U.S. patent application number 12/308695 was filed with the patent office on 2010-04-15 for diagnosis and treatment of cancer using anti-desmoglein-3 antibodies.
This patent application is currently assigned to Forerunner Pharma Research Co., Ltd.. Invention is credited to Hiroyuki Aburatani, Shunpei Ishikawa, Hirotaka Ito, Shigeto Kawai, Kiyotaka Nakano.
Application Number | 20100092457 12/308695 |
Document ID | / |
Family ID | 39082111 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100092457 |
Kind Code |
A1 |
Aburatani; Hiroyuki ; et
al. |
April 15, 2010 |
Diagnosis and Treatment of Cancer Using Anti-Desmoglein-3
Antibodies
Abstract
Methods that involve detection of a DSG3 protein for diagnosing
cancer are disclosed. In lung cancer, the expression of DSG3 was
found to be enhanced at very high frequency at the gene level and
protein level. Methods of the present invention can be carried out
using an antibody that recognizes a DSG3 protein. Pharmaceutical
compositions, cell growth inhibitors, and anticancer agents
containing a DSG3-binding antibody as an active ingredient are also
disclosed. Methods of inducing cell damage in DSG3-expressing cells
and methods of suppressing proliferation of DSG3-expressing cells
by contacting the DSG3-expressing cells with DSG3-binding
antibodies are also disclosed.
Inventors: |
Aburatani; Hiroyuki; (Tokyo,
JP) ; Ishikawa; Shunpei; (Tokyo, JP) ; Ito;
Hirotaka; (Tokyo, JP) ; Nakano; Kiyotaka;
(Tokyo, JP) ; Kawai; Shigeto; (Tokyo, JP) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Forerunner Pharma Research Co.,
Ltd.
Tokyo
JP
The University of Tokyo
Tokyo
JP
|
Family ID: |
39082111 |
Appl. No.: |
12/308695 |
Filed: |
August 14, 2007 |
PCT Filed: |
August 14, 2007 |
PCT NO: |
PCT/JP2007/065834 |
371 Date: |
June 29, 2009 |
Current U.S.
Class: |
424/130.1 ;
435/325; 435/6.14; 530/387.1; 530/391.7 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/28 20130101; A61P 1/18 20180101; C07K 2317/24 20130101;
G01N 2333/705 20130101; A61P 1/04 20180101; C07K 2317/732 20130101;
C07K 2319/30 20130101; A61P 11/00 20180101; C07K 2317/734 20130101;
G01N 33/57423 20130101; C07K 2317/565 20130101; A61P 35/00
20180101; C07K 2317/73 20130101 |
Class at
Publication: |
424/130.1 ;
530/387.1; 435/325; 530/391.7; 435/6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 5/00 20060101
C12N005/00; C12Q 1/68 20060101 C12Q001/68; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 14, 2006 |
JP |
2006-221230 |
Jan 30, 2007 |
JP |
2007-019108 |
Claims
1. A pharmaceutical composition comprising as an active ingredient
an antibody that binds to a DSG3 protein.
2. A cell growth inhibitor comprising as an active ingredient an
antibody that binds to a DSG3 protein.
3. An anticancer agent comprising as an active ingredient an
antibody that binds to a DSG3 protein.
4. The anticancer agent of claim 3, wherein the antibody that binds
to a DSG3 protein is an antibody that has cytotoxic activity.
5. The anticancer agent of claim 3, wherein the antibody that binds
to a DSG3 protein is an antibody described in any of (1) to (47)
below: (1) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 2 as CDR1, the amino acid sequence of SEQ ID
NO: 4 as CDR2, and the amino acid sequence of SEQ ID NO: 6 as CDR3;
(2) an antibody comprising the H chain of (1), wherein the H chain
has the amino acid sequence of SEQ ID NO: 8 as CH; (3) an antibody
comprising the H chain of (1), wherein the H chain has the amino
acid sequence of SEQ ID NO: 10 as CH; (4) an antibody comprising an
L chain having the amino acid sequence of SEQ ID NO: 12 as CDR1,
the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino
acid sequence of SEQ ID NO: 16 as CDR3; (5) an antibody comprising
the L chain of (4), wherein the L chain has the amino acid sequence
of SEQ ID NO: 18 as CL; (6) an antibody comprising the L chain of
(4), wherein the L chain has the amino acid sequence of SEQ ID NO:
20 as CL; (7) an antibody comprising the H chain of (1) and the L
chain of (4); (8) an antibody comprising the H chain of (2) and the
L chain of (5); (9) an antibody comprising the H chain of (3) and
the L chain of (6); (10) an antibody comprising an H chain having
the amino acid sequence of SEQ ID NO: 22 as CDR1, the amino acid
sequence of SEQ ID NO: 24 as CDR2, and the amino acid sequence of
SEQ ID NO: 26 as CDR3; (11) an antibody comprising the H chain of
(10), wherein the H chain has the amino acid sequence of SEQ ID NO:
28 as CH; (12) an antibody comprising the H chain of (10), wherein
the H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
(13) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 30 as CDR1, the amino acid sequence of SEQ
ID NO: 32 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as
CDR3; (14) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL; (15) an
antibody comprising the L chain of (13), wherein the L chain has
the amino acid sequence of SEQ ID NO: 20 as CL; (16) an antibody
comprising the H chain of (10) and the L chain of (13); (17) an
antibody comprising the H chain of (11) and the L chain of (14);
(18) an antibody comprising the H chain of (12) and the L chain of
(15); (19) an antibody comprising the H chain of (1) and the L
chain of (13); (20) an antibody comprising the H chain of (2) and
the L chain of (14); (21) an antibody comprising the H chain of (3)
and the L chain of (15); (22) an antibody comprising the H chain of
(10) and the L chain of (4); (23) an antibody comprising the H
chain of (11) and the L chain of (5); (24) an antibody comprising
the H chain of (12) and the L chain of (6); (25) an antibody
comprising an H chain having the amino acid sequence of SEQ ID NO:
81 as CDR1, the amino acid sequence of SEQ ID NO: 83 as CDR2, and
the amino acid sequence of SEQ ID NO: 85 as CDR3; (26) an antibody
comprising the H chain of (25), wherein the H chain has the amino
acid sequence of SEQ ID NO: 28 as CH; (27) an antibody comprising
the H chain of (25), wherein the H chain has the amino acid
sequence of SEQ ID NO: 10 as CH; (28) an antibody comprising an L
chain having the amino acid sequence of SEQ ID NO: 87 as CDR1, the
amino acid sequence of SEQ ID NO: 89 as CDR2, and the amino acid
sequence of SEQ ID NO: 91 as CDR3; (29) an antibody comprising the
L chain of (28), wherein the L chain has the amino acid sequence of
SEQ ID NO: 36 as CL; (30) an antibody comprising the L chain of
(28), wherein the L chain has the amino acid sequence of SEQ ID NO:
20 as CL; (31) an antibody comprising the H chain of (25) and the L
chain of (28); (32) an antibody comprising the H chain of (26) and
the L chain of (29); (33) an antibody comprising the H chain of
(27) and the L chain of (30); (34) an antibody comprising the H
chain of (1) and the L chain of (28); (35) an antibody comprising
the H chain of (2) and the L chain of (29); (36) an antibody
comprising the H chain of (3) and the L chain of (30); (37) an
antibody comprising the H chain of (10) and the L chain of (28);
(38) an antibody comprising the H chain of (11) and the L chain of
(29); (39) an antibody comprising the H chain of (12) and the L
chain of (30); (40) an antibody comprising the H chain of (25) and
the L chain of (4); (41) an antibody comprising the H chain of (26)
and the L chain of (5); (42) an antibody comprising the H chain of
(27) and the L chain of (6); (43) an antibody comprising the H
chain of (25) and the L chain of (13); (44) an antibody comprising
the H chain of (26) and the L chain of (14); (45) an antibody
comprising the H chain of (27) and the L chain of (15); (46) an
antibody comprising one or more amino acid substitutions,
deletions, additions, and/or insertions in the antibody of any of
(1) to (45), which has equivalent activity as the antibody of any
of (1) to (45); and (47) an antibody that binds to the same DSG3
protein epitope as the antibody of any of (1) to (45).
6. The anticancer agent of claim 3, wherein the cancer is selected
from the group consisting of lung cancer, colon cancer, esophageal
cancer, stomach cancer, pancreatic cancer, skin cancer, and uterine
cancer.
7. The anticancer agent of claim 6, wherein said lung cancer is
non-small-cell lung cancer.
8. A method of inducing cell damage in DSG3-expressing cells by
contacting cells that express a DSG3 protein with an antibody that
binds to the DSG3 protein.
9. A method of suppressing growth of DSG3-expressing cells by
contacting cells that express a DSG3 protein with an antibody that
binds to the DSG3 protein.
10. The method of claim 8, wherein the antibody that binds to the
DSG3 protein has cytotoxic activity.
11. The method of claim 8, wherein the antibody that binds to the
DSG3 protein is an antibody of any of (1) to (47) below: (1) an
antibody comprising an H chain having the amino acid sequence of
SEQ ID NO: 2 as CDR1, the amino acid sequence of SEQ ID NO: 4 as
CDR2, and the amino acid sequence of SEQ ID NO: 6 as CDR3; (2) an
antibody comprising the H chain of (1), wherein the H chain has the
amino acid sequence of SEQ ID NO: 8 as CH; (3) an antibody
comprising the H chain of (1), wherein the H chain has the amino
acid sequence of SEQ ID NO: 10 as CH; (4) an antibody comprising an
L chain having the amino acid sequence of SEQ ID NO: 12 as CDR1,
the amino acid sequence of SEQ ID NO: 14 as CDR2, and the amino
acid sequence of SEQ ID NO: 16 as CDR3; (5) an antibody comprising
the L chain of (4), wherein the L chain has the amino acid sequence
of SEQ ID NO: 18 as CL; (6) an antibody comprising the L chain of
(4), wherein the L chain has the amino acid sequence of SEQ ID NO:
20 as CL; (7) an antibody comprising the H chain of (1) and the L
chain of (4); (8) an antibody comprising the H chain of (2) and the
L chain of (5); (9) an antibody comprising the H chain of (3) and
the L chain of (6); (10) an antibody comprising an H chain having
the amino acid sequence of SEQ ID NO: 22 as CDR1, the amino acid
sequence of SEQ ID NO: 24 as CDR2, and the amino acid sequence of
SEQ ID NO: 26 as CDR3; (11) an antibody comprising the H chain of
(10) having the amino acid sequence of SEQ ID NO: 28 as CH; (12) an
antibody comprising the H chain of (10) having the amino acid
sequence of SEQ ID NO: 10 as CH; (13) an antibody comprising an L
chain having the amino acid sequence of SEQ ID NO: 30 as CDR1, the
amino acid sequence of SEQ ID NO: 32 as CDR2, and the amino acid
sequence of SEQ ID NO: 34 as CDR3; (14) an antibody comprising the
L chain of (13), wherein the L chain has the amino acid sequence of
SEQ ID NO: 36 as CL; (15) an antibody comprising the L chain of
(13), wherein the L chain has the amino acid sequence of SEQ ID NO:
20 as CL; (16) an antibody comprising the H chain of (10) and the L
chain of (13); (17) an antibody comprising the H chain of (11) and
the L chain of (14); (18) an antibody comprising the H chain of
(12) and the L chain of (15); (19) an antibody comprising the H
chain of (1) and the L chain of (13); (20) an antibody comprising
the H chain of (2) and the L chain of (14); (21) an antibody
comprising the H chain of (3) and the L chain of (15); (22) an
antibody comprising the H chain of (10) and the L chain of (4);
(23) an antibody comprising the H chain of (11) and the L chain of
(5); (24) an antibody comprising the H chain of (12) and the L
chain of (6); (25) an antibody comprising an H chain having the
amino acid sequence of SEQ ID NO: 81 as CDR1, the amino acid
sequence of SEQ ID NO: 83 as CDR2, and the amino acid sequence of
SEQ ID NO: 85 as CDR3; (26) an antibody comprising the H chain of
(25), wherein the H chain has the amino acid sequence of SEQ ID NO:
28 as CH; (27) an antibody comprising the H chain of (25), wherein
the H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
(28) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 87 as CDR1, the amino acid sequence of SEQ
ID NO: 89 as CDR2, and the amino acid sequence of SEQ ID NO: 91 as
CDR3; (29) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL; (30) an
antibody comprising the L chain of (28), wherein the L chain has
the amino acid sequence of SEQ ID NO: 20 as CL; (31) an antibody
comprising the H chain of (25) and the L chain of (28); (32) an
antibody comprising the H chain of (26) and the L chain of (29);
(33) an antibody comprising the H chain of (27) and the L chain of
(30); (34) an antibody comprising the H chain of (1) and the L
chain of (28); (35) an antibody comprising the H chain of (2) and
the L chain of (29); (36) an antibody comprising the H chain of (3)
and the L chain of (30); (37) an antibody comprising the H chain of
(10) and the L chain of (28); (38) an antibody comprising the H
chain of (11) and the L chain of (29); (39) an antibody comprising
the H chain of (12) and the L chain of (30); (40) an antibody
comprising the H chain of (25) and the L chain of (4); (41) an
antibody comprising the H chain of (26) and the L chain of (5);
(42) an antibody comprising the H chain of (27) and the L chain of
(6); (43) an antibody comprising the H chain of (25) and the L
chain of (13); (44) an antibody comprising the H chain of (26) and
the L chain of (14); (45) an antibody comprising the H chain of
(27) and the L chain of (15); (46) an antibody comprising one or
more amino acid substitutions, deletions, additions, and/or
insertions in the antibody of any of (1) to (45), which has
equivalent activity as the antibody of any of (1) to (45); and (47)
an antibody that binds to the same DSG3 protein epitope as the
antibody of any of (1) to (45).
12. The method of claim 8, wherein the cells that express a DSG3
protein are cancer cells.
13. An antibody that binds to a DSG3 protein and has cytotoxic
activity against cells that express a DSG3 protein.
14. The antibody of claim 13, wherein the cytotoxic activity is
ADCC activity.
15. The antibody of claim 13, wherein the cytotoxic activity is CDC
activity.
16. The antibody of claim 13, wherein a low-molecular-weight
chemotherapeutic agent or a toxic peptide is bound to the
antibody.
17. An antibody binding to a DSG3 protein, wherein a
low-molecular-weight chemotherapeutic agent or a toxic peptide is
bound to the antibody.
18. The antibody of claim 13, wherein the antibody is an antibody
of any of (1) to (47) below: (1) an antibody comprising an H chain
having the amino acid sequence of SEQ ID NO: 2 as CDR1, the amino
acid sequence of SEQ ID NO: 4 as CDR2, and the amino acid sequence
of SEQ ID NO: 6 as CDR3; (2) an antibody comprising the H chain of
(1), wherein the H chain has the amino acid sequence of SEQ ID NO:
8 as CH; (3) an antibody comprising the H chain of (1), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH; (4) an
antibody comprising an L chain having the amino acid sequence of
SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ ID NO: 14 as
CDR2, and the amino acid sequence of SEQ ID NO: 16 as CDR3; (5) an
antibody comprising the L chain of (4) having the amino acid
sequence of SEQ ID NO: 18 as CL; (6) an antibody comprising the L
chain of (4) having the amino acid sequence of SEQ ID NO: 20 as CL;
(7) an antibody comprising the H chain of (1) and the L chain of
(4); (8) an antibody comprising the H chain of (2) and the L chain
of (5); (9) an antibody comprising the H chain of (3) and the L
chain of (6); (10) an antibody comprising an H chain having the
amino acid sequence of SEQ ID NO: 22 as CDR1, the amino acid
sequence of SEQ ID NO: 24 as CDR2, and the amino acid sequence of
SEQ ID NO: 26 as CDR3; (11) an antibody comprising the H chain of
(10), wherein the H chain has the amino acid sequence of SEQ ID NO:
28 as CH; (12) an antibody comprising the H chain of (10), wherein
the H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
(13) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 30 as CDR1, the amino acid sequence of SEQ
ID NO: 32 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as
CDR3; (14) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL; (15) an
antibody comprising the L chain of (13), wherein the L chain has
the amino acid sequence of SEQ ID NO: 20 as CL; (16) an antibody
comprising the H chain of (10) and the L chain of (13); (17) an
antibody comprising the H chain of (11) and the L chain of (14);
(18) an antibody comprising the H chain of (12) and the L chain of
(15); (19) an antibody comprising the H chain of (1) and the L
chain of (13); (20) an antibody comprising the H chain of (2) and
the L chain of (14); (21) an antibody comprising the H chain of (3)
and the L chain of (15); (22) an antibody comprising the H chain of
(10) and the L chain of (4); (23) an antibody comprising the H
chain of (11) and the L chain of (5); (24) an antibody comprising
the H chain of (12) and the L chain of (6); (25) an antibody
comprising an H chain having the amino acid sequence of SEQ ID NO:
81 as CDR1, the amino acid sequence of SEQ ID NO: 83 as CDR2, and
the amino acid sequence of SEQ ID NO: 85 as CDR3; (26) an antibody
comprising the H chain of (25), wherein the H chain has the amino
acid sequence of SEQ ID NO: 28 as CH; (27) an antibody comprising
the H chain of (25), wherein the H chain has the amino acid
sequence of SEQ ID NO: 10 as CH; (28) an antibody comprising an L
chain having the amino acid sequence of SEQ ID NO: 87 as CDR1, the
amino acid sequence of SEQ ID NO: 89 as CDR2, and the amino acid
sequence of SEQ ID NO: 91 as CDR3; (29) an antibody comprising the
L chain of (28), wherein the L chain has the amino acid sequence of
SEQ ID NO: 36 as CL; (30) an antibody comprising the L chain of
(28), wherein the L chain has the amino acid sequence of SEQ ID NO:
20 as CL; (31) an antibody comprising the H chain of (25) and the L
chain of (28); (32) an antibody comprising the H chain of (26) and
the L chain of (29); (33) an antibody comprising the H chain of
(27) and the L chain of (30); (34) an antibody comprising the H
chain of (1) and the L chain of (28); (35) an antibody comprising
the H chain of (2) and the L chain of (29); (36) an antibody
comprising the H chain of (3) and the L chain of (30); (37) an
antibody comprising the H chain of (10) and the L chain of (28);
(38) an antibody comprising the H chain of (11) and the L chain of
(29); (39) an antibody comprising the H chain of (12) and the L
chain of (30); (40) an antibody comprising the H chain of (25) and
the L chain of (4); (41) an antibody comprising the H chain of (26)
and the L chain of (5); (42) an antibody comprising the H chain of
(27) and the L chain of (6); (43) an antibody comprising the H
chain of (25) and the L chain of (13); (44) an antibody comprising
the H chain of (26) and the L chain of (14); (45) an antibody
comprising the H chain of (27) and the L chain of (15); (46) an
antibody comprising one or more amino acid substitutions,
deletions, additions, and/or insertions in the antibody of any of
(1) to (45), which has equivalent activity as the antibody of any
of (1) to (45); and (47) an antibody that binds to the same DSG3
protein epitope as the antibody of any of (1) to (45).
19. (canceled)
20. A method of diagnosing cancer, comprising detecting a DSG3
protein using an antibody that binds to the DSG3 protein.
21. A method of diagnosing cancer, comprising: (a) collecting a
sample from a subject; and (b) detecting a DSG3 protein contained
in the collected sample using an antibody that binds to the DSG3
protein.
22. The method of diagnosis of claim 20, wherein the antibody that
binds to the DSG3 protein is labeled with a positron-emitting
nuclide.
23. The method of diagnosis of claim 22, wherein the
positron-emitting nuclide is a nuclide selected from the group
consisting of .sup.11C, .sup.13N, .sup.15O, .sup.18F, .sup.45Ti,
.sup.55Co, .sup.64Cu, .sup.66Ga, .sup.68Ga, .sup.76Br, .sup.89Zr,
and .sup.124I.
24. A method of diagnosing cancer, comprising detecting expression
of a gene encoding a DSG3 protein.
25. The method of diagnosis of claim 20, wherein the cancer is
selected from the group consisting of lung cancer, colon cancer,
esophageal cancer, stomach cancer, pancreatic cancer, skin cancer,
and uterine cancer.
26. The method of diagnosis of claim 25, wherein said lung cancer
is non-small-cell lung cancer.
27.-30. (canceled)
31. A method of suppressing cell growth, comprising the step of
administering to a subject an antibody that binds to a DSG3
protein.
32. A method of preventing or treating cancer, comprising the step
of administering to a subject an antibody that binds to a DSG3
protein.
Description
TECHNICAL FIELD
[0001] The present invention relates to methods for diagnosing and
treating cancer, cell proliferation inhibitors, and anticancer
agents.
BACKGROUND ART
[0002] Desmoglein 3 (hereinafter referred to as DSG3) was first
identified as a glycoprotein having a molecular weight of 130 kDa
by immunoprecipitation of keratinocyte extracts with an
autoantibody obtained from the serum of patients affected by
pemphigus vulgaris (hereinafter referred to as PV), which is an
autoimmune blister-forming disease of the skin and mucosa, and was
named the PV antigen (hereinafter referred to as PVA) (Non-patent
Document 1 and J. Clin. Invest. 74, 313-320, 1984). Then, antibody
molecules that react with the above-mentioned 130-kDa protein were
isolated from the serum of PV patients by affinity purification.
Next, an expression library was constructed using poly(A) RNA
isolated from human keratinocytes and was screened using the
isolated antibodies, and a cDNA encoding PVA was isolated. Based on
analysis of the nucleotide sequence of the isolated cDNA, the PVA
molecule was found to be highly homologous to the sequences of a
group of molecules belonging to the cadherin gene superfamily which
encodes intercellular adhesion factors (Non-patent Document 2).
[0003] Cadherin molecules are expressed in a wide variety of
tissues and they are involved in cell adhesion in vivo. Within the
cadherin group, a group of molecules are expressed in desmosomes,
which are adhesion sites between cells on the cell membrane, and
are called desmosomal cadherins or desmogleins. Keratinocytes,
which were used for isolation and cloning of the DSG3 molecule (a
member of the desmoglein family), are cells that occupy a large
portion of the epidermis. They are tightly adhered to adjacent
cells via desmosomes and the DSG3 molecule is considered to be
involved in this adhesion. Anti-DSG3 autoantibodies present in PV
patients' sera are thought to cause PV lesions by binding to the
DSG3 molecule and inhibiting intercellular adhesion mediated by the
DSG3 molecule.
[0004] As described above, PV lesions are induced by polyclonal
anti-DSG3 autoantibodies present in PV patients' sera. Monoclonal
anti-DSG3 antibodies that have the ability to induce PV-like
lesions upon transplantation of hybridomas into mice have also been
isolated (Non-patent Document 3), and they have been shown to have
a cell-dissociating activity that inhibits cell adhesion of
keratinocytes in the test tube as well (Non-patent Document 4). As
described above, the cell-dissociating activity of anti-DSG3
antibodies observed in the test tube has been suggested to be the
activity that induces PV lesions in vivo.
[0005] As described above, it is known that the DSG3 protein has an
important function in keratinocyte adhesion, and that anti-DSG3
antibodies are involved in the development of PV lesions. However,
involvement of the DSG3 protein in other diseases, or functions of
anti-DSG3 antibodies other than the cell-dissociating activity have
not been elucidated. In particular, connection of the DSG3 molecule
with the development of cancer, especially lung cancer, and
proliferation, invasion, metastasis, or transformation of lung
cancer cells in mammals, in particular, humans, has not been
elucidated.
[0006] Of the various types of cancers, lung cancer has the highest
mortality rate in both men and women. The mortality rate of lung
cancer in Japan has increased after 1950; as a result, the number
of lung cancer deaths in 1998 was 50,871 individuals, which was
approximately 18% of all malignant tumor deaths, and after 1993,
the number of deaths has exceeded that of stomach cancer and is
ranked number one among malignant tumors for men (Health and
Welfare Statistics Association, Kokumin eisei no doko/kousei no
shihyou (Trends of national health/indicators of welfare), 47,
52-53, 2000). Furthermore, on a global scale, approximately
3,000,000 people a year are dying of lung cancer. Basic
histological types of lung cancer include adenocarcinoma, squamous
cell carcinoma, adenosquamous carcinoma, large cell carcinoma, and
small cell carcinoma. Since the former four do not show large
differences in prognosis or therapeutic strategy, they are
collectively referred to as non-small cell lung cancer.
[0007] The number of non-small cell lung cancer cases accounts for
80% to 85% of the total number of lung cancer cases. Examples of
the characteristics of non-small cell lung cancer are slow
progression compared to small-cell cancers, and insufficient
response to chemotherapy and radiation therapy. Therefore, when the
tumor is localized, surgical resection is the number one choice,
but the treatment outcome is very poor compared to other carcinomas
such as stomach cancer at the same disease stage by TNM
classification. While recent attempts have been actively pursued to
improve the outcome by multimodal treatment, effective therapeutic
methods that lead to complete remission have not been established.
In non-small cell lung cancer, surgical therapy is considered for
up to stage IIIa, while in subsequent clinical disease stages,
surgery is rarely applied, and chemotherapy and radiation therapy
are the main therapies. SCC (squamous cell carcinoma related
antigen), Cyfra (cytokeratin 19 fragment), CEA (carcinoembryonic
antigen), and SLX (sialyl Lewis x-i antigen) are selected as
markers for serodiagnosis, and they are used separately or in
combination, but the positive rate for early stage cancers is still
low, and development of diagnostic markers that will assure
early-stage diagnosis of non-small cell lung cancer by
serodiagnosis is anticipated (Shuyo maka no yomikata no jissai;
haigan (Practical method for reading tumor markers; lung cancer)
Rinsho to Kenkyu (Clinic and Research) 78, 35-40, 2001).
[0008] Small cell lung cancer tumors constitute approximately 15%
to 20% of all lung cancers in Japan, and their speed of
proliferation is fast compared to other lung cancers, but they are
highly sensitive to anticancer agents and radiation therapy, and
have significantly different clinical characteristics from those of
adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and
such. For small cell cancer, surgical therapy is considered only in
stage Ia (tumor diameter is 20 mm or less, and no invasion or
metastasis to lymph nodes and nearby organs is shown), and
chemotherapy and radiation therapy are basically the main
therapeutic methods employed. As diagnostic markers, NSE
(neuron-specific enolase) and proGRP (pro gastrin-releasing
peptide) are used as tumor markers with relatively high specificity
to small cell cancer, and their positive rates are reported to be
approximately 60% and 70%, respectively.
[0009] Although there are still no examples of application in
clinical practice for lung cancers, the therapeutic response rate
in breast cancer, lymphoma, and such is increasing, because
targeted therapy using monoclonal antibodies against
cancer-specific tumor antigens exhibits a mode of action different
from conventional therapy which uses chemotherapeutic agents. In
targeted therapy that uses the above-mentioned antibody
pharmaceuticals, when the antibodies are functional and effective,
their activities include: antibody-dependent cell-mediated
cytotoxicity (ADCC) activity via effector cells;
complement-dependent cytotoxicity (CDC) activity via complements;
and cytotoxic activity as a result of construction of conjugate
molecules with chemotherapeutic agents, toxic peptides, or
radioactive chemical substances. Additional activities besides
those mentioned above include, for example, agonistic activity in
which the antibody itself catalyzes an agonistic effect on the
antigenic molecule; and neutralizing activity that blocks signals
for cell activation, proliferation, or the like. In order to apply
molecular-targeting therapy that uses antibodies exhibiting
activities such as those mentioned above in the treatment of lung
cancer, which has low positive rate of diagnosis, low disease cure
rate, and still has room for complete remission, identification of
tumor-specific molecules expressed in lung cancer cells and
production of antibodies that exhibit desirable activity by
targeting such molecules are strongly anticipated.
[0010] Prior art literature information relating to the present
invention is the following:
[Patent Document 1] WO 99/57149.
[Patent Document 2] WO 02/86443.
[Patent Document 3] WO 03/20769.
[0011] [Non-patent Document 1] J. Clin. Invest. 70, 281-288,
1982.
[Non-patent Document 2] Cell 67, 869-877, 1991.
[Non-patent Document 3] J. Immunology 170, 2170-2178, 2003.
[0012] [Non-patent Document 4] J. Invest. Dermatol., 124, 939-946,
2005.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0013] An objective of the present invention is to provide
anti-DSG3 antibodies and uses thereof. More specifically, an
objective of the present invention is to provide novel methods for
diagnosing and treating cancer using anti-DSG3 antibodies, novel
cell proliferation inhibitors and anticancer agents comprising
anti-DSG3 antibodies, and novel anti-DSG3 antibodies.
Means for Solving the Problems
[0014] The present inventors discovered that DSG3 is highly
expressed in cancer cells such as lung cancer cells. Furthermore,
when complement-dependent cytotoxicity (CDC) activity and
antibody-dependent cellular cytotoxicity (ADCC) activity of
anti-DSG3 antibodies were measured, the anti-DSG3 antibodies were
found to have CDC activity and ADCC activity towards
DSG3-expressing cells. Furthermore, from the above-mentioned
findings, the present inventors discovered that the anti-DSG3
antibodies were effective for diagnosing, preventing, and treating
cancers in which the DSG3 expression is elevated, including lung
cancer, and thereby completed the present invention.
[0015] The present invention provides pharmaceutical compositions
comprising a DSG3 protein-binding antibody as an active ingredient.
The present invention also provides cell proliferation inhibitors
comprising a DSG3 protein-binding antibody as an active ingredient.
The present invention further provides anticancer agents comprising
a DSG3 protein-binding antibody as an active ingredient.
Preferably, the DSG3 protein-binding antibody has cytotoxic
activity. Preferably, the cancer is lung cancer. More preferably,
the cancer is non-small cell lung cancer.
[0016] In another embodiment, the present invention provides
methods for inducing cell injury towards cells that express the
DSG3 protein by contacting DSG3-expressing cells with a DSG3
protein-binding antibody. The present invention also provides
methods for suppressing proliferation of cells that express a DSG3
protein by contacting cells that express the DSG3 protein with a
DSG3 protein-binding antibody. The DSG3 protein-binding antibody
preferably has cytotoxic activity. Cells that express a DSG3
protein are preferably cancer cells.
[0017] Furthermore, in another embodiment, the present invention
provides antibodies that bind to a DSG3 protein and have cytotoxic
activity towards cells that express the DSG3 protein. Preferably,
the cytotoxic activity is ADCC activity. Preferably, the cytotoxic
activity is CDC activity. The present invention also provides
antibodies to which a low-molecular-weight chemotherapeutic agent
or a toxic peptide is bound, or antibodies having cytotoxic
activity to which a low-molecular-weight chemotherapeutic agent or
a toxic peptide is bound.
[0018] The present invention further provides antibodies that bind
to a DSG3 protein, and have cytotoxic activity but not
cell-dissociating activity towards cells expressing the DSG3
protein.
[0019] In another embodiment, the present invention provides uses
of the DSG3 protein as a cancer diagnostic marker.
[0020] Furthermore, in another embodiment, the present invention
provides methods for diagnosing cancer, which comprise detecting a
DSG3 protein using an antibody that binds to the DSG3 protein. In
the methods of the present invention, preferably the extracellular
region of the DSG3 protein is detected. Preferably, the methods of
the present invention are carried out using an antibody that
recognizes the DSG3 protein. Preferably, the methods of the present
invention detect the DSG3 protein in the blood, serum, or plasma,
or DSG3 protein isolated from cells.
[0021] In another embodiment, the present invention provides
methods for diagnosing cancer which comprise the steps of:
(a) collecting a sample from a subject; and (b) using a DSG3
protein-binding antibody to detect the DSG3 protein contained in
the collected sample. In the present invention, any substance can
be used as the above-mentioned sample so long as it can be
collected from the subject. Serum collected from a subject is used
in one embodiment, and a biopsy sample collected from a subject is
used in another embodiment. In the methods of diagnosis, the cancer
may be any cancer so long as the subject cancer cells express a
DSG3 protein, but it is preferably lung cancer, and more preferably
non-small cell lung cancer. In the present invention, the step of
collecting a sample from a subject can also be expressed as the
step of providing a sample collected from a subject.
[0022] Furthermore, in another embodiment, the present invention
provides methods for diagnosing cancer, in which the DSG3
protein-binding antibody is labeled with a nuclide selected from
any one of 11C, 13N, 15O, 18F, 45Ti, 55Co, 64Cu, 66Ga, 68Ga, 76Br,
89Zr, and 124I.
[0023] Furthermore, in another embodiment, the present invention
provides methods for diagnosing cancer, in which the expression of
a gene encoding the DSG3 protein is detected.
[0024] Furthermore, in another embodiment, the present invention
provides diagnostic agents and kits to be used in the methods of
diagnosis of the present invention
[0025] Thus, the present application provides the following:
[1] a pharmaceutical composition comprising as an active ingredient
an antibody that binds to a DSG3 protein; [2] a cell growth
inhibitor comprising as an active ingredient an antibody that binds
to a DSG3 protein; [3] an anticancer agent comprising as an active
ingredient an antibody that binds to a DSG3 protein; [4] the
anticancer agent of [3], wherein the antibody that binds to a DSG3
protein is an antibody that has cytotoxic activity; [5] the
anticancer agent of [3] or [4], wherein the antibody that binds to
a DSG3 protein is an antibody described in any of (1) to (47)
below:
[0026] (1) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 2 as CDR1, the amino acid sequence of SEQ ID
NO: 4 as CDR2, and the amino acid sequence of SEQ ID NO: 6 as
CDR3;
[0027] (2) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 8 as CH;
[0028] (3) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0029] (4) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ
ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as
CDR3;
[0030] (5) an antibody comprising the L chain of (4), wherein the L
chain has the amino acid sequence of SEQ ID NO: 18 as CL;
[0031] (6) an antibody comprising the L chain of (4), wherein the L
chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0032] (7) an antibody comprising the H chain of (1) and the L
chain of (4);
[0033] (8) an antibody comprising the H chain of (2) and the L
chain of (5);
[0034] (9) an antibody comprising the H chain of (3) and the L
chain of (6);
[0035] (10) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 22 as CDR1, the amino acid sequence of SEQ
ID NO: 24 as CDR2, and the amino acid sequence of SEQ ID NO: 26 as
CDR3;
[0036] (11) an antibody comprising the H chain of (10), wherein the
H chain has the amino acid sequence of SEQ ID NO: 28 as CH;
[0037] (12) an antibody comprising the H chain of (10), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0038] (13) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 30 as CDR1, the amino acid sequence of SEQ
ID NO: 32 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as
CDR3;
[0039] (14) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0040] (15) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0041] (16) an antibody comprising the H chain of (10) and the L
chain of (13);
[0042] (17) an antibody comprising the H chain of (11) and the L
chain of (14);
[0043] (18) an antibody comprising the H chain of (12) and the L
chain of (15);
[0044] (19) an antibody comprising the H chain of (1) and the L
chain of (13);
[0045] (20) an antibody comprising the H chain of (2) and the L
chain of (14);
[0046] (21) an antibody comprising the H chain of (3) and the L
chain of (15);
[0047] (22) an antibody comprising the H chain of (10) and the L
chain of (4);
[0048] (23) an antibody comprising the H chain of (11) and the L
chain of (5);
[0049] (24) an antibody comprising the H chain of (12) and the L
chain of (6);
[0050] (25) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 81 as CDR1, the amino acid sequence of SEQ
ID NO: 83 as CDR2, and the amino acid sequence of SEQ ID NO: 85 as
CDR3;
[0051] (26) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 28 as CH;
[0052] (27) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0053] (28) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 87 as CDR1, the amino acid sequence of SEQ
ID NO: 89 as CDR2, and the amino acid sequence of SEQ ID NO: 91 as
CDR3;
[0054] (29) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0055] (30) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0056] (31) an antibody comprising the H chain of (25) and the L
chain of (28);
[0057] (32) an antibody comprising the H chain of (26) and the L
chain of (29);
[0058] (33) an antibody comprising the H chain of (27) and the L
chain of (30);
[0059] (34) an antibody comprising the H chain of (1) and the L
chain of (28);
[0060] (35) an antibody comprising the H chain of (2) and the L
chain of (29);
[0061] (36) an antibody comprising the H chain of (3) and the L
chain of (30);
[0062] (37) an antibody comprising the H chain of (10) and the L
chain of (28);
[0063] (38) an antibody comprising the H chain of (11) and the L
chain of (29);
[0064] (39) an antibody comprising the H chain of (12) and the L
chain of (30);
[0065] (40) an antibody comprising the H chain of (25) and the L
chain of (4);
[0066] (41) an antibody comprising the H chain of (26) and the L
chain of (5);
[0067] (42) an antibody comprising the H chain of (27) and the L
chain of (6);
[0068] (43) an antibody comprising the H chain of (25) and the L
chain of (13);
[0069] (44) an antibody comprising the H chain of (26) and the L
chain of (14);
[0070] (45) an antibody comprising the H chain of (27) and the L
chain of (15);
[0071] (46) an antibody comprising one or more amino acid
substitutions, deletions, additions, and/or insertions in the
antibody of any of (1) to (45), which has equivalent activity as
the antibody of any of (1) to (45); and
[0072] (47) an antibody that binds to the same DSG3 protein epitope
as the antibody of any of (1) to (45);
[6] the anticancer agent of any one of [3] to [5], wherein the
cancer is lung cancer, colon cancer, esophageal cancer, stomach
cancer, pancreatic cancer, skin cancer, or uterine cancer; [7] the
anticancer agent of [6], wherein the lung cancer is non-small-cell
lung cancer; [8] a method of inducing cell damage in
DSG3-expressing cells by contacting cells that express a DSG3
protein with an antibody that binds to the DSG3 protein; [9] a
method of suppressing growth of DSG3-expressing cells by contacting
cells that express a DSG3 protein with an antibody that binds to
the DSG3 protein; [10] the method of [8] or [9], wherein the DSG3
protein-binding antibody has cytotoxic activity; [11] the method of
any one of [8] to [10], wherein the DSG3 protein-binding antibody
is an antibody of any of (1) to (47) below:
[0073] (1) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 2 as CDR1, the amino acid sequence of SEQ ID
NO: 4 as CDR2, and the amino acid sequence of SEQ ID NO: 6 as
CDR3;
[0074] (2) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 8 as CH;
[0075] (3) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0076] (4) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ
ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as
CDR3;
[0077] (5) an antibody comprising the L chain of (4), wherein the L
chain has the amino acid sequence of SEQ ID NO: 18 as CL;
[0078] (6) an antibody comprising the L chain of (4), wherein the L
chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0079] (7) an antibody comprising the H chain of (1) and the L
chain of (4);
[0080] (8) an antibody comprising the H chain of (2) and the L
chain of (5);
[0081] (9) an antibody comprising the H chain of (3) and the L
chain of (6);
[0082] (10) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 22 as CDR1, the amino acid sequence of SEQ
ID NO: 24 as CDR2, and the amino acid sequence of SEQ ID NO: 26 as
CDR3;
[0083] (11) an antibody comprising the H chain of (10) having the
amino acid sequence of SEQ ID NO: 28 as CH;
[0084] (12) an antibody comprising the H chain of (10) having the
amino acid sequence of SEQ ID NO: 10 as CH;
[0085] (13) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 30 as CDR1, the amino acid sequence of SEQ
ID NO: 32 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as
CDR3;
[0086] (14) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0087] (15) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0088] (16) an antibody comprising the H chain of (10) and the L
chain of (13);
[0089] (17) an antibody comprising the H chain of (11) and the L
chain of (14);
[0090] (18) an antibody comprising the H chain of (12) and the L
chain of (15);
[0091] (19) an antibody comprising the H chain of (1) and the L
chain of (13);
[0092] (20) an antibody comprising the H chain of (2) and the L
chain of (14);
[0093] (21) an antibody comprising the H chain of (3) and the L
chain of (15);
[0094] (22) an antibody comprising the H chain of (10) and the L
chain of (4);
[0095] (23) an antibody comprising the H chain of (11) and the L
chain of (5);
[0096] (24) an antibody comprising the H chain of (12) and the L
chain of (6);
[0097] (25) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 81 as CDR1, the amino acid sequence of SEQ
ID NO: 83 as CDR2, and the amino acid sequence of SEQ ID NO: 85 as
CDR3;
[0098] (26) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 28 as CH;
[0099] (27) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0100] (28) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 87 as CDR1, the amino acid sequence of SEQ
ID NO: 89 as CDR2, and the amino acid sequence of SEQ ID NO: 91 as
CDR3;
[0101] (29) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0102] (30) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0103] (31) an antibody comprising the H chain of (25) and the L
chain of (28);
[0104] (32) an antibody comprising the H chain of (26) and the L
chain of (29);
[0105] (33) an antibody comprising the H chain of (27) and the L
chain of (30);
[0106] (34) an antibody comprising the H chain of (1) and the L
chain of (28);
[0107] (35) an antibody comprising the H chain of (2) and the L
chain of (29);
[0108] (36) an antibody comprising the H chain of (3) and the L
chain of (30);
[0109] (37) an antibody comprising the H chain of (10) and the L
chain of (28);
[0110] (38) an antibody comprising the H chain of (11) and the L
chain of (29);
[0111] (39) an antibody comprising the H chain of (12) and the L
chain of (30);
[0112] (40) an antibody comprising the H chain of (25) and the L
chain of (4);
[0113] (41) an antibody comprising the H chain of (26) and the L
chain of (5);
[0114] (42) an antibody comprising the H chain of (27) and the L
chain of (6);
[0115] (43) an antibody comprising the H chain of (25) and the L
chain of (13);
[0116] (44) an antibody comprising the H chain of (26) and the L
chain of (14);
[0117] (45) an antibody comprising the H chain of (27) and the L
chain of (15);
[0118] (46) an antibody comprising one or more amino acid
substitutions, deletions, additions, and/or insertions in the
antibody of any of (1) to (45), which has equivalent activity as
the antibody of any of (1) to (45); and
[0119] (47) an antibody that binds to the same DSG3 protein epitope
as the antibody of any of (1) to (45);
[12] the method of any one of [8] to [11], wherein the cells that
express a DSG3 protein are cancer cells; [13] an antibody that
binds to a DSG3 protein and has cytotoxic activity against cells
that express a DSG3 protein; [14] the antibody of [13], wherein the
cytotoxic activity is ADCC activity; [15] the antibody of [13],
wherein the cytotoxic activity is CDC activity; [16] the antibody
of any one of [13] to [15], wherein a low-molecular-weight
chemotherapeutic agent or a toxic peptide is bound to the antibody;
[17] an antibody binding to a DSG3 protein, wherein a
low-molecular-weight chemotherapeutic agent or a toxic peptide is
bound to the antibody; [18] the antibody of any one of [13] to
[17], wherein the antibody is an antibody of any of (1) to (47)
below:
[0120] (1) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 2 as CDR1, the amino acid sequence of SEQ ID
NO: 4 as CDR2, and the amino acid sequence of SEQ ID NO: 6 as
CDR3;
[0121] (2) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 8 as CH;
[0122] (3) an antibody comprising the H chain of (1), wherein the H
chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0123] (4) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 12 as CDR1, the amino acid sequence of SEQ
ID NO: 14 as CDR2, and the amino acid sequence of SEQ ID NO: 16 as
CDR3;
[0124] (5) an antibody comprising the L chain of (4) having the
amino acid sequence of SEQ ID NO: 18 as CL;
[0125] (6) an antibody comprising the L chain of (4) having the
amino acid sequence of SEQ ID NO: 20 as CL;
[0126] (7) an antibody comprising the H chain of (1) and the L
chain of (4);
[0127] (8) an antibody comprising the H chain of (2) and the L
chain of (5);
[0128] (9) an antibody comprising the H chain of (3) and the L
chain of (6);
[0129] (10) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 22 as CDR1, the amino acid sequence of SEQ
ID NO: 24 as CDR2, and the amino acid sequence of SEQ ID NO: 26 as
CDR3;
[0130] (11) an antibody comprising the H chain of (10), wherein the
H chain has the amino acid sequence of SEQ ID NO: 28 as CH;
[0131] (12) an antibody comprising the H chain of (10), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0132] (13) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 30 as CDR1, the amino acid sequence of SEQ
ID NO: 32 as CDR2, and the amino acid sequence of SEQ ID NO: 34 as
CDR3;
[0133] (14) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0134] (15) an antibody comprising the L chain of (13), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0135] (16) an antibody comprising the H chain of (10) and the L
chain of (13);
[0136] (17) an antibody comprising the H chain of (11) and the L
chain of (14);
[0137] (18) an antibody comprising the H chain of (12) and the L
chain of (15);
[0138] (19) an antibody comprising the H chain of (1) and the L
chain of (13);
[0139] (20) an antibody comprising the H chain of (2) and the L
chain of (14);
[0140] (21) an antibody comprising the H chain of (3) and the L
chain of (15);
[0141] (22) an antibody comprising the H chain of (10) and the L
chain of (4);
[0142] (23) an antibody comprising the H chain of (11) and the L
chain of (5);
[0143] (24) an antibody comprising the H chain of (12) and the L
chain of (6);
[0144] (25) an antibody comprising an H chain having the amino acid
sequence of SEQ ID NO: 81 as CDR1, the amino acid sequence of SEQ
ID NO: 83 as CDR2, and the amino acid sequence of SEQ ID NO: 85 as
CDR3;
[0145] (26) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 28 as CH;
[0146] (27) an antibody comprising the H chain of (25), wherein the
H chain has the amino acid sequence of SEQ ID NO: 10 as CH;
[0147] (28) an antibody comprising an L chain having the amino acid
sequence of SEQ ID NO: 87 as CDR1, the amino acid sequence of SEQ
ID NO: 89 as CDR2, and the amino acid sequence of SEQ ID NO: 91 as
CDR3;
[0148] (29) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 36 as CL;
[0149] (30) an antibody comprising the L chain of (28), wherein the
L chain has the amino acid sequence of SEQ ID NO: 20 as CL;
[0150] (31) an antibody comprising the H chain of (25) and the L
chain of (28);
[0151] (32) an antibody comprising the H chain of (26) and the L
chain of (29);
[0152] (33) an antibody comprising the H chain of (27) and the L
chain of (30);
[0153] (34) an antibody comprising the H chain of (1) and the L
chain of (28);
[0154] (35) an antibody comprising the H chain of (2) and the L
chain of (29);
[0155] (36) an antibody comprising the H chain of (3) and the L
chain of (30);
[0156] (37) an antibody comprising the H chain of (10) and the L
chain of (28);
[0157] (38) an antibody comprising the H chain of (11) and the L
chain of (29);
[0158] (39) an antibody comprising the H chain of (12) and the L
chain of (30);
[0159] (40) an antibody comprising the H chain of (25) and the L
chain of (4);
[0160] (41) an antibody comprising the H chain of (26) and the L
chain of (5);
[0161] (42) an antibody comprising the H chain of (27) and the L
chain of (6);
[0162] (43) an antibody comprising the H chain of (25) and the L
chain of (13);
[0163] (44) an antibody comprising the H chain of (26) and the L
chain of (14);
[0164] (45) an antibody comprising the H chain of (27) and the L
chain of (15);
[0165] (46) an antibody comprising one or more amino acid
substitutions, deletions, additions, and/or insertions in the
antibody of any of (1) to (45), which has equivalent activity as
the antibody of any of (1) to (45); and
[0166] (47) an antibody that binds to the same DSG3 protein epitope
as the antibody of any of (1) to (45);
[19] use of a DSG3 protein as a cancer diagnostic marker; [20] a
method of diagnosing cancer, comprising detecting a DSG3 protein
using an antibody that binds to the DSG3 protein; [21] a method of
diagnosing cancer, comprising the steps of: (a) collecting a sample
from a subject; and (b) detecting a DSG3 protein contained in the
collected sample using an antibody that binds to the DSG3 protein;
[22] the method of diagnosis of [20] or [21], wherein the DSG3
protein-binding antibody is an antibody labeled with a
positron-emitting nuclide; [23] the method of diagnosis of [22],
wherein the positron-emitting nuclide is a nuclide selected from
any of 11C, 13N, 15O, 18F, 45Ti, 55Co, 64Cu, 66Ga, 68Ga, 76Br,
89Zr, and 124I; [24] a method of diagnosing cancer, comprising
detecting expression of a gene encoding a DSG3 protein; [25] the
method of diagnosis of any one of [20] to [24], wherein the cancer
is lung cancer, colon cancer, esophageal cancer, stomach cancer,
pancreatic cancer, skin cancer, or uterine cancer; [26] the method
of diagnosis of [25], wherein the lung cancer is non-small-cell
lung cancer; [27] a diagnostic agent to be used for the diagnostic
method of any one of [20] to [26]; [28] a kit to be used for the
diagnostic method of any one of [20] to [26]; [29] use of an
antibody that binds to a DSG3 protein in the production of a cell
growth inhibitor; [30] use of an antibody that binds to a DSG3
protein in the production of an anticancer agent; [31] a method of
suppressing cell growth, comprising the step of administering to a
subject an antibody that binds to a DSG3 protein; and [32] a method
of preventing or treating cancer, comprising the step of
administering to a subject an antibody that binds to a DSG3
protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0167] FIG. 1 depicts the result of DSG3 gene expression analysis
in normal tissues and cancer tissues using GeneChip U133.
[0168] FIG. 2 depicts the result of DSG3 expression analysis in
cancer cell lines using GeneChip U133.
[0169] FIG. 3 shows photographs of immunohistological staining in
which expression of the DSG3 protein in lung squamous cell
carcinoma is visualized by immunostaining. Elevation of the DSG3
protein expression is shown in all five clinical samples.
[0170] FIG. 4 depicts the result of flow cytometric analysis which
shows binding of all the anti-DSG3 monoclonal antibodies DF120,
DF122, DF148, DF151, DF153, DF168, DF331, DF364, and DF366 to a CHO
cell line that constitutively expresses full-length DSG3.
[0171] FIG. 5 depicts the CDC activity of anti-DSG3 monoclonal
antibodies DF120, DF122, DF148, DF151, DF153, DF168, and DF331
towards a CHO cell line that constitutively expresses full-length
DSG3.
[0172] FIG. 6 depicts the CDC activity of the DF151 anti-DSG3
monoclonal antibody towards the human epidermoid carcinoma cell
line A431, and the DSG3-A549 cell line, which is a human lung
carcinoma cell line that constitutively expresses DSG3.
[0173] FIG. 7 depicts the ADCC activity of anti-DSG3 monoclonal
antibodies DF151, DF364, and DF366 towards the DSG3-A549 cell line
which is a human lung carcinoma cell line that constitutively
expresses DSG3. FIG. 7A depicts the result of analysis using bone
marrow-derived effector cells, and FIG. 7B shows the result of
analysis using mouse spleen-derived effector cells.
[0174] FIG. 8 depicts the CDC activity of anti-DSG3 mouse-human
chimeric antibodies DF151c, DF364c, and DF366c towards the
DSG3-Ba/F3 cell line, which is a Ba/F3 cell line that
constitutively expresses DSG3.
[0175] FIG. 9 depicts the ADCC activity of anti-DSG3 mouse-human
chimeric antibodies DF364c and DF366c, and low-fucose anti-DSG3
mouse-human chimeric antibodies YB-DF364c and YB-DF366c, towards
the DSG3-Ba/F3 cell line, which is a Ba/F3 cell line that
constitutively expresses DSG3.
[0176] FIG. 10 depicts the ADCC activity of anti-DSG3 antibodies
DF366m (mouse IgG2a chimeric antibody), low-fucose DF366m
(low-fucose mouse IgG2a chimeric antibody), DF366c (mouse-human
chimeric antibody) and YB-DF366c (low-fucose mouse-human chimeric
antibody) towards the DSG3-Ba/F3 cell line, which is a Ba/F3 cell
line that constitutively expresses DSG3. Mouse spleen cells that
have added interleukin-2 were used as effector cells.
[0177] FIG. 11 depicts the ADCC activity of anti-DSG3 antibodies
DF366m (mouse IgG2a chimeric antibody), low-fucose DF366m
(low-fucose mouse IgG2a chimeric antibody), DF366c (mouse-human
chimeric antibody) and YB-DF366c (low-fucose mouse-human chimeric
antibody) towards the DSG3-Ba/F3 cell line, which is a Ba/F3 cell
line that constitutively expresses DSG3. Mouse spleen cells
cultured for four days in the presence of interleukin-2 were used
as effector cells.
[0178] FIG. 12 depicts the antitumor activity of anti-DSG3
antibodies DF366m (mouse IgG2a chimeric antibody) and low-fucose
DF366m (low-fucose mouse IgG2a chimeric antibody).
BEST MODE FOR CARRYING OUT THE INVENTION
[0179] DSG3 (Desmoglein 3) is an axon guidance receptor protein,
and its amino acid sequence and its encoding gene sequence are
disclosed in GenBank Accession Number NP.sub.--001935 (SEQ ID NO:
40) and NM.sub.--001944 (SEQ ID NO: 39), respectively. In the
present invention, the DSG3 protein refers to both the full-length
protein and fragments thereof. "Fragments" refers to polypeptides
comprising any region of the DSG3 protein, and may not have the
function of the naturally-occurring DSG3 protein. Without being
limited thereto, an example of the fragments is a fragment
comprising the extracellular region of the DSG3 protein. Positions
1 to 616 in the amino acid sequence of SEQ ID NO: 40 correspond to
the extracellular region of the DSG3 protein. Positions 617 to 641
in the amino acid sequence of SEQ ID NO: 40 correspond to the
transmembrane region.
[0180] In the present invention, DSG3 expression was found to be
elevated at very high frequency in lung cancer tissues at the gene
and protein levels. Furthermore, analyses of clinical samples and
cancer cell lines of other cancer types showed that the expression
was elevated not only in lung cancer, but also in colon cancer,
esophageal cancer, stomach cancer, pancreatic cancer, skin cancer,
and uterine cancer. Furthermore, it was shown that
immunohistological diagnosis is possible by using DSG3
protein-specific monoclonal antibodies. In other words, the DSG3
protein is useful as a diagnostic marker for cancer.
Detection of DSG3 Gene Expression
[0181] Methods of the present invention comprise detecting the DSG3
gene expression. In an embodiment of the methods of the present
invention, the DSG3 protein expression is detected.
[0182] In the present invention, detection includes quantitative
and qualitative detections. Examples of qualitative detection
include simple measurement for the presence or absence of the DSG3
protein, measurement to see whether or not the DSG3 protein is
present above a certain amount, and measurement that compares the
amount of the DSG3 protein with that of other samples (for example,
a control sample). On the other hand, examples of quantitative
detection include measurement of the DSG3 protein concentration,
and measurement of the amount of the DSG3 protein.
[0183] Test samples are not particularly limited so long as they
are samples that may contain DSG3 protein, and are preferably
samples collected from the body of organisms such as mammals, and
more preferably samples collected from humans. Specific examples of
the test samples include blood, interstitial fluid, plasma,
extravascular fluid, cerebrospinal fluid, synovial fluid, pleural
fluid, serum, lymphatic fluid, saliva, and urine, but the test
samples are preferably blood, serum, or plasma. Test samples of the
present invention also include samples obtained from test samples,
such as cell culture solutions and specimens of immobilized tissues
or cells collected from the body of an organism.
[0184] The cancers that are diagnosed are not particularly limited
and may be any cancer, but specific examples include lung cancer,
colon cancer, esophageal cancer, stomach cancer, pancreatic cancer,
skin cancer, and uterine cancer. Lung cancer is preferable and
non-small cell lung cancer is particularly preferable.
[0185] In the present invention, when a DSG3 protein is detected in
a test sample and if the test sample is judged to have a greater
amount of the DSG3 protein than a negative control or a healthy
individual, it can be determined that the subject has cancer or has
a high risk of being affected with cancer in the future.
[0186] Subjects in the present invention may be animal species that
genetically carry a DSG3 protein, and many non-human mammals such
as monkeys, cattle, sheep, mice, dogs, cats, and hamsters are known
as such animal species. Subjects that are suitably used are, in
particular, humans, but are not limited thereto.
[0187] Preferred embodiments of the diagnostic methods of the
present invention include diagnostic methods that comprise
detecting a DSG3 protein on a section of immobilized tissue or
cells obtained from a patient affected with an aforementioned
cancer. Furthermore, other embodiments of the present invention
include diagnostic methods comprising detecting cell-released DSG3
protein in the blood. In particular, the present invention is
preferably a diagnostic method that detects a fragment comprising
the extracellular domain of the DSG3 protein present in the
blood.
[0188] Methods for detecting a DSG3 protein contained in a test
sample are not particularly limited, but an immunological method
that uses an anti-DSG3 antibody for detection is preferred. The
immunological method includes, for example, radioimmunoassay (RIA),
enzyme immunoassay (EIA), fluorescence immunoassay (FIA),
luminescence immunoassay (LIA), immunoprecipitation (IP),
turbidimetric immunoassay (TIA), Western blotting (WB),
immunohistochemical staining (IHC), and single radial
immunodiffusion (SRID), and is preferably enzyme immunoassay, in
particular, enzyme-linked immunosorbent assay (ELISA), for example,
sandwich ELISA as an embodiment thereof. The above-mentioned
immunological methods such as ELISA can be performed by methods
known to those skilled in the art.
[0189] The following method is, for example, a common detection
method that uses an anti-DSG3 antibody. After immobilizing an
anti-DSG3 antibody to a support, the support is blocked with bovine
serum albumin (BSA), gelatin, albumin, or such to avoid
non-specific binding of proteins to the support. Next, a test
sample is added to the support for incubation, and the DSG3
proteins are left to bind to the anti-DSG3 antibody bound to the
support. Subsequently, by washing the complex formed between the
DSG3 proteins and the anti-DSG3 antibody bound to the support with
a washing solution, DSG3 proteins other than those bound to the
anti-DSG3 antibody on the support that bound non-specifically to
the support are removed. Examples of detection methods that use an
anti-DSG3 antibody include methods for detecting a DSG3 protein in
a test sample by qualitatively or quantitatively detecting the DSG3
protein bound to the anti-DSG3 antibody on the support, and several
specific examples described below.
[0190] In the present invention, a support used to immobilize an
anti-DSG3 antibody is, for example, insoluble polysaccharides such
as agarose and cellulose, synthetic resins such as silicon resin,
polystyrene resin, polyacrylamide resin, nylon resin, and
polycarbonate resin, and insoluble support such as glass. Such a
support is used in the form of beads or plates. In the case of
beads, a column or the like filled with beads can be used. In the
case of a plate, a multi-well plate (96-well multi-well plate, or
such), or a biosensor chip can be used. For binding between an
anti-DSG3 antibody and a support, an anti-DSG3 antibody can be
bound to a support by generally used methods such as chemical
bonding or physical adsorption. Commercially available supports can
be used suitably.
[0191] Binding between an anti-DSG3 antibody and a DSG3 protein is
generally performed in a buffer. For example, phosphate buffer,
Tris buffer, citric acid buffer, borate buffer, carbonate buffer,
or such is used as the buffer. Furthermore, incubation can be
suitably carried out using conditions that are already commonly
used, such as incubation at a temperature between 4.degree. C. and
room temperature for one hour to 24 hours. So long as the binding
between the DSG3 protein and anti-DSG3 antibody is not interrupted,
anything can be used for washing after incubation, and for example,
a buffer containing a surfactant such as Tween 20 or such can be
used suitably.
[0192] In the DSG3 protein detection method of the present
invention, a control sample can be prepared suitably in addition to
the test sample in which the DSG3 protein content will be detected.
The control sample includes, for example, a negative control sample
containing no DSG3 protein and a positive control sample containing
the DSG3 protein. In this case, by comparing the results obtained
from a negative control sample containing no DSG3 protein with the
results obtained from a positive control sample containing the DSG3
protein, the presence or absence of the DSG3 protein in the test
sample can be confirmed. Furthermore, after preparing a series of
control samples with stepwise changes in concentration, and
obtaining detection results for each control sample as a numerical
value, the DSG3 protein contained in a test sample can be
quantitatively detected according to a standard curve produced
based on the values of the DSG3 protein concentration and their
corresponding measured values.
[0193] In a preferred embodiment, an example of detection of the
DSG3 protein bound to a support via an anti-DSG3 antibody is a
method that uses an anti-DSG3 antibody labeled with a labeling
substance. For example, the DSG3 protein can be detected by
contacting a test sample with the anti-DSG3 antibody immobilized
onto a support, washing it, and then using a labeled antibody that
specifically recognizes the DSG3 protein bound to the anti-DSG3
antibody.
[0194] Anti-DSG3 antibodies can be labeled by generally known
methods. A labeling substance known to those skilled in the art
such as fluorescent dyes, enzymes, coenzymes, chemiluminescent
substances, and radioactive substances can be used as the labeling
substance, and specific examples include radioisotopes (32P, 14C,
125I, 3H, 131I, and such), fluorescein, rhodamine, dansyl chloride,
umbelliferone, luciferase, peroxidase, alkaline phosphatase,
.beta.-galactosidase, .beta.-glucosidase, horseradish peroxidase,
glucoamylase, lysozyme, saccharide oxidase, micro peroxidase, and
biotin. When using biotin as a labeling substance, addition of
biotin-labeled antibodies is preferably followed by addition of
avidin bound to an enzyme such as alkaline phosphatase. For the
binding of labeling substance with an anti-DSG3 antibody, known
methods such as the glutaraldehyde method, maleimide method,
pyridyl disulfide method, or periodic acid method can be used.
[0195] Specifically, an anti-DSG3 antibody is immobilized onto a
support by addition of a solution containing the anti-DSG3 antibody
to the support such as a plate. After the plate is washed, it is
blocked with, for example, bovine serum albumin (BSA), gelatin,
albumin, or such to avoid non-specific protein binding. After the
plate is washed again, incubation is carried out by adding a test
sample to the plate. After incubation, the plate is washed, and the
labeled anti-DSG3 antibody is added. After appropriate incubation,
the plate is washed, and then the labeled anti-DSG3 antibody that
remains on the plate can be detected. Detection can be performed by
methods known to those skilled in the art, and for example, when
detecting an anti-DSG3 antibody labeled with a radioactive
substance, the labeled anti-DSG3 antibody can be detected by liquid
scintillation or an RIA method. When detecting an enzyme-labeled
anti-DSG3 antibody, addition of substrate to the labeled anti-DSG3
antibody can be followed by detecting the substrate's enzymatic
change, such as color development, using a spectrophotometer.
Specific examples of a substrate include
2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine), and
3,3',5,5'-tetramethylbenzidine (TMB). When the substrate is a
fluorescence emitting substance, enzymatic change of the substrate
can be detected using a spectrofluorometer.
[0196] In the present invention, a particularly preferred
embodiment of the method for detecting the DSG3 protein is, for
example, a method that uses a biotin-labeled anti-DSG3 antibody and
avidin. Specifically, addition of a solution containing an
anti-DSG3 antibody to a support such as a plate enables
immobilization of the anti-DSG3 antibody to the plate. After the
plate is washed, it is blocked with, for example, BSA to avoid
non-specific protein binding. The plate is washed again, and then a
test sample is added to the plate. After incubation, the plate is
washed, and a biotin-labeled anti-DSG3 antibody is added to the
plate. After suitable incubation, the plate is washed, and avidin
bound to an enzyme such as alkaline phosphatