or prophylaxis of cancers, and/or prevention
of postoperative recurrence thereof, as well as methods for
inducing CTLs, methods for inducing anti-tumor immunity, using the
INHBB polypeptides, polynucleotides encoding the polypeptides,
exosomes or antigen-presenting cells presenting the polypeptides,
or the pharmaceutical agents of the present invention.
Inventors: |
Tsunoda; Takuya; (Kanagawa,
JP) ; Ohsawa; Ryuji; (Kanagawa, JP) ;
Yoshimura; Sachiko; (Kanagawa, JP) |
Assignee: |
Oncotherapy Science, Inc.
Kawasaki-shi, KANAGAWA
JP
|
Family ID: |
41707002 |
Appl. No.: |
13/059617 |
Filed: |
August 14, 2009 |
PCT Filed: |
August 14, 2009 |
PCT NO: |
PCT/JP2009/003894 |
371 Date: |
June 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61089973 |
Aug 19, 2008 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
424/184.1; 424/277.1; 435/366; 435/372.3; 435/375; 530/328;
536/23.5 |
Current CPC
Class: |
G01N 2500/00 20130101;
C07K 14/47 20130101; C07K 4/12 20130101; A61K 39/0011 20130101;
A61K 39/001122 20180801; A61K 2039/572 20130101; A61P 37/04
20180101; A61K 31/7088 20130101; A61K 38/00 20130101; A61P 35/00
20180101; C07K 7/06 20130101; A61K 39/00 20130101 |
Class at
Publication: |
424/185.1 ;
530/328; 536/23.5; 424/184.1; 435/375; 435/372.3; 424/277.1;
435/366 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; A61P 35/00 20060101
A61P035/00; C12N 5/0783 20100101 C12N005/0783; C12N 5/071 20100101
C12N005/071; C07K 7/06 20060101 C07K007/06; C12N 5/00 20060101
C12N005/00 |
Claims
1. An isolated peptide derived from SEQ ID NO: 16, wherein said
peptide comprises an amino acid sequence selected from the group
consisting of: (a) SEQ ID NOs: 1 to 14; and (b) SEQ ID NOs: 1 to
14, in which 1, 2, or several amino acids are substituted,
inserted, deleted and/or added, and has cytotoxic T lymphocyte
(CTL) inducibility.
2. The peptide as set forth in claim 1, wherein the peptide
consists of less than 15 amino acid residues.
3. The peptide as set forth in claim 2, wherein the peptide is a
nonapeptide or a decapeptide.
4. The peptide as set forth in claims 1, in which the peptide,
comprising the amino acid sequence selected from the group
consisting of SEQ ID NOs: 1 to 14, has one or both of the following
characteristics: (a) the second amino acid from the N-terminus is
selected from the group of leucine or methionine, and (b) the
C-terminal amino acid is selected from the group of valine or
leucine.
5. An isolated polynucleotide encoding one or more peptides as set
forth in claim 1.
6. A pharmaceutical agent comprising an active ingredient selected
from the group consisting of: (a) one or more peptides as set forth
in claim 1; (b) one or more polynucleotides encoding the peptide
(c) one or more antigen-presenting cells and/or exosomes, which
present a complex formed between a HLA antigen and a peptide as set
forth in claim 1 on its surface, (d) one or more CTLs induced
against a peptide as set forth in claim 1; and (e) combinations
thereof, in combination with a pharmacologically acceptable carrier
formulated for a purpose selected from the group consisting of: (i)
treatment of cancer, (ii) prophylaxis of cancer, (iii) preventing
postoperative recurrence of cancer, and (iv) combinations
thereof.
7. The pharmaceutical agent as set forth in claim 6, formulated for
administration to a subject whose HLA antigen is HLA-A02.
8. The pharmaceutical agent of claim 6, wherein said cancer is
selected from the group consisting of cholangio cellular carcinoma,
esophageal cancer, non-small cell lung cancer (NSCLC), renal
carcinoma, small cell lung cancer (SCLC) and soft tissue tumor.
9. The pharmaceutical agent of claim 6, wherein said agent is
formulated as a vaccine.
10. A method for inducing an antigen-presenting cell with high CTL
inducibility, wherein the method comprises the step selected from
the group consisting of: (a) contacting an antigen-presenting cell
with a peptide set forth in claim 1; and (b) introducing a
polynucleotide encoding the peptide in an expressible form into an
antigen-presenting cell.
11. A method for inducing CTL, wherein the method comprises the
step selected from the group consisting of: (a) contacting
CD8-positive T cells with antigen-presenting cells and/or exosomes
which present a complex formed between a HLA antigen and a peptide
as set forth in claim 1; and (b) introducing a polynucleotide
encoding a polypeptide which is capable of forming a TCR subunit
recognizing a complex formed between a HLA antigen and a peptide as
set forth in claim 1 into a CD8-positive T cell.
12. An isolated CTL which targets any of the peptides set forth in
claim 1.
13. An isolated CTL which is induced by a peptide as set forth in
claim 1.
14. The CTL as set forth in claim 12, which is capable of
recognizing on a cell surface, a complex formed between a HLA
antigen and a peptide which comprises an amino acid sequence
selected from the group consisting of: (a) SEQ ID NOs: 1 to 14: and
(b) SEQ ID NOs: 1 to 14, wherein 1, 2, or several amino acids are
substituted, inserted, deleted and/or added, and has cytotoxic T
lymphocyte (CTL) inducibility.
15. An isolated CTL which targets a peptide which comprises an
amino acid sequence selected from the group consisting of: (a) SEQ
ID NOs: 1 to 14; and (b) SEQ ID NOs: 1 to 14, wherein 1, 2, or
several amino acids are substituted, inserted, deleted and/or
added, and has cytotoxic T lymphocyte (CTL) inducibility; which is
induced by the method as set forth in claim 11.
16. An isolated antigen-presenting cell, which presents on its
surface a complex formed between a HLA antigen and a peptide as set
forth in claim 1.
17. The antigen-presenting cell of claim 16, which is induced by
the method of inducing an antigen-presenting cell with high CTL
inducibility, wherein the method comprises the step selected from
the group consisting of: (a) contacting an antigen-presenting cell
with a peptide which comprises an amino acid sequence selected from
the group consisting of: (i) SEQ ID NOs: 1 to 14; and (ii) SEQ ID
NOs: 1 to 14, wherein 1, 2, or several amino acids are substituted,
inserted, deleted and/or added, and has cytotoxic T lymphocyte
(CTL) inducibility; and (b) introducing a polynucleotide encoding
the peptide in an expressible form into an antigen-presenting
cell.
18. The antigen-presenting cell as set forth in claim 16, wherein
the HLA antigen is HLA-A02.
19. An agent for inducing an immune response against a cancer in a
subject, wherein said agent comprises an active ingredient selected
from the group consisting of: (a) one or more peptides as set forth
in claim 1; (b) one or more polynucleotides encoding the peptide in
an expressible form; (c) one or more antigen-presenting cells
and/or exosomes, which antigen-presenting cells and exosomes
present a complex formed between a HLA antigen and a peptide as set
forth in claim 1 on its surface; (d) one or more CTLs induced
against a peptide as set forth in claim 1; and (e) combinations
thereof.
20. A method of inducing an immune response against a cancer in a
subject, said method comprising the step of administering to said
subject an agent comprising an active ingredient selected from the
group consisting of: (a) one or more peptides as set forth in claim
1; (b) one or more polynucleotides encoding the peptide in an
expressible form; (c) one or more antigen-presenting cells and/or
exosomes, which antigen-presenting cells and exosomes present a
complex formed between a HLA antigen and a peptide as set forth in
claim 1 on its surface; (d) one or more CTLs induced against a
peptide as set forth in claim 1; and (e) combinations thereof, and
a pharmaceutically acceptable carrier
21. The method as set forth in claim 20, wherein said cancer is
selected from the group consisting of cholangio cellular carcinoma,
esophageal cancer, non-small cell lung cancer (NSCLC), renal
carcinoma, small cell lung cancer (SCLC) and soft tissue tumor.
22. The method as set forth in claim 20, wherein the subject has
HLA A02.
23. An agent for inducing CTL, wherein the agent comprises one or
more peptides comprising an amino acid sequence selected from the
group consisting of: (a) SEQ ID NOs: 1 to 14; and (b) SEQ ID NOs: 1
to 14, wherein 1, 2, or several amino acids are substituted.,
inserted, deleted and/or added, and has cytotoxic T lymphocyte
(CTL) inducibility; a polynucleotide encoding the peptide, or an
isolated antigen-presenting cell of claim 16.
24. The CTL as set forth in claim 13, which is capable of
recognizing on a cell surface, a complex formed between a HLA
antigen and a peptide derived from SEQ ID NO: 16, wherein said
peptide comprises an amino acid sequence selected from the group
consisting of: (a) SEQ ID NOs: 1 to 14; and (b) SEQ ID NOs: 1 to
14, wherein 1, 2, or several amino acids are substituted, inserted,
deleted and/or added, and has cytotoxic T lymphocyte (CTL)
inducibility.
25. An isolated CTL which is induced by a peptide derived from SEQ
ID NO: 16, wherein said peptide comprises an amino acid sequence
selected from the group consisting of: (a) SEQ ID NOs: 1 to 14; and
(b) SEQ ID NOs: 1 to 14, wherein 1, 2, or several amino acids are
substituted, inserted, deleted and/or added, and has cytotoxic T
lymphocyte (CTL) inducibility; which is induced by the method as
set forth in claim 11.
26. The antigen-presenting cell as set forth in claim 17, wherein
the HLA antigen is HLA-A02.
Description
TECHNICAL FIELD
[0001] 1. Priority
[0002] The present application claims the benefit of U.S.
Provisional Application No. 61/089,973, filed on Aug. 19, 2008, the
entire content of which is incorporated by reference herein.
[0003] 2. Technical Field
[0004] The present invention relates to the field of biological
science, more specifically to the field of cancer therapy. In
particular, the present invention relates to novel peptides that
are extremely effective as cancer vaccines, and drugs for treating
and preventing tumors.
BACKGROUND ART
[0005] It has been demonstrated that CD8 positive CTLs recognize
epitope peptides derived from the tumor-associated antigens (TAAs)
found on major histocompatibility complex (MHC) class I molecule,
and then kill the tumor cells. Since the discovery of the melanoma
antigen (MAGE) family as the first example of TAAs, many other TAAs
have been discovered, primarily through immunological approaches
(NPL 1: Boon T, Int J Cancer 1993 May 8, 54(2): 177-80; NPL 2: Boon
T & van der Bruggen P, J Exp Med 1996 Mar. 1, 183(3): 725-9).
Some of the TAAs are now currently undergoing clinical development
as immunotherapeutic targets.
[0006] Identification of new TAAs capable of inducing potent and
specific anti-tumor immune responses, warrants further development
and clinical application of peptide vaccination strategies for
various types of cancer (NPL 3: Harris C C, J Natl Cancer Inst 1996
Oct. 16, 88(20): 1442-55; NPL 4 : Butterfield L H et al., Cancer
Res 1999 Jul. 1, 59(13): 3134-42; NPL 5: Vissers J L et al., Cancer
Res 1999 Nov. 1, 59(21): 5554-9; NPL 6: van der Burg S H et al., J
Immunol 1996 May 1, 156(9): 3308-14; NPL 7: Tanaka F et al., Cancer
Res 1997 Oct. 15, 57(20): 4465-8; NPL 8: Fujie T et al., Int J
Cancer 1999 Jan. 18, 80(2): 169-72; NPL 9: Kikuchi M et al., Int J
Cancer 1999 May 5, 81(3): 459-66; NPL 10: Oiso M et al., Int J
Cancer 1999 May 5, 81(3): 387-94). To date, there have been several
reports of clinical trials using these tumor-associated antigen
derived peptides. Unfortunately, only a low objective response rate
has been observed in these cancer vaccine trials so far (NPL 11:
Belli F et al., J Clin Oncol 2002 Oct. 15, 20(20): 4169-80; NPL 12:
Coulie P G et al., Immunol Rev 2002 Oct, 188: 33-42; NPL 13:
Rosenberg S A et al., Nat Med 2004 Sep, 10(9): 909-15).
[0007] Recently, algorithms for predicting HLA class I-binding
peptide sequences have been developed (NPL 14: Journal of
Immunological Methods, (1995), Vol. 185, pp. 181-190, NPL 15: J.
Immunol., (1994), Vol. 152, pp. 163-175, NPL 16: protein science,
(2000), Vol. 9, pp. 1838-1846). However, it is difficult to
estimate if a predicted epitope peptide can be processed naturally
in the target cells and expressed on the target cell surface with
HLA molecule. Moreover, the algorithms, for example BIMAS
(bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform) (NPL 17:
Parker K C, et al., (1994) J Immunol.; 152(1):163-75.; NPL 18:
Kuzushima K, et al., (2001) Blood.; 98(6):1872-81.) can suggest
less than rigorous HLA molecule-binding peptides (NPL 19: Bachinsky
M M, et al., Cancer Immun. 2005 Mar 22; 5:6.). Thus TAA screening
remains challenging and difficult.
[0008] Inhibins are heterodimeric glycoproteins composed of an
alpha subunit (INHA) and one of two beta subunits (beta-A or
beta-B). Inhibin, beta B (INHBB) is a subunit of both inhibin and
activin, two closely related glycoproteins with opposing biological
effects. Through gene expression profile analysis using a
genome-wide cDNA microarray containing 23,040 genes, INHBB was
recently shown to be up-regulated in several cancers such as
non-small cell lung cancer (NSCLC), renal cell carcinoma (PTL 1:
WO2005/019475; PTL 2: WO2007/013575) and esophageal cancer (See PTL
3: WO2004/031413, PTL 4: WO2005/019475, PTL 5: WO2007/013575, and
PTL 6: WO2007/013671, the disclosures of which are incorporated by
reference herein). Accordingly, INHBB is an interesting target for
cancer immunotherapy and CTL inducing epitope peptides derived
therefrom are sought by those in the art.
CITATION LIST
Non Patent Literature
[0009] [NPL 1] Boon T, Int J Cancer 1993 May 8, 54(2): 177-80
[0010] [NPL 2] Boon T & amp; van der Bruggen P, J Exp Med 1996
Mar. 1, 183(3): 725-9 [0011] [NPL 3] Harris C C, J Natl Cancer Inst
1996 Oct. 16, 88(20): 1442-55 [0012] [NPL 4] Butterfield L H et
al., Cancer Res 1999 Jul. 1, 59(13): 3134-42 [0013] [NPL 5] Vissers
J L et al., Cancer Res 1999 Nov. 1, 59(21): 5554-9 [0014] [NPL 6]
van der Burg S H et al., J Immunol 1996 May 1, 156(9): 3308-14
[0015] [NPL 7] Tanaka F et al., Cancer Res 1997 Oct. 15, 57(20):
4465-8 [0016] [NPL 8] Fujie T et al., Int J Cancer 1999 Jan. 18,
80(2): 169-72 [0017] [NPL 9] Kikuchi M et al., Int J Cancer 1999
May 5, 81(3): 459-66 [0018] [NPL 10] Oiso M et al., Int J Cancer
1999 May 5, 81(3): 387-94 [0019] [NPL 11] Belli F et al., J Clin
Oncol 2002 Oct. 15, 20(20): 4169-80 [0020] [NPL 12] Coulie P G et
al., Immunol Rev 2002 Oct, 188: 33-42 [0021] [NPL 13] Rosenberg S A
et al., Nat Med 2004 Sep, 10(9): 909-15 [0022] [NPL 14] Journal of
Immunological Methods, (1995), Vol. 185, pp. 181-190 [0023] [NPL
15] J. Immunol., (1994), Vol. 152, pp. 163-175 [0024] [NPL 16]
protein science, (2000), Vol. 9, pp. 1838-1846 [0025] [NPL 17]
Parker K C, et al., (1994) J Immunol.; 152(1):163-75. [0026] [NPL
18] Kuzushima K, et al., (2001) Blood.; 98(6):1872-81. [0027] [NPL
19] Bachinsky M M, et al., Cancer Immun. 2005 Mar. 22; 5:6.
Patent Literature
[0027] [0028] [PTL 1] WO2005/019475 [0029] [PTL 2] WO2007/013575
[0030] [PTL 3] WO2004/031413 [0031] [PTL 4] WO2005/019475 [0032]
[PTL 5] WO2007/013575 [0033] [PTL 6] WO2007/013671
SUMMARY OF INVENTION
[0034] The present invention is based in part on the discovery of
suitable targets of immunotherapy. Because TAAs often induce immune
tolerance and therefore elicit poor immunogenicity, the discovery
of appropriate targets is of extreme importance. Recognizing that
INHBB has been identified as up-regulated in cancer tissues of
cholangio cellular carcinoma, esophageal cancer, non-small cell
lung cancer (NSCLC), renal carcinoma, small cell lung cancer (SCLC)
and soft tissue tumor, the present invention targets the Homo
sapiens inhibin, beta B (INHBB) protein (SEQ ID NO: 16) encoded by
the gene of GenBank Accession No. NM.sub.--002193 (SEQ ID NO: 15))
for further analysis. In particular, INHBB gene products containing
epitope peptides that elicit CTLs specific to the corresponding
molecules were selected for study. Peripheral blood mononuclear
cells (PBMCs) obtained from a healthy donor were stimulated using
HLA-A*0201 binding candidate peptides derived from INHBB. CTLs that
specifically recognize HLA-A02 positive target cells pulsed with
the respective candidate peptides were established, and HLA-A02
restricted epitope peptides that can induce potent and specific
immune responses against INHBB expressed on the surface of tumor
cells were identified. These results demonstrate that INHBB is
strongly immunogenic and the epitopes thereof are effective targets
for tumor immunotherapy.
[0035] Accordingly, it is an object of the present invention to
provide peptides having CTL inducibility as well as an amino acid
sequence selected from the group of SEQ ID NOs: 1 to 14, and which
have CTL inducibility. In addition, the present invention
contemplates modified peptides, wherein one, two or more amino
acids are substituted or added, so long as the modified peptides
retain the original CTL inducibility.
[0036] When administered to a subject, the present peptides are
presented on the surface of antigen-expressing cells and then
induce CTLs targeting the respective peptides. Therefore, it is an
object of the present invention to provide antigen-presenting cells
and exosomes that present any of the present peptides, as well as
methods for inducing antigen-presenting cells.
[0037] An anti-tumor immune response is induced by the
administration of the present INHBB polypeptides or polynucleotide
encoding the polypeptides, as well as exosomes and
antigen-presenting cells which present the INHBB polypeptides.
Therefore, it is yet another object of the present invention to
provide pharmaceutical agents containing the polypeptides or
polynucleotides encoding them, as well as the exosomes and
antigen-presenting cells as their active ingredients. The
pharmaceutical agents of the present invention find use as
vaccines.
[0038] Moreover, it is a further object of the present invention to
provide methods for the treatment and/or prophylaxis of (i.e.,
prevention) cancers (tumors), and/or prevention of postoperative
recurrence thereof, as well as methods for inducing CTLs, methods
for inducing an immune response against tumor-associated endothelia
and also anti-tumor immunity, which methods include the step of
administering the INHBB polypeptides, polynucleotides encoding
INHBB polypeptides, exosomes or the antigen-presenting cells
presenting INHBB polypeptides or the pharmaceutical agents of the
invention. In addition, the CTLs of the invention also find use as
vaccines against cancer. Examples of the cancer include, but are
not limited to, cholangio cellular carcinoma, esophageal cancer,
non-small cell lung cancer (NSCLC), renal carcinoma, small cell
lung cancer (SCLC) and soft tissue tumor.
[0039] It is to be understood that both the foregoing summary of
the invention and the following detailed description are of
exemplified embodiments, and not restrictive of the invention or
other alternate embodiments of the invention.
BRIEF DESCRIPTION OF DRAWINGS
[0040] Various aspects and applications of the present invention
will become apparent to the skilled artisan upon consideration of
the brief description of the figures and the detailed description
of the present invention and its preferred embodiments which
follows:
[0041] FIG. 1 includes a series of photographs, (a)-(n), depicting
the results of IFN-gamma ELISPOT assay on CTLs that were induced
with peptides derived from INHBB. The CTLs in well #4 stimulated
with INHBB-A02-9-213 (SEQ ID NO: 1) (a), well #5 and #7 stimulated
with INHBB-A02-9-174 (SEQ ID NO: 2) (b), well #8 stimulated with
INHBB-A02-9-257 (SEQ ID NO: 3) (c), well #1 and #8 stimulated with
INHBB-A02-9-313 (SEQ ID NO: 4) (d), well #1, #4 and #8 stimulated
with INHBB-A02-9-139 (SEQ ID NO: 5) (e), well #4 stimulated with
INHBB-A02-9-8 (SEQ ID NO: 6) (f), well #6 stimulated with
INHBB-A02-9-250 (SEQ ID NO: 7) (g), well #5 stimulated with
INHBB-A02-10-179 (SEQ ID NO: 8) (h), well #3 stimulated with
INHBB-A02-10-237 (SEQ ID NO: 9) (i), well #5 stimulated with
INHBB-A02-10-313 (SEQ ID NO: 10) (j), well #3 and #7 stimulated
with INHBB-A02-10-173 (SEQ ID NO: 11) (k), well #4 stimulated with
INHBB-A02-10-256 (SEQ ID NO: 12) (l), well #7 stimulated with
INHBB-A02-10-162 (SEQ ID NO: 13) (m) and well #7 stimulated with
INHBB-A02-10-85 (SEQ ID NO: 14) (n) showed potent IFN-gamma
production as compared with the control respectively. In the
figures, "+" indicates that the target cells in the well were
pulsed with the appropriate peptide, and "-" indicates that the
target cells had not been pulsed with any peptides.
[0042] FIG. 2 depicts a line graph showing the results of
establishment of CTL lines stimulated with INHBB-A02-9-174 (SEQ ID
NO: 2) with IFN-gamma ELISA assay. The depicted results demonstrate
that CTL line established by stimulation with the peptide showed
potent IFN-gamma production as compared with the control. In the
figures, "+" indicates that the target cells were pulsed with the
appropriate peptide and "-" indicates that the target cells had not
been pulsed with any peptides.
DESCRIPTION OF EMBODIMENTS
[0043] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
embodiments of the present invention, the preferred methods,
devices, and materials are now described. However, before the
present materials and methods are described, it is to be understood
that the present invention is not limited to the particular sizes,
shapes, dimensions, materials, methodologies, protocols, etc.
described herein, as these may vary in accordance with routine
experimentation and optimization. It is also to be understood that
the terminology used in the description is for the purpose of
describing the particular versions or embodiments only, and is not
intended to limit the scope of the present invention which will be
limited only by the appended claims.
[0044] The disclosure of each publication, patent or patent
application mentioned in this specification is specifically
incorporated by reference herein in its entirety. However, nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0045] In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
I. Definitions
[0046] The words "a", "an", and "the" as used herein mean "at least
one" unless otherwise specifically indicated.
[0047] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is a modified residue, or a non-naturally
occurring residue, such as an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0048] The term "amino acid" as used herein refers to naturally
occurring and synthetic amino acids, as well as amino acid analogs
and amino acid mimetics that similarly function to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those modified after
translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate,
and O-phosphoserine). The phrase "amino acid analog" refers to
compounds that have the same basic chemical structure (an alpha
carbon bound to a hydrogen, a carboxy group, an amino group, and an
R group) as a naturally occurring amino acid but have a modified R
group or modified backbones (e.g., homoserine, norleucine,
methionine, sulfoxide, methionine methyl sulfonium). The phrase
"amino acid mimetic" refers to chemical compounds that have
different structures but similar functions to general amino
acids.
[0049] Amino acids may be referred to herein by their commonly
known three letter symbols or the one-letter symbols recommended by
the IUPAC-IUB Biochemical Nomenclature Commission.
[0050] The terms "gene", "polynucleotides", "nucleotides" and
"nucleic acids" are used interchangeably herein unless otherwise
specifically indicated and are similarly to the amino acids
referred to by their commonly accepted single-letter codes.
[0051] Unless otherwise defined, the terms "cancer" refers to
cancers over-expressing the INHBB gene, examples of which include,
but are not limited to, cholangio cellular carcinoma, esophageal
cancer, non-small cell lung cancer (NSCLC), renal carcinoma, small
cell lung cancer (SCLC) and soft tissue tumor.
[0052] Unless otherwise defined, the terms "cytotoxic T
lymphocyte", "cytotoxic T cell" and "CTL" are used interchangeably
herein and, unless otherwise specifically indicated, refer to a
sub-group of T lymphocytes that are capable of recognizing non-self
cells (e.g., tumor cells, virus-infected cells) and inducing the
death of such cells.
[0053] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
II. Peptides
[0054] To demonstrate that peptides derived from INHBB function as
an antigen recognized by cytotoxic T lymphocytes (CTLs), peptides
derived from INHBB (SEQ ID NO: 16) were analyzed to determine
whether they were antigen epitopes restricted by HLA-A02 which are
commonly encountered HLA alleles (Date Y et al., Tissue Antigens
47: 93-101, 1996; Kondo A et al., J Immunol 155: 4307-12, 1995;
Kubo R T et al., J Immunol 152: 3913-24, 1994). Candidates of
HLA-A02 binding peptides derived from INHBB were identified based
on their binding affinities to HLA-A02. After in vitro stimulation
of T-cells by dendritic cells (DCs) loaded with these peptides,
CTLs were successfully established using each of the peptides of
SEQ ID NOs: 1 to 14, particularly following peptides. [0055]
INHBB-A02-9-213 (SEQ ID NO: 1), [0056] INHBB-A02-9-174 (SEQ ID NO:
2), [0057] INHBB-A02-9-257 (SEQ ID NO: 3), [0058] INHBB-A02-9-313
(SEQ ID NO: 4), [0059] INHBB-A02-9-139 (SEQ ID NO: 5), [0060]
INHBB-A02-9-8 (SEQ ID NO: 6), [0061] INHBB-A02-9-250 (SEQ ID NO:
7), [0062] INHBB-A02-10-179 (SEQ ID NO: 8), [0063] INHBB-A02-10-237
(SEQ ID NO: 9), [0064] INHBB-A02-10-313 (SEQ ID NO: 10), [0065]
INHBB-A02-10-173 (SEQ ID NO: 11), [0066] INHBB-A02-10-256 (SEQ ID
NO: 12), [0067] INHBB-A02-10-162 (SEQ ID NO: 13) [0068] and [0069]
INHBB-A02-10-85 (SEQ ID NO: 14).
[0070] These established CTLs show potent specific CTL activity
against target cells pulsed with respective peptides. The results
herein demonstrate that INHBB is an antigen recognized by CTL and
that the peptides may be epitope peptides of INHBB restricted by
HLA-A02.
[0071] Since the INHBB gene is over expressed in most cancer
tissues, such as cholangio cellular carcinoma, esophageal cancer,
non-small cell lung cancer (NSCLC), renal carcinoma, small cell
lung cancer (SCLC) and soft tissue tumor, it is a good target for
immunotherapy. Thus, the present invention provides nonapeptides
(peptides consisting of nine amino acid residues) and decapeptides
(peptides consisting of ten amino acid residues) corresponding to
CTL-recognized epitopes of INHBB. Particularly preferred examples
of nonapeptides and decapeptides of the present invention include
those peptides having an amino acid sequence selected from among
SEQ ID NOs: 1 to 14.
[0072] Generally, software programs presently available on the
Internet, such as those described in Parker K C et al., J Immunol
1994 Jan. 1, 152(1): 163-75, can be used to calculate the binding
affinities between various peptides and HLA antigens in silico.
Binding affinity with HLA antigens can be measured as described,
for example, in the references Parker K C et al., J Immunol 1994
Jan. 1, 152(1): 163-75; and Kuzushima K et al., Blood 2001, 98(6):
1872-81. The methods for determining binding affinity are
described, for example, in; Journal of Immunological Methods, 1995,
185: 181-190; Protein Science, 2000, 9: 1838-1846. Thus, the
present invention encompasses peptides of INHBB which bind with HLA
antigens identified using such known programs.
[0073] The nonapeptides and decapeptides of the present invention
can be flanked with additional amino acid residues so long as the
resulting peptides retain their CTL inducibility. Such peptides
having CTL inducibility are typically less than about 40 amino
acids, often less than about 20 amino acids, usually less than
about 15 amino acids. The particular amino acid sequences flanking
the nonapeptides and decapeptides of the present invention (i.e.,
peptides consisting of the amino acid sequence selected from among
SEQ ID NOs: 1 to 14) is not limited and can be composed of any kind
of amino acids so long as it does not impair the CTL inducibility
of the original peptide. Thus, the present invention also provides
peptides having CTL inducibility and the amino acid sequence
selected from among SEQ ID NOs: 1 to 14.
[0074] In general, modification of one, two, or more amino acids in
a protein will not influence the function of the protein, and, in
some cases will even enhance the desired function of the original
protein. In fact, modified peptides (i.e., peptides composed of an
amino acid sequence in which one, two or several amino acid
residues have been modified (i.e., substituted, deleted, added or
inserted) as compared to an original reference sequence) have been
known to retain the biological activity of the original peptide
(Mark et al., Proc Natl Acad Sci USA 1984, 81: 5662-6; Zoller and
Smith, Nucleic Acids Res 1982, 10: 6487-500; Dalbadie-McFarland et
al., Proc Natl Acad Sci USA 1982, 79: 6409-13). Thus, in one
embodiment, the peptides of the present invention may have both CTL
inducibility and an amino acid sequence selected from among SEQ ID
NO: 1 to 14 wherein one, two or even more amino acids are deleted,
inserted, added and/or substituted.
[0075] Those of skill in the art recognize that individual
additions or substitutions to an amino acid sequence which alters a
single amino acid or a small percentage of amino acids tend to
result in the conservation of the properties of the original amino
acid side-chain. As such, they are conventionally referred to as
"conservative substitutions" or "conservative modifications",
wherein the alteration of a protein results in a modified protein
having properties and functions analogous to the original protein.
Conservative substitution tables providing functionally similar
amino acids are well known in the art. Examples of amino acid side
chain characteristics that are desirable to conserve include, for
example, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and side
chains having the following functional groups or characteristics in
common: an aliphatic side-chain (G, A, V, L, I, P); a hydroxyl
group containing side-chain (S, T, Y); a sulfur atom containing
side-chain (C, M); a carboxylic acid and amide containing
side-chain (D, N, E, Q); a base containing side-chain (R, K, H);
and an aromatic containing side-chain (H, F, Y, W). In addition,
the following eight groups each contain amino acids that are
accepted in the art as conservative substitutions for one another:
[0076] 1) Alanine (A), Glycine (G); [0077] 2) Aspartic acid (D),
Glutamic acid (E); [0078] 3) Aspargine (N), Glutamine (Q); [0079]
4) Arginine (R), Lysine (K); [0080] 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); [0081] 6) Phenylalanine (F), Tyrosine
(Y), Tryptophan (W); [0082] 7) Serine (S), Threonine (T); and
[0083] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton,
Proteins 1984).
[0084] Such conservatively modified peptides are also considered to
be peptides of the present invention. However, peptides of the
present invention are not restricted thereto and can include
non-conservative modifications, so long as the modified peptide
retains the CTL inducibility of the original peptide. Furthermore,
modified peptides should not exclude CTL inducible peptides of
polymorphic variants, interspecies homologues, and alleles of
INHBB.
[0085] To retain the requisite CTL inducibility one can modify
(insert, delete, add and/or substitute) a small number (for
example, 1, 2 or several) or a small percentage of amino acids.
Herein, the term "several" means 5 or fewer amino acids, for
example, 3 or fewer. The percentage of amino acids to be modified
is preferably 20% or less, more preferably 15% of less, even more
preferably 10% or less or 1 to 5%.
[0086] Homology analysis of preferred peptides of the present
invention, INHBB-A02-9-213 (SEQ ID NO: 1), INHBB-A02-9-174 (SEQ ID
NO: 2), INHBB-A02-9-257 (SEQ ID NO: 3), INHBB-A02-9-313 (SEQ ID NO:
4), INHBB-A02-9-139 (SEQ ID NO: 5), INHBB-A02-9-8 (SEQ ID NO: 6),
INHBB-A02-9-250 (SEQ ID NO: 7), INHBB-A02-10-179 (SEQ ID NO: 8),
INHBB-A02-10-237 (SEQ ID NO: 9), INHBB-A02-10-313 (SEQ ID NO: 10),
INHBB-A02-10-173 (SEQ ID NO: 11), INHBB-A02-10-256 (SEQ ID NO: 12),
INHBB-A02-10-162 (SEQ ID NO: 13) and INHBB-A02-10-85 (SEQ ID NO:
14) confirmed that these peptides do not have significant homology
with peptides derived from any other known human gene products.
Thus, the possibility of these peptides generating unknown or
undesired immune responses when used for immunotherapy is
significantly lowered. Accordingly, these peptides are expected to
be highly useful for eliciting immunity in tumor patients against
INHBB on cancer cells, such as cholangio cellular carcinoma,
esophageal cancer, non-small cell lung cancer (NSCLC), renal
carcinoma, small cell lung cancer (SCLC) and soft tissue tumor.
[0087] When used in the context of immunotherapy, peptides of the
present invention should be presented on the surface of a cell or
exosome, preferably as a complex with an HLA antigen. Therefore, it
is preferable to select peptides that not only induce CTLs but also
that possess high binding affinity to the HLA antigen. To that end,
the peptides can be modified by substitution, insertion, deletion,
and/or addition of the amino acid residues to yield a modified
peptide having improved binding affinity. In addition to peptides
that are naturally displayed, since the regularity of the sequences
of peptides displayed by binding to HLA antigens is already known
(J Immunol 1994, 152: 3913; Immunogenetics 1995, 41: 178; J Immunol
1994, 155: 4307), modifications based on such regularity can be
introduced into the immunogenic peptides of the invention. For
example, it may be desirable to substitute the second amino acid
from the N-terminus substituted with leucine or methionine, and/or
the amino acid at C-terminus with valine or leucine in order to
increase the HLA-A0201 binding. Thus, peptides having the amino
acid sequences of SEQ ID NOs: 1 to 14 wherein the second amino acid
from the N-terminus of the amino acid sequence of the SEQ ID NOs is
substituted with leucine or methionine and/or wherein the
C-terminus of the amino acid sequence of the SEQ ID NOs is
substituted with valine or leucine, are encompassed by the present
invention. Substitutions can be introduced not only at the terminal
amino acids but also at the position of potential TCR recognition
of peptides. Several studies have demonstrated that amino acid
substitutions in a peptide can be equal to or better than the
original, for example, CAP1, p53.sub.(264-272),
Her-2/neu.sub.(369-377) or gp100.sub.(209-217) (Zaremba et al.,
Cancer Res. 57, 4570-4577, 1997, T. K. Hoffmann et al., J Immunol.
(2002) Feb 1; 168(3):1338-47., S. O. Dionne et al., Cancer Immunol
immunother. (2003) 52: 199-206 and S. O. Dionne et al., Cancer
Immunology, Immunotherapy (2004) 53, 307-314).
[0088] The present invention also contemplates the addition one to
two amino acids to the N and/or C-terminus of the present peptides.
Such modified peptides having high HLA antigen binding affinity and
retained CTL inducibility are also included in the present
invention.
[0089] However, when the peptide sequence is identical to a portion
of the amino acid sequence of an endogenous or exogenous protein
having a different function, side effects such as autoimmune
disorders and/or allergic symptoms against specific substances may
be induced. Therefore, it is preferable to first perform homology
searches using available databases to avoid situations in which the
sequence of the peptide matches the amino acid sequence of another
protein. When it becomes clear from the homology searches that
there exists not even a peptide with 1 or 2 amino acid differences
as compared to the objective peptide, the objective peptide can be
modified in order to increase its binding affinity with HLA
antigens, and/or increase its CTL inducibility without any danger
of such side effects.
[0090] Although peptides having high binding affinity to the HLA
antigens as described above are expected to be highly effective,
the candidate peptides, which are selected according to the
presence of high binding affinity as an indicator, are further
examined for the presence of CTL inducibility. Herein, the phrase
"CTL inducibility" indicates the ability of the peptide to induce
cytotoxic lymphocytes (CTLs) when presented on antigen-presenting
cells. Further, "CTL inducibility" includes the ability of the
peptide to induce CTL activation and/or CTL proliferation, promote
CTL lysis of target cells, and to increase CTL IFN-gamma
production.
[0091] Confirmation of CTL inducibility is accomplished by inducing
antigen-presenting cells carrying human MHC antigens (for example,
B-lymphocytes, macrophages, and dendritic cells (DCs)), or more
specifically DCs derived from human peripheral blood mononuclear
leukocytes, and after stimulation with the peptides, mixing with
CD8-positive cells, and then measuring the IFN-gamma produced and
released by CTL against the target cells. As the reaction system,
transgenic animals that have been produced to express a human HLA
antigen (for example, those described in BenMohamed L, Krishnan R,
Longmate J, Auge C, Low L, Primus J, Diamond D J, Hum Immunol 2000
Aug, 61(8): 764-79, Related Articles, Books, Linkout Induction of
CTL response by a minimal epitope vaccine in HLA A*0201/DR1
transgenic mice: dependence on HLA class II restricted T(H)
response) can be used. For example, the target cells can be
radiolabeled with .sup.51Cr and such, and cytotoxic activity can be
calculated from radioactivity released from the target cells.
Alternatively, CTL inducibility can be assessed by measuring
IFN-gamma produced and released by CTL in the presence of
antigen-presenting cells (APCs) that carry immobilized peptides,
and visualizing the inhibition zone on the media using
anti-IFN-gamma monoclonal antibodies.
[0092] As a result of examining the CTL inducibility of the
peptides as described above, it was discovered that those peptides
having high binding affinity to an HLA antigen did not necessarily
have high CTL inducibility. However, of those peptides identified
and assessed, nonapeptides or decapeptides selected from peptides
having an amino acid sequence selected from among SEQ ID NOs: 1 to
14 were found to exhibit particularly high CTL inducibility as well
as high binding affinity to an HLA antigen. Thus, these peptides
are exemplified as preferred embodiments of the present
invention.
[0093] In addition to the above-described modifications, the
peptides of the present invention can also be linked to other
substances, so long as the resulting linked peptide retains the
requisite CTL inducibility of the original peptide. Examples of
suitable substances include, but are not limited to: peptides,
lipids, sugar and sugar chains, acetyl groups, natural and
synthetic polymers, etc. The peptides can contain modifications
such as glycosylation, side chain oxidation, or phosphorylation,
etc., provided the modifications do not destroy the biological
activity of the original peptide. These kinds of modifications can
be performed to confer additional functions (e.g., targeting
function, and delivery function) or to stabilize the
polypeptide.
[0094] For example, to increase the in vivo stability of a
polypeptide, it is known in the art to introduce D-amino acids,
amino acid mimetics or unnatural amino acids; this concept can also
be adapted to the present polypeptides. The stability of a
polypeptide can be assayed in a number of ways. For instance,
peptidases and various biological media, such as human plasma and
serum, can be used to test stability (see, e.g., Verhoef et al.,
Eur J Drug Metab Pharmacokin 1986, 11: 291-302).
[0095] Further, the peptides of the present invention may be linked
to other peptides via spacers or linkers. Examples of other
peptides include, but are not limited to, CTL inducible peptides
derived from other TAAs. Alternatively, two or more peptides of the
present invention may be linked via spacers or linkers. The
peptides linked via spacers or linkers may be the same or different
each other. Spacers or linkers are not specifically limited, but
are preferably peptides, more preferably peptides having one or
more cleavage sites which are capable of being cleaved by enzymes
such as peptidases, proteases and proteasomes. Examples of linkers
or spacers include, but are not limited to: AAY (P. M. Daftarian et
al., J Trans Med 2007, 5:26), AAA, NKRK (R. P. M. Sutmuller et al.,
J Immunol. 2000, 165: 7308-7315) or, one to several lysine
redsidues (S. Ota et al., Can Res. 62, 1471-1476, K. S. Kawamura et
al., J Immunol. 2002, 168: 5709-5715). The peptide of the present
invention encompass those peptides linked to other peptides via
spacers or linkers.
[0096] The peptides of the present invention may be existed on the
surface of a cell carrying human MHC antigens (e.g. antigen
presenting cell) or an exosome as complexes in combination with MHC
molecules and then induce CTLs. The cells and the exosomes can be
prepared by well-known methods in the art, for example, the cells
may be prepared by contacting with the peptides of the present
invention, and the exosomes may be prepared by collecting an
exosome-containing fraction from the cells contacted with the
peptides of the present invention (see, e.g., Japanese Patent
Application Kohyo Publications Nos. Hei 11-510507 and WO99/03499).
The peptides of the present invention encompass those peptides
existed on the surface of a cell or an exosome as complexes in
combination with MHC molecules.
[0097] Herein, the peptides of the present invention can also be
described as "INHBB peptide(s)" or "INHBB polypeptide(s)".
III. Preparation of INHBB Peptides
[0098] The peptides of the present invention can be prepared using
well known techniques. For example, the peptides can be prepared
synthetically, using recombinant DNA technology or chemical
synthesis. Peptides of the present invention can be synthesized
individually or as longer polypeptides composed of two or more
peptides. The peptides can then be isolated i.e., purified or
isolated so as to be substantially free of other naturally
occurring host cell proteins and fragments thereof, or any other
chemical substances.
[0099] A peptide of the present invention can be obtained through
chemical synthesis based on the selected amino acid sequence.
Examples of conventional peptide synthesis methods that can be
adapted to the synthesis include, but are not limited to:
[0100] (i) Peptide Synthesis, Interscience, New York, 1966;
[0101] (ii) The Proteins, Vol. 2, Academic Press, New York,
1976;
[0102] (iii) Peptide Synthesis (in Japanese), Maruzen Co.,
1975;
[0103] (iv) Basics and Experiment of Peptide Synthesis (in
Japanese), Maruzen Co., 1985;
[0104] (v) Development of Pharmaceuticals (second volume) (in
Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
[0105] (vi) WO99/67288; and
[0106] (vii) Barany G. & Merrifield R. B., Peptides Vol. 2,
"Solid Phase Peptide Synthesis", Academic Press, New York, 1980,
100-118.
[0107] Alternatively, the present peptides can be obtained adapting
any known genetic engineering methods for producing peptides (e.g.,
Morrison J, J Bacteriology 1977, 132: 349-51; Clark-Curtiss &
Curtiss, Methods in Enzymology (eds. Wu et al.) 1983, 101: 347-62).
For example, first, a suitable vector harboring a polynucleotide
encoding the objective peptide in an expressible form (e.g.,
downstream of a regulatory sequence corresponding to a promoter
sequence) is prepared and transformed into a suitable host cell.
The host cell is then cultured to produce the peptide of interest.
The peptide can also be produced in vitro adopting an in vitro
translation system.
IV. Polynucleotides
[0108] The present invention also provides a polynucleotide which
encodes any of the aforementioned peptides of the present
invention. These include polynucleotides derived from the natural
occurring INHBB gene (GenBank Accession No. NM.sub.--002193 (SEQ ID
NO: 15)) as well as those having a conservatively modified
nucleotide sequence thereof. Herein, the phrase "conservatively
modified nucleotide sequence" refers to sequences which encode
identical or essentially identical amino acid sequences. Due to the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a peptide also describes every possible silent
variation of the nucleic acid. One of ordinary skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a peptide is implicitly described in each
disclosed sequence.
[0109] The polynucleotide of the present invention can be composed
of DNA, RNA, and derivatives thereof. A DNA is suitably composed of
bases such as A, T, C, and G, and T is replaced by U in an RNA.
[0110] The polynucleotide of the present invention can encode
multiple peptides of the present invention, with or without
intervening amino acid sequences in between. For example, the
intervening amino acid sequence can provide a cleavage site (e.g.,
enzyme recognition sequence) of the polynucleotide or the
translated peptides. Furthermore, the polynucleotide can include
any additional sequences to the coding sequence encoding the
peptide of the present invention. For example, the polynucleotide
can be a recombinant polynucleotide that includes regulatory
sequences required for the expression of the peptide or can be an
expression vector (plasmid) with marker genes and such. In general,
such recombinant polynucleotides can be prepared by the
manipulation of polynucleotides through conventional recombinant
techniques using, for example, polymerases and endonucleases.
[0111] Both recombinant and chemical synthesis techniques can be
used to produce the polynucleotides of the present invention. For
example, a polynucleotide can be produced by insertion into an
appropriate vector, which can be expressed when transfected into a
competent cell. Alternatively, a polynucleotide can be amplified
using PCR techniques or expression in suitable hosts (see, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1989). Alternatively, a
polynucleotide can be synthesized using the solid phase techniques,
as described in Beaucage S L & Iyer R P, Tetrahedron 1992, 48:
2223-311; Matthes et al., EMBO J 1984, 3: 801-5.
[0112] Vectors containing the polynucleotide of the present
invention and host cells harboring the vectors are also included in
the present invention.
V. Exosomes
[0113] The present invention further provides intracellular
vesicles called exosomes, which present complexes formed between
the peptides of the present invention and HLA antigens on their
surface. Exosomes can be prepared, for example, by using the
methods detailed in Japanese Patent Application Kohyo Publications
Nos. Hei 11-510507 and WO99/03499, and can be prepared using APCs
obtained from patients who are subject to treatment and/or
prevention. The exosomes of this invention can be inoculated as
vaccines, in a fashion similar to the peptides of this
invention.
[0114] The type of HLA antigens included in the complexes must
match that of the subject requiring treatment and/or prevention.
For example, in the Japanese and Caucasian populations, HLA-A02
type is prevalent. Thus, the use of the A02 type is favorable for
obtaining effective results in these populations, with subtypes
such as A0201 also finding use. Typically, in the clinic, the type
of HLA antigen of the patient requiring treatment is investigated
in advance, which enables the appropriate selection of peptides
having high levels of binding affinity to the particular antigen,
or having CTL inducibility by antigen presentation. Furthermore, in
order to obtain peptides having both high binding affinity and CTL
inducibility, substitution, insertion and/or addition of 1, 2, or
several amino acids can be performed based on the amino acid
sequence of the naturally occurring INHBB partial peptide.
[0115] When using A02 type HLA antigen for the exosome of the
present invention, the peptides having the sequence selected from
among SEQ ID NO: 1 to 14 find use.
VI. Antigen-Presenting Cells (APCs)
[0116] The present invention also provides isolated APCs that
present complexes formed between HLA antigens and the peptides of
this invention on its surface. The APCs that are obtained by
contacting the peptides of this invention, or introducing the
nucleotides encoding the peptides of this invention in an
expressible form can be derived from patients who are subject to
treatment and/or prevention, and can be administered as vaccines by
themselves or in combination with other drugs including the
peptides of this invention, exosomes, or cytotoxic T cells.
[0117] The APCs are not limited to a particular kind of cells and
include dendritic cells (DCs), Langerhans cells, macrophages, B
cells, and activated T cells, which are known to present
proteinaceous antigens on their cell surface so as to be recognized
by lymphocytes. Since DC is a representative APC having the
strongest CTL inducing action among APCs, DCs find use as the APCs
of the present invention.
[0118] For example, an APC can be obtained by inducing DCs from
peripheral blood monocytes and then contacting (stimulating) them
with the peptides of this invention in vitro, ex vivo or in vivo.
When the peptides of this invention are administered to the
subjects, APCs that present the peptides of this invention are
induced in the body of the subject. The phrase "inducing APC"
includes contacting (stimulating) a cell with the peptides of this
invention, or nucleotides encoding the peptides of this invention
to present complexes formed between HLA antigens and the peptides
of this invention on cell's surface. Alternatively, after
introducing the peptides of this invention to the APCs to allow the
APCs to present the peptides, the APCs can be administered to the
subject as a vaccine. For example, the ex vivo administration can
include the steps of: [0119] a: collecting APCs from a first
subject, [0120] b: contacting with the APCs of step a, with the
peptide and [0121] c: administering the peptide-loaded APCs to a
second subject.
[0122] The first subject and the second subject can be the same
individual, or may be different individuals. Alternatively,
according to the present invention, use of the peptides of the
present invention for manufacturing a pharmaceutical composition
inducing antigen-presenting cells is provided. In addition, the
present invention provides a method or process for manufacturing a
pharmaceutical composition inducing antigen-presenting cells,
wherein the method includes the step of admixing or formulating the
peptide of the present invention with a pharmaceutically acceptable
carrier. Further, the present invention also provides the peptides
of the present invention for inducing antigen-presenting cells. The
APCs obtained by step b can be administered to the subject as a
vaccine.
[0123] According to an aspect of the present invention, the APCs of
the present invention have a high level of CTL inducibility. In the
term of "high level of CTL inducibility", the high level is
relative to the level of that by APCs contacted with no peptide or
peptides which can not induce the CTL. Such APCs having a high
level of CTL inducibility can be prepared by a method which
includes the step of transferring genes containing polynucleotides
that encode the peptides of this invention to APCs in vitro. The
introduced genes can be in the form of DNAs or RNAs. Examples of
methods for introduction include, without particular limitations,
various methods conventionally performed in this field, such as
lipofection, electroporation, and calcium phosphate method can be
used. More specifically, it can be performed as described in Cancer
Res 1996, 56: 5672-7; J Immunol 1998, 161: 5607-13; J Exp Med 1996,
184: 465-72; Published Japanese Translation of International
Publication No. 2000-509281. By transferring the gene into APCs,
the gene undergoes transcription, translation, and such in the
cell, and then the obtained protein is processed by MHC Class I or
Class II, and proceeds through a presentation pathway to present
peptides.
VII. Cytotoxic T Cells (CTLs)
[0124] A cytotoxic T cell induced against any of the peptides of
the present invention strengthens the immune response targeting
tumor-associated endothelia in vivo and thus can be used as
vaccines, in a fashion similar to the peptides per se. Thus, the
present invention also provides isolated cytotoxic T cells that are
specifically induced or activated by any of the present
peptides.
[0125] Such cytotoxic T cells can be obtained by (1) administering
the peptide of the present invention to a subject, and then
collecting cytotoxic T cells from the subject, or (2) contacting
(stimulating) subject-derived APCs, and CD8-positive cells, or
peripheral blood mononuclear leukocytes in vitro with the peptides
of the present invention and then isolating cytotoxic T cells.
[0126] The cytotoxic T cells, which have been induced by
stimulation with APCs that present the peptides of this invention,
can be derived from patients who are subject to treatment and/or
prevention, and can be administered by themselves or in combination
with other drugs including the peptides of this invention or
exosomes for the purpose of regulating effects. The obtained
cytotoxic T cells act specifically against target cells presenting
the peptides of this invention, or for example, the same peptides
used for induction. In the other word, the cytotoxic T cells can
recognize (i.e., binding to) a complex formed between a HLA antigen
and the peptide of the present invention on a target cell surface
with the T cell receptor and then attack the target cell to induce
the death of the target cell. The target cells can be cells that
endogenously express INHBB, or cells that are transfected with the
INHBB gene; and cells that present a peptide of this invention on
the cell surface due to stimulation by the peptide can also serve
as targets of activated CTL attack.
VIII. T Cell Receptor (TCR)
[0127] The present invention also provides a composition composed
of a nucleic acid sequence encoding polypeptides that are capable
of forming a subunit of a T cell receptor (TCR), and methods of
using the same. The TCR subunits have the ability to form TCRs that
confer specificity to T cells against tumor cells presenting INHBB.
By using the known methods in the art, the nucleic acid sequence of
alpha- and beta-chains of the TCR expressed in the CTL induced with
one or more peptides of this invention can be identified
(WO2007/032255 and Morgan et al., J Immunol, 171, 3288 (2003)). The
derivative TCRs can bind to the INHBB peptide displaying on the
target cells with high avidity, and optionally mediate efficient
killing of target cells presenting the INHBB peptide in vivo and in
vitro.
[0128] The nucleic acids sequence encoding the TCR subunits can be
incorporated into suitable vectors e.g. retroviral vectors. These
vectors are well known in the art. The nucleic acids or the vectors
containing them usefully can be transferred into a T cell, for
example, a T cell from a patient. Advantageously, the invention
provides an off-the-shelf composition allowing rapid modification
of a patient's own T cells (or those of another mammal) to rapidly
and easily produce modified T cells having excellent cancer cell
killing properties.
[0129] Also, the present invention provides CTLs which are prepared
by transduction with the nucleic acids encoding the TCR subunit
polypeptides that bind to the INHBB peptide e.g. SEQ ID NOs: 1 to
14 in the context of HLA-A02. The transduced CTLs are capable of
homing to cancer cells in vivo, and can be expanded by well known
culturing methods in vitro (e.g., Kawakami et al., J Immunol., 142,
3452-3461 (1989)). The T cells of the invention can be used to form
an immunogenic composition useful in treating or the prevention of
cancer in a patient in need of therapy or protection
(WO2006/031221).
IX. Pharmaceutical Agents or Composition
[0130] Prevention and prophylaxis include any activity which
reduces the burden of mortality or morbidity from disease.
Prevention and prophylaxis can occur "at primary, secondary and
tertiary prevention levels." While primary prevention and
prophylaxis avoid the development of a disease, secondary and
tertiary levels of prevention and prophylaxis encompass activities
aimed at the prevention and prophylaxis of the progression of a
disease and the emergence of symptoms as well as reducing the
negative impact of an already established disease by restoring
function and reducing disease-related complications. Alternatively,
prevention and prophylaxis include a wide range of prophylactic
therapies aimed at alleviating the severity of the particular
disorder, e.g. reducing the proliferation and metastasis of
tumors.
[0131] Treating and/or for the prophylaxis of cancer or tumor
and/or the prevention of postoperative recurrence thereof includes
any of the following steps, such as surgical removal of cancer
cells, inhibition of the growth of cancerous cells, involution or
regression of a tumor, induction of remission and suppression of
occurrence of cancer, tumor regression, and reduction or inhibition
of metastasis. Effectively treating and/or the prophylaxis of
cancer decreases mortality and improves the prognosis of
individuals having cancer, decreases the levels of tumor markers in
the blood, and alleviates detectable symptoms accompanying cancer.
For example, reduction or improvement of symptoms constitutes
effectively treating and/or the prophylaxis include 10%, 20%, 30%
or more reduction, or stable disease.
[0132] Since INHBB expression is up-regulated in several cancers as
compared with normal tissue, the peptides of this invention or
polynucleotides encoding such peptides can be used for the
treatment and/or for the prophylaxis of cancer, and/or prevention
of postoperative recurrence thereof. Thus, the present invention
provides a pharmaceutical agent or composition for treating and/or
preventing cancer, and/or preventing the postoperative recurrence
thereof, which includes one or more of the peptides of this
invention, or polynucleotides encoding the peptides as an active
ingredient. Alternatively, the present peptides can be expressed on
the surface of any of the foregoing exosomes or cells, such as APCs
for the use as pharmaceutical agents or compositions. In addition,
the aforementioned cytotoxic T cells which target any of the
peptides of the present invention can also be used as the active
ingredient of the present pharmaceutical agents or compositions. In
the context of the present invention, the phrase "targeting a
peptide" refers to recognizing (i.e., binding to) a complex formed
between a HLA antigen and a peptide on a target cell surface with
the T cell receptor, and then attacking the target cell to induce
the death of the target cell.
[0133] In another embodiment, the present invention also provides
the use of an active ingredient selected from among:
[0134] (a) a peptide of the present invention,
[0135] (b) a nucleic acid encoding such a peptide as disclosed
herein in an expressible form,
[0136] (c) an APC of the present invention, and
[0137] (d) a cytotoxic T cells of the present invention in
manufacturing a pharmaceutical composition or agent for treating
cancer.
[0138] Alternatively, the present invention further provides an
active ingredient selected from among:
[0139] (a) a peptide of the present invention,
[0140] (b) a nucleic acid encoding such a peptide as disclosed
herein in an expressible form,
[0141] (c) an APC of the present invention, and
[0142] (d) a cytotoxic T cells of the present invention for use in
treating cancer.
[0143] Alternatively, the present invention further provides a
method or process for manufacturing a pharmaceutical composition or
agent for treating cancer, wherein the method or process includes
the step of formulating a pharmaceutically or physiologically
acceptable carrier with an active ingredient selected from
among:
[0144] (a) a peptide of the present invention,
[0145] (b) a nucleic acid encoding such a peptide as disclosed
herein in an expressible form,
[0146] (c) an APC of the present invention, and
[0147] (d) a cytotoxic T cells of the present invention as active
ingredients.
[0148] In another embodiment, the present invention also provides a
method or process for manufacturing a pharmaceutical composition or
agent for treating cancer, wherein the method or process includes
the step of admixing an active ingredient with a pharmaceutically
or physiologically acceptable carrier, wherein the active
ingredient is selected from among:
[0149] (a) a peptide of the present invention,
[0150] (b) a nucleic acid encoding such a peptide as disclosed
herein in an expressible form,
[0151] (c) an APC of the present invention, and
[0152] (d) a cytotoxic T cells of the present invention.
[0153] Alternatively, the pharmaceutical composition or agent of
the present invention may be used for either or both the
prophylaxis of cancer and prevention of postoperative recurrence
thereof.
[0154] The present pharmaceutical agents or compositions find use
as a vaccine. In the context of the present invention, the phrase
"vaccine" (also referred to as an "immunogenic composition") refers
to a substance that has the function to induce anti-tumor immunity
upon inoculation into animals.
[0155] The pharmaceutical agents or compositions of the present
invention can be used to treat and/or prevent cancers, and/or
prevention of postoperative recurrence thereof in subjects or
patients including human and any other mammal including, but not
limited to, mouse, rat, guinea-pig, rabbit, cat, dog, sheep, goat,
pig, cattle, horse, monkey, baboon, and chimpanzee, particularly a
commercially important animal or a domesticated animal.
[0156] According to the present invention, polypeptides having an
amino acid sequence selected from among SEQ ID NOs: 1 to 14 or
polypeptides having an amino acid sequence selected from among SEQ
ID NOs: 1 to 14 have been found to be HLA-A02 restricted epitope
peptides or candidates, respectively, that can induce potent and
specific immune response. Therefore, the present pharmaceutical
agents or compositions which include any of these polypeptides with
the amino acid sequences selected from among SEQ ID NOs: 1 to 14
are particularly suited for the administration to subjects whose
HLA antigen is HLA-A02. The same applies to pharmaceutical agents
or compositions which include polynucleotides encoding any of these
polypeptides.
[0157] Cancers to be treated by the pharmaceutical agents or
compositions of the present invention are not limited and include
all kinds of cancers wherein INHBB is involved, including, for
example, cholangio cellular carcinoma, esophageal cancer, non-small
cell lung cancer (NSCLC), renal carcinoma, small cell lung cancer
(SCLC) and soft tissue tumor.
[0158] The present pharmaceutical agents or compositions can
contain in addition to the aforementioned active ingredients, other
peptides which have the ability to induce CTLs against cancerous
cells, other polynucleotides encoding the other peptides, other
cells that present the other peptides, or such. Herein, the other
peptides that have the ability to induce CTLs against cancerous
cells are exemplified by cancer specific antigens (e.g., identified
TAAs), but are not limited thereto.
[0159] If needed, the pharmaceutical agents or compositions of the
present invention can optionally include other therapeutic
substances as an active ingredient, so long as the substance does
not inhibit the antitumoral effect of the active ingredient, e.g.,
any of the present peptides. For example, formulations can include
anti-inflammatory agents or compositions, pain killers,
chemotherapeutics, and the like. In addition to including other
therapeutic substances in the medicament itself, the medicaments of
the present invention can also be administered sequentially or
concurrently with the one or more other pharmacologic agents or
compositions. The amounts of medicament and pharmacologic agent or
composition depend, for example, on what type of pharmacologic
agent(s) or composition(s) is/are used, the disease being treated,
and the scheduling and routes of administration.
[0160] It should be understood that in addition to the ingredients
particularly mentioned herein, the pharmaceutical agents or
compositions of this invention can include other agents or
compositions conventional in the art having regard to the type of
formulation in question.
[0161] In one embodiment of the present invention, the present
pharmaceutical agents or compositions can be included in articles
of manufacture and kits containing materials useful for treating
the pathological conditions of the disease to be treated, e.g.,
cancer. The article of manufacture can include a container of any
of the present pharmaceutical agents or compositions with a label.
Suitable containers include bottles, vials, and test tubes. The
containers can be formed from a variety of materials, such as glass
or plastic. The label on the container should indicate the agent or
compositions is used for treating or prevention of one or more
conditions of the disease. The label can also indicate directions
for administration and so on.
[0162] In addition to the container described above, a kit
including a pharmaceutical agent or compositions of the present
invention can optionally further include a second container housing
a pharmaceutically-acceptable diluent. It can further include other
materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, syringes, and
package inserts with instructions for use.
[0163] The pharmaceutical compositions can, if desired, be
presented in a pack or dispenser device which can contain one or
more unit dosage forms containing the active ingredient. The pack
can, for example, include metal or plastic foil, such as a blister
pack. The pack or dispenser device can be accompanied by
instructions for administration.
[0164] (1) Pharmaceutical Agents or Compositions Containing the
Peptides as the Active Ingredient
[0165] The peptides of this invention can be administered directly
as a pharmaceutical agent or composition, or if necessary, that has
been formulated by conventional formulation methods. In the latter
case, in addition to the peptides of this invention, carriers,
excipients, and such that are ordinarily used for drugs can be
included as appropriate without particular limitations. Examples of
such carriers are sterilized water, physiological saline, phosphate
buffer, culture fluid and such. Furthermore, the pharmaceutical
agents or compositions can contain as necessary, stabilizers,
suspensions, preservatives, surfactants and such. The
pharmaceutical agents or compositions of this invention can be used
for anticancer purposes.
[0166] The peptides of this invention can be prepared as a
combination composed of two or more of peptides of the invention,
to induce CTL in vivo. The peptide combination can take the form of
a cocktail or can be conjugated to each other using standard
techniques. For example, the peptides can be chemically linked or
expressed as a single fusion polypeptide sequence. The peptides in
the combination can be the same or different. By administering the
peptides of this invention, the peptides are presented at a high
density by the HLA antigens on APCs, then CTLs that specifically
react toward the complex formed between the displayed peptide and
the HLA antigen are induced. Alternatively, APCs that present any
of the peptides of this invention on their cell surface, which may
be obtained by stimulating APCs (e.g., DCs) derived from a subject
with the peptides of this invention, may be administered to the
subject, and as a result, CTLs are induced in the subject and
aggressiveness towards the cancer cells, such as cholangio cellular
carcinoma, esophageal cancer, non-small cell lung cancer (NSCLC),
renal carcinoma, small cell lung cancer (SCLC) and soft tissue
tumor can be increased.
[0167] The pharmaceutical agents or compositions for the treatment
and/or prevention of cancer, which include a peptide of this
invention as the active ingredient, can also include an adjuvant
known to effectively establish cellular immunity. Alternatively,
they can be administered with other active ingredients, and they
can be administered by formulation into granules. An adjuvant
refers to a compound that enhances the immune response against the
protein when administered together (or successively) with the
protein having immunological activity. Adjuvants contemplated
herein include those described in the literature (Clin Microbiol
Rev 1994, 7: 277-89). Examples of suitable adjuvants include, but
are not limited to, aluminum phosphate, aluminum hydroxide, alum,
cholera toxin, salmonella toxin, and such, but are not limited
thereto.
[0168] Furthermore, liposome formulations, granular formulations in
which the peptide is bound to few-micrometers diameter beads, and
formulations in which a lipid is bound to the peptide may be
conveniently used.
[0169] In some embodiments, the pharmaceutical agents or
compositions of the invention may further include a component which
primes CTL. Lipids have been identified as agents or compositions
capable of priming CTL in vivo against viral antigens. For example,
palmitic acid residues can be attached to the epsilon- and
alpha-amino groups of a lysine residue and then linked to a peptide
of the invention. The lipidated peptide can then be administered
either directly in a micelle or particle, incorporated into a
liposome, or emulsified in an adjuvant. As another example of lipid
priming of CTL responses, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to
prime CTL when covalently attached to an appropriate peptide (see,
e.g., Deres et al., Nature 1989, 342: 561-4).
[0170] The method of administration can be oral, intradermal,
subcutaneous, intravenous injection, or such, and systemic
administration or local administration to the vicinity of the
targeted sites. The administration can be performed by single
administration or boosted by multiple administrations. The dose of
the peptides of this invention can be adjusted appropriately
according to the disease to be treated, age of the patient, weight,
method of administration, and such, and is ordinarily 0.001 mg to
1000 mg, for example, 0.001 mg to 1000 mg, for example, 0.1 mg to
10 mg, and can be administered once in a few days to few months.
One skilled in the art can appropriately select a suitable
dose.
[0171] (2) Pharmaceutical Agents or Compositions Containing
Polynucleotides as the Active Ingredient
[0172] The pharmaceutical agents or compositions of the invention
can also contain nucleic acids encoding the peptides disclosed
herein in an expressible form. Herein, the phrase "in an
expressible form" means that the polynucleotide, when introduced
into a cell, will be expressed in vivo as a polypeptide that
induces anti-tumor immunity. In an exemplified embodiment, the
nucleic acid sequence of the polynucleotide of interest includes
regulatory elements necessary for expression of the polynucleotide.
The polynucleotide(s) can be equipped so to achieve stable
insertion into the genome of the target cell (see, e.g., Thomas K R
& Capecchi M R, Cell 1987, 51: 503-12 for a description of
homologous recombination cassette vectors). See, e.g., Wolff et
al., Science 1990, 247: 1465-8; U.S. Pat. Nos. 5,580,859;
5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO
98/04720. Examples of DNA-based delivery technologies include
"naked DNA", facilitated (bupivacaine, polymers, peptide-mediated)
delivery, cationic lipid complexes, and particle-mediated ("gene
gun") or pressure-mediated delivery (see, e.g., U.S. Pat. No.
5,922,687).
[0173] The peptides of the present invention can also be expressed
by viral or bacterial vectors. Examples of expression vectors
include attenuated viral hosts, such as vaccinia or fowlpox. This
approach involves the use of vaccinia virus, e.g., as a vector to
express nucleotide sequences that encode the peptide. Upon
introduction into a host, the recombinant vaccinia virus expresses
the immunogenic peptide, and thereby elicits an immune response.
Vaccinia vectors and methods useful in immunization protocols are
described in, e.g., U.S. Pat. No. 4,722,848. Examples of another
vector include BCG (Bacille Calmette Guerin). BCG vectors are
described in Stover et al., Nature 1991, 351: 456-60. A wide
variety of other vectors useful for therapeutic administration or
immunization, e.g., adeno and adeno-associated virus vectors,
retroviral vectors, Salmonella typhi vectors, detoxified anthrax
toxin vectors, and the like, will be apparent. See, e.g., Shata et
al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol
2000, 68: 793-806; Hipp et al., In Vivo 2000, 14: 571-85.
[0174] Delivery of a polynucleotide into a subject can be either
direct, in which case the subject is directly exposed to a
polynucleotide-carrying vector, or indirect, in which case, cells
are first transformed with the polynucleotide of interest in vitro,
then the cells are transplanted into the subject. Theses two
approaches are known, respectively, as in vivo and ex vivo gene
therapies.
[0175] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical
[0176] Pharmacy 1993, 12: 488-505; Wu and Wu, Biotherapy 1991, 3:
87-95; Tolstoshev, Ann Rev Pharmacol Toxicol 1993, 33: 573-96;
Mulligan, Science 1993, 260: 926-32; Morgan & Anderson, Ann Rev
Biochem 1993, 62: 191-217; Trends in Biotechnology 1993, 11(5):
155-215). Methods commonly known in the art of recombinant DNA
technology which can also be used for the present invention are
described in eds. Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, NY, 1993; and Krieger, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY,
1990.
[0177] The method of administration can be oral, intradermal,
subcutaneous, intravenous injection, or such, and systemic
administration or local administration to the vicinity of the
targeted sites finds use. The administration can be performed by
single administration or boosted by multiple administrations. The
dose of the polynucleotide in the suitable carrier or cells
transformed with the polynucleotide encoding the peptides of this
invention can be adjusted appropriately according to the disease to
be treated, age of the patient, weight, method of administration,
and such, and is ordinarily 0.001 mg to 1000 mg, for example, 0.001
mg to 1000 mg, for example, 0.1 mg to 10 mg, and can be
administered once every a few days to once every few months. One
skilled in the art can appropriately select the suitable dose.
X. Methods Using the Peptides, Exosomes, APCs and CTLs
[0178] The peptides of the present invention and polynucleotides
encoding such peptides can be used for inducing APCs and CTLs. The
exosomes and APCs of the present invention can be also used for
inducing CTLs. The peptides, polynucleotides, exosomes and APCs can
be used in combination with any other compounds so long as the
compounds do not inhibit their CTL inducibility. Thus, any of the
aforementioned pharmaceutical agents or compositions of the present
invention can be used for inducing CTLs, and in addition thereto,
those including the peptides and polynucleotides can be also be
used for inducing APCs as discussed below.
[0179] (1) Method of Inducing Antigen-Presenting Cells (APCs)
[0180] The present invention provides methods of inducing APCs
using the peptides of this invention or polynucleotides encoding
the peptides. The induction of APCs can be performed as described
above in section "VI. Antigen-presenting cells". This invention
also provides a method for inducing APCs having a high level of CTL
inducibility, the induction of which has been also mentioned under
the item of "VI. Antigen-presenting cells", supra.
Preferably, the methods for inducing APCs include at least one step
selected from among: [0181] a: contacting APCs with the peptides of
the present invention, and [0182] b: introducing the polypeptides
of the present invention in an expressible form into APCs. Such
methods for inducing APCs are preferably performed in vitro or ex
vivo. When the methods performed in vitro or ex vivo, APCs to be
induced may be obtained from a subject to be treated or others
whose HLA antigens are the same as the subject.
[0183] (2) Method of Inducing CTLs
[0184] Furthermore, the present invention provides methods for
inducing CTLs using the peptides of this invention, polynucleotides
encoding the peptides, or exosomes or APCs presenting the
peptides.
[0185] The present invention also provides methods for inducing
CTLs using a polynucleotide encoding a polypeptide that is capable
of forming a T cell receptor (TCR) subunit recognizing (i.e.,
binding to) a complex of the peptides of the present invention and
HLA antigens on a cell surface. Preferably, the methods for
inducing CTLs include at least one step selected from among:
[0186] a: contacting a CD8-positive T cell with an
antigen-presenting cell and/or an exosome that presents on its
surface a complex of an HLA antigen and a peptide of the present
invention, and
[0187] b: introducing a polynucleotide encoding a polypeptide that
is capable of forming a TCR subunit recognizing a complex of a
peptide of the present invention and an HLA antigen into a CD8
positive T cell.
[0188] When the peptides of this invention are administered to a
subject, CTL is induced in the body of the subject, and the
strength of the immune response targeting the tumor-associated
endothelia is enhanced. Alternatively, the peptides and
polynucleotides encoding the peptides can be used for an ex vivo
therapeutic method, in which subject-derived APCs, and CD8-positive
cells, or peripheral blood mononuclear leukocytes are contacted
(stimulated) with the peptides of this invention in vitro, and
after inducing CTL, the activated CTL cells are returned to the
subject. For example, the method can include steps of:
[0189] a: collecting APCs from subject,
[0190] b: contacting with the APCs of step a, with the peptide,
[0191] c: mixing the APCs of step b with CD.sup.8+ T cells, and
co-culturing for inducing CTLs, and
[0192] d: collecting CD.sup.8+ T cells from the co-culture of step
c.
[0193] Alternatively, according to the present invention, use of
the peptides of this invention for manufacturing a pharmaceutical
composition inducing CTLs is provided. In addition, the present
invention provides a method or process for manufacturing a
pharmaceutical agent or composition inducing CTLs, wherein the
method includes the step of admixing or formulating the peptide of
the present invention with a pharmaceutically acceptable carrier.
Further, the present invention also provides the peptide of the
present invention for inducing CTLs.
[0194] The CD.sup.8+ T cells having cytotoxic activity obtained by
step d can be administered to the subject as a vaccine. The APCs to
be mixed with the CD.sup.8+ T cells in above step c can also be
prepared by transferring genes coding for the present peptides into
the APCs as detailed above in section "VI. Antigen-presenting
cells"; but are not limited thereto and any APC or exosome which
effectively presents the present peptides to the T cells can be
used for the present method.
[0195] The following examples are presented to illustrate the
present invention and to assist one of ordinary skill in making and
using the same. The examples are not intended in any way to
otherwise limit the scope of the invention.
EXAMPLES
Materials and Methods
[0196] Cell Lines
[0197] H2 (HLA-A02), human B-lymphoblastoid cell line, and COS7
were purchased from ATCC.
[0198] Candidate Selection of Peptides Derived From INHBB
[0199] 9-mer and 10-mer peptides derived from INHBB that bind to
HLA-A*0201 molecules were predicted using binding prediction
software "BIMAS" (www-bimas.cit.nih.gov/molbio/hla_bind), which
algorithms had been described by Parker K C et al.(J Immunol 1994,
152(1): 163-75) and Kuzushima K et al.(Blood 2001, 98(6): 1872-81).
These peptides were synthesized by Sigma (Sapporo, Japan) or
Biosynthesis Inc. (Lewisville, Tex.) according to a standard solid
phase synthesis method and purified by reversed phase high
performance liquid chromatography (HPLC). The purity (>90%) and
the identity of the peptides were determined by analytical HPLC and
mass spectrometry analysis, respectively. Peptides were dissolved
in dimethylsulfoxide (DMSO) at 20 mg/ml and stored at -80 degrees
C.
[0200] In Vitro CTL Induction
[0201] Monocyte-derived dendritic cells (DCs) were used as
antigen-presenting cells (APCs) to induce cytotoxic T lymphocyte
(CTL) responses against peptides presented on human leukocyte
antigen (HLA). DCs were generated in vitro as described elsewhere
(Nakahara S et al., Cancer Res 2003 Jul 15, 63(14): 4112-8).
Specifically, peripheral blood mononuclear cells (PBMCs) isolated
from a normal volunteer (HLA-A*0201 positive) by Ficoll-Plaque
(Pharmacia) solution were separated by adherence to a plastic
tissue culture dish (Becton Dickinson) so as to enrich them as the
monocyte fraction. The monocyte-enriched population was cultured in
the presence of 1000 U/ml of granulocyte-macrophage
colony-stimulating factor (GM-CSF) (R&D System) and 1000 U/ml
of interleukin (IL)-4 (R&D System) in AIM-V Medium (Invitrogen)
containing 2% heat-inactivated autologous serum (AS). After 7 days
of culture, the cytokine-induced DCs were pulsed with 20 mcg/ml of
each of the synthesized peptides in the presence of 3 mcg/ml of
beta2-microglobulin for 3 hr at 37 degrees C. in AIM-V Medium. The
generated cells appeared to express DC-associated molecules, such
as CD80, CD83, CD86 and HLA class II, on their cell surfaces (data
not shown). These peptide-pulsed DCs were then inactivated by
Mitomycin C (MMC) (30 mcg/ml for 30 min) and mixed at a 1:20 ratio
with autologous CD8+ T cells, obtained by positive selection with
CD8 Positive Isolation Kit (Dynal). These cultures were set up in
48-well plates (Corning); each well contained 1.5.times.10.sup.4
peptide-pulsed DCs, 3.times.10.sup.5 CD8+ T cells and 10 ng/ml of
IL-7 (R&D System) in 0.5 ml of AIM-V/2% AS medium. Three days
later, these cultures were supplemented with IL-2 (CHIRON) to a
final concentration of 20 IU/ml. On day 7 and 14, the T cells were
further stimulated with the autologous peptide-pulsed DCs. The DCs
were prepared each time by the same way described above. CTL was
tested against peptide-pulsed T2 cells after the 3rd round of
peptide stimulation on day 21 (Tanaka H et al., Br J Cancer 2001
Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer 2001 Apr. 20,
84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004 Dec. 15,
10(24): 8577-86; Suda T et al., Cancer Sci 2006 May, 97(5): 411-9;
Watanabe T et al., Cancer Sci 2005 Aug, 96(8): 498-506).
[0202] CTL Expansion Procedure
[0203] CTLs were expanded in culture using the method similar to
the one described by Riddell et al. (Walter E A et al., N Engl J
Med 1995 Oct. 19, 333(16): 1038-44; Riddell S R et al., Nat Med
1996 Feb., 2(2): 216-23). A total of 5.times.10.sup.4 CTLs were
suspended in 25 ml of AIM-V/5% AS medium with 2 kinds of human
B-lymphoblastoid cell lines, inactivated by MMC, in the presence of
40 ng/ml of anti-CD3 monoclonal antibody (Pharmingen). One day
after initiating the cultures, 120 IU/ml of IL-2 were added to the
cultures. The cultures were fed with fresh AIM-V/5% AS medium
containing 30 IU/ml of IL-2 on days 5, 8 and 11 (Tanaka H et al.,
Br J Cancer 2001 Jan. 5, 84(1): 94-9; Umano Y et al., Br J Cancer
2001 Apr. 20, 84(8): 1052-7; Uchida N et al., Clin Cancer Res 2004
Dec. 15, 10(24): 8577-86; Suda T et al., Cancer Sci 2006 May,
97(5): 411-9; Watanabe T et al., Cancer Sci 2005 Aug, 96(8):
498-506).
[0204] Specific CTL Activity
[0205] To examine specific CTL activity, interferon (IFN)-gamma
enzyme-linked immunospot (ELISPOT) assay and IFN-gamma
enzyme-linked immunosorbent assay (ELISA) were performed.
Specifically, peptide-pulsed T2 (1.times.10.sup.4/well) was
prepared as stimulator cells. Cultured cells in 48 wells were used
as responder cells. IFN-gamma ELISPOT assay and IFN-gamma ELISA
assay were performed under manufacture procedure.
Results
[0206] Enhanced INHBB Expression in Cancers
[0207] INHBB expression was validly elevated in the following
cancers: 10 out of 21 in cholangiocellular carcinoma, 12 out of 12
in esophageal cancer, 10 out of 13 in NSCLC, 22 out of 24 in renal
carcinoma, 8 out of 14 in SCLC cancer and 45 out of 49 in soft
tissue tumor, in comparing with corresponding normal tissue.
[0208] Stimulation of the T Cells Using the Predicted Peptides From
INHBB Restricted With HLA-A0201 and Establishment for CTL Lines
Stimulated With INHBB Derived Peptides
[0209] CTLs for those peptides derived from INHBB were generated
according to the protocols set forth in "Materials and Methods"
section above. Resulting CTLs having detectable specific CTL
activity, as determined by IFN-gamma ELISPOT assay, are shown in
FIG. 1.
[0210] INHBB-A02-9-213 (SEQ ID NO: 1), INHBB-A02-9-174 (SEQ ID NO:
2), INHBB-A02-9-257 (SEQ ID NO: 3), INHBB-A02-9-313 (SEQ ID NO: 4),
INHBB-A02-9-139 (SEQ ID NO: 5), INHBB-A02-9-8 (SEQ ID NO: 6),
INHBB-A02-9-250 (SEQ ID NO: 7), INHBB-A02-10-179 (SEQ ID NO: 8),
INHBB-A02-10-237 (SEQ ID NO: 9), INHBB-A02-10-313 (SEQ ID NO: 10),
INHBB-A02-10-173 (SEQ ID NO: 11), INHBB-A02-10-256 (SEQ ID NO: 12),
INHBB-A02-10-162 (SEQ ID NO: 13) and INHBB-A02-10-85 (SEQ ID NO:
14) demonstrated potent IFN-gamma production as compared to the
control by IFN-gamma ELISPOT assay. Furthermore, the cells in the
positive well number #7 stimulated with SEQ ID NO: 2, were expanded
and CTL line was established. The CTL line having higher specific
CTL activity against the peptide-pulsed target as compared to the
activity against target without peptide pulse was determined by
IFN-gamma ELISA (FIG. 2).
[0211] The results herein demonstrate that the CTL line
demonstrated potent IFN-gamma production against the target cells
pulsed with corresponding peptide as compared to target cells
without peptide pulse. In the context of the present invention, the
peptides which could establish CTL line were selected as potent CTL
stimulation peptide.
[0212] In conclusion, novel HLA-A02 epitope peptides derived from
INHBB were identified and demonstrated to be applicable for cancer
immunotherapy.
INDUSTRIAL APPLICABILITY
[0213] The present invention describes new TAAs, particularly those
derived from INHBB, that induce potent and specific anti-tumor
immune responses and have applicability to a wide array of cancer
types. Such TAAs warrant further development as peptide vaccines
against diseases associated with INHBB, e.g., cancer, more
particularly, cholangio cellular carcinoma, esophageal cancer,
non-small cell lung cancer (NSCLC), renal carcinoma, small cell
lung cancer (SCLC) and soft tissue tumor.
[0214] While the invention is herein described in detail and with
reference to specific embodiments thereof, it is to be understood
that the foregoing description is exemplary and explanatory in
nature and is intended to illustrate the invention and its
preferred embodiments. Through routine experimentation, one skilled
in the art will readily recognize that various changes and
modifications can be made therein without departing from the spirit
and scope of the invention, the metes and bounds of which are
defined by the appended claims.
Sequence CWU 1
1
1619PRTArtificialAn artificially synthesized peptide 1Asn Met Val
Glu Lys Arg Val Asp Leu1 529PRTArtificialAn artificially
synthesized peptide 2Val Gln Ala Ser Leu Trp Leu Tyr Leu1
539PRTArtificialAn artificially synthesized peptide 3Glu Leu Ala
Val Val Pro Val Phe Val1 549PRTArtificialAn artificially
synthesized peptide 4Arg Leu Ile Gly Trp Asn Asp Trp Ile1
559PRTArtificialAn artificially synthesized peptide 5Arg Val Ser
Glu Ile Ile Ser Phe Ala1 569PRTArtificialAn artificially
synthesized peptide 6Ala Leu Gly Ala Ala Cys Leu Leu Leu1
579PRTArtificialAn artificially synthesized peptide 7Val Gln Cys
Asp Ser Cys Gln Glu Leu1 5810PRTArtificialAn artificially
synthesized peptide 8Trp Leu Tyr Leu Lys Leu Leu Pro Tyr Val1 5
10910PRTArtificialAn artificially synthesized peptide 9Ala Leu Phe
Glu Arg Gly Glu Arg Arg Leu1 5 101010PRTArtificialAn artificially
synthesized peptide 10Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile1 5
101110PRTArtificialAn artificially synthesized peptide 11Val Val
Gln Ala Ser Leu Trp Leu Tyr Leu1 5 101210PRTArtificialAn
artificially synthesized peptide 12Gln Glu Leu Ala Val Val Pro Val
Phe Val1 5 101310PRTArtificialAn artificially synthesized peptide
13Phe Ile Ser Asn Glu Gly Asn Gln Asn Leu1 5 101410PRTArtificialAn
artificially synthesized peptide 14Arg Leu Gln Met Arg Gly Arg Pro
Asn Ile1 5 10153218DNAHomo sapiensCDS(47)..(1270) 15actcggctcg
cctcgcggcg ggcgccctcg tcgccagcgg cgcacc atg gac ggg 55 Met Asp Gly
1ctg ccc ggt cgg gcg ctg ggg gcc gcc tgc ctt ctg ctg ctg gcg gcc
103Leu Pro Gly Arg Ala Leu Gly Ala Ala Cys Leu Leu Leu Leu Ala Ala
5 10 15ggc tgg ctg ggg cct gag gcc tgg ggc tca ccc acg ccc ccg ccg
acg 151Gly Trp Leu Gly Pro Glu Ala Trp Gly Ser Pro Thr Pro Pro Pro
Thr20 25 30 35cct gcc gcg ccg ccg cca ccc ccg cca ccc gga tcc ccg
ggt ggc tcg 199Pro Ala Ala Pro Pro Pro Pro Pro Pro Pro Gly Ser Pro
Gly Gly Ser 40 45 50cag gac acc tgt acg tcg tgc ggc ggc ttc cgg cgg
cca gag gag ctc 247Gln Asp Thr Cys Thr Ser Cys Gly Gly Phe Arg Arg
Pro Glu Glu Leu 55 60 65ggc cga gtg gac ggc gac ttc ctg gag gcg gtg
aag cgg cac atc ttg 295Gly Arg Val Asp Gly Asp Phe Leu Glu Ala Val
Lys Arg His Ile Leu 70 75 80agc cgc ctg cag atg cgg ggc cgg ccc aac
atc acg cac gcc gtg cct 343Ser Arg Leu Gln Met Arg Gly Arg Pro Asn
Ile Thr His Ala Val Pro 85 90 95aag gcc gcc atg gtc acg gcc ctg cgc
aag ctg cac gcg ggc aag gtg 391Lys Ala Ala Met Val Thr Ala Leu Arg
Lys Leu His Ala Gly Lys Val100 105 110 115cgc gag gac ggc cgc gtg
gag atc ccg cac ctc gac ggc cac gcc agc 439Arg Glu Asp Gly Arg Val
Glu Ile Pro His Leu Asp Gly His Ala Ser 120 125 130ccg ggc gcc gac
ggc cag gag cgc gtt tcc gaa atc atc agc ttc gcc 487Pro Gly Ala Asp
Gly Gln Glu Arg Val Ser Glu Ile Ile Ser Phe Ala 135 140 145gag aca
gat ggc ctc gcc tcc tcc cgg gtc cgc cta tac ttc ttc atc 535Glu Thr
Asp Gly Leu Ala Ser Ser Arg Val Arg Leu Tyr Phe Phe Ile 150 155
160tcc aac gaa ggc aac cag aac ctg ttt gtg gtc cag gcc agc ctg tgg
583Ser Asn Glu Gly Asn Gln Asn Leu Phe Val Val Gln Ala Ser Leu Trp
165 170 175ctt tac ctg aaa ctc ctg ccc tac gtc ctg gag aag ggc agc
cgg cgg 631Leu Tyr Leu Lys Leu Leu Pro Tyr Val Leu Glu Lys Gly Ser
Arg Arg180 185 190 195aag gtg cgg gtc aaa gtg tac ttc cag gag cag
ggc cac ggt gac agg 679Lys Val Arg Val Lys Val Tyr Phe Gln Glu Gln
Gly His Gly Asp Arg 200 205 210tgg aac atg gtg gag aag agg gtg gac
ctc aag cgc agc ggc tgg cat 727Trp Asn Met Val Glu Lys Arg Val Asp
Leu Lys Arg Ser Gly Trp His 215 220 225acc ttc cca ctc acg gag gcc
atc cag gcc ttg ttt gag cgg ggc gag 775Thr Phe Pro Leu Thr Glu Ala
Ile Gln Ala Leu Phe Glu Arg Gly Glu 230 235 240cgg cga ctc aac cta
gac gtg cag tgt gac agc tgc cag gag ctg gcc 823Arg Arg Leu Asn Leu
Asp Val Gln Cys Asp Ser Cys Gln Glu Leu Ala 245 250 255gtg gtg ccg
gtg ttc gtg gac cca ggc gaa gag tcg cac cgg ccc ttt 871Val Val Pro
Val Phe Val Asp Pro Gly Glu Glu Ser His Arg Pro Phe260 265 270
275gtg gtg gtg cag gct cgg ctg ggc gac agc agg cac cgc att cgc aag
919Val Val Val Gln Ala Arg Leu Gly Asp Ser Arg His Arg Ile Arg Lys
280 285 290cga ggc ctg gag tgc gat ggc cgg acc aac ctc tgt tgc agg
caa cag 967Arg Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg
Gln Gln 295 300 305ttc ttc att gac ttc cgc ctc atc ggc tgg aac gac
tgg atc ata gca 1015Phe Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp
Trp Ile Ile Ala 310 315 320ccc acc ggc tac tac ggg aac tac tgt gag
ggc agc tgc cca gcc tac 1063Pro Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu
Gly Ser Cys Pro Ala Tyr 325 330 335ctg gca ggg gtc ccc ggc tct gcc
tcc tcc ttc cac acg gct gtg gtg 1111Leu Ala Gly Val Pro Gly Ser Ala
Ser Ser Phe His Thr Ala Val Val340 345 350 355aac cag tac cgc atg
cgg ggt ctg aac ccc ggc acg gtg aac tcc tgc 1159Asn Gln Tyr Arg Met
Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys 360 365 370tgc att ccc
acc aag ctg agc acc atg tcc atg ctg tac ttc gat gat 1207Cys Ile Pro
Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe Asp Asp 375 380 385gag
tac aac atc gtc aag cgg gac gtg ccc aac atg att gtg gag gag 1255Glu
Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met Ile Val Glu Glu 390 395
400tgc ggc tgc gcc tga cagtgcaagg caggggcacg gtggtggggc acggagggca
1310Cys Gly Cys Ala 405gtcccgggtg ggcttcttcc agcccccgcg ggaacggggg
tacacggtgg gctgagtaca 1370gtcattctgt tgggctgtgg agatagtgcc
agggtgcggc ctgagatatt tttctacagc 1430ttcatagagc aaccagtcaa
aaccagagcg agaaccctca actgacatga aatactttaa 1490aatgcacacg
tagccacgca cagccagacg catcctgcca cccacacagc agcctccagg
1550ataccagcaa atggatgcgg tgacaaatgg cagcttagct acaaatgcct
gtcagtcgga 1610gagaatgggg tgagcagcca ccattcccac cagctggccc
ggccactctg aattgcgcct 1670tccgagcaca cataaaagca caaagacaga
gacgcagaga gagagagaga gccacggaga 1730ggaaaagcag atgcaggggt
ggggagcgca gctcggcgga ggctgcgtgt gccccgtggc 1790ttttaccagg
cctgctctgc ctggctcgat gtctgcttct tccccagcct gggatccttc
1850gtgcttcaag gcctggggag cctgtccttc catgcccttg tcgagggaaa
gagacccaga 1910aaggacacaa cccgtcagag acctgggagc aggggcaatg
accgtttgac tgtttgtggc 1970ttgggcctct gacatgactt atgtgtgtgt
gtgtttttgg ggtggggagg gagggagaga 2030agagggggct aaatttgatg
ctttaactga tctccaacag ttgacaggtc atccttgcca 2090gttgtataac
tgaaaaagga cttttctacc aggtatgacc ttttaagtga aaatctgaat
2150tgttctaaat ggaaagaaaa aaagttgcaa tctgtgccct tcattgggga
cattcctcta 2210ggactggttt ggggacgggt gggaatgacc cctaggcaag
gggatgagac cgcaggagga 2270aatggcgggg aggaggcatt cttgaactgc
tgaggatggg gggtgtcccc tcagcggagg 2330ccaagggagg ggagcagcct
agttggtctt ggagagatgg ggaaggcttt cagctgattt 2390gcagaagttg
cccatgtggg ccccagccat cagggctggc cgtggacgtg gcccctgccc
2450actcacctgc ccgcctgccc gcccgcccgc atagcacttg cagacctgcc
tgaacgcaca 2510tgacatagca cttgccgatc tgcgtgtgtc cagaagtggc
ccttggccga gcgccgaact 2570cgctcgccct ctagatgtcc aagtgccacg
tgaactatgc aatttaaagg gttgacccac 2630actagacgaa actggactcg
tacgactctt tttatatttt ttatacttga aatgaaatcc 2690tttgcttctt
ttttaagcga atgattgctt ttaatgtttg cactgattta gttgcatgat
2750tagtcagaaa ctgccatttg aaaaaaagtt atttttatag cagcaaaaaa
aaaaaaaaaa 2810gaatacagtt aaatgtatta tacataattt tggaaccaaa
gaggccaaca gatcagtttt 2870aattttatta gacggtgagg ccatctgaga
tgaggtggac gttctgagca gtcccttgag 2930tggcctgcca acgtttcagg
gtatgaatgg attttgttta ttcggtttga tgtgtctttt 2990ccatccttac
acacccagaa ggtagagtaa aaatgactat gatagaatgc aggtgtgtat
3050ccttaaatcc tcatctttat gtttatttaa taaagctccc cttagattct
gtttcataat 3110aatttaaaac caaacaattt tcccatagac ttgctgttaa
agtattgtac gtttgtgtac 3170agtttaagaa aataaaagat tgagtgccac
gggaaaaaaa aaaaaaaa 321816407PRTHomo sapiens 16Met Asp Gly Leu Pro
Gly Arg Ala Leu Gly Ala Ala Cys Leu Leu Leu1 5 10 15Leu Ala Ala Gly
Trp Leu Gly Pro Glu Ala Trp Gly Ser Pro Thr Pro 20 25 30Pro Pro Thr
Pro Ala Ala Pro Pro Pro Pro Pro Pro Pro Gly Ser Pro 35 40 45Gly Gly
Ser Gln Asp Thr Cys Thr Ser Cys Gly Gly Phe Arg Arg Pro 50 55 60Glu
Glu Leu Gly Arg Val Asp Gly Asp Phe Leu Glu Ala Val Lys Arg65 70 75
80His Ile Leu Ser Arg Leu Gln Met Arg Gly Arg Pro Asn Ile Thr His
85 90 95Ala Val Pro Lys Ala Ala Met Val Thr Ala Leu Arg Lys Leu His
Ala 100 105 110Gly Lys Val Arg Glu Asp Gly Arg Val Glu Ile Pro His
Leu Asp Gly 115 120 125His Ala Ser Pro Gly Ala Asp Gly Gln Glu Arg
Val Ser Glu Ile Ile 130 135 140Ser Phe Ala Glu Thr Asp Gly Leu Ala
Ser Ser Arg Val Arg Leu Tyr145 150 155 160Phe Phe Ile Ser Asn Glu
Gly Asn Gln Asn Leu Phe Val Val Gln Ala 165 170 175Ser Leu Trp Leu
Tyr Leu Lys Leu Leu Pro Tyr Val Leu Glu Lys Gly 180 185 190Ser Arg
Arg Lys Val Arg Val Lys Val Tyr Phe Gln Glu Gln Gly His 195 200
205Gly Asp Arg Trp Asn Met Val Glu Lys Arg Val Asp Leu Lys Arg Ser
210 215 220Gly Trp His Thr Phe Pro Leu Thr Glu Ala Ile Gln Ala Leu
Phe Glu225 230 235 240Arg Gly Glu Arg Arg Leu Asn Leu Asp Val Gln
Cys Asp Ser Cys Gln 245 250 255Glu Leu Ala Val Val Pro Val Phe Val
Asp Pro Gly Glu Glu Ser His 260 265 270Arg Pro Phe Val Val Val Gln
Ala Arg Leu Gly Asp Ser Arg His Arg 275 280 285Ile Arg Lys Arg Gly
Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys 290 295 300Arg Gln Gln
Phe Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp305 310 315
320Ile Ile Ala Pro Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys
325 330 335Pro Ala Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser Ser Phe
His Thr 340 345 350Ala Val Val Asn Gln Tyr Arg Met Arg Gly Leu Asn
Pro Gly Thr Val 355 360 365Asn Ser Cys Cys Ile Pro Thr Lys Leu Ser
Thr Met Ser Met Leu Tyr 370 375 380Phe Asp Asp Glu Tyr Asn Ile Val
Lys Arg Asp Val Pro Asn Met Ile385 390 395 400Val Glu Glu Cys Gly
Cys Ala 405
* * * * *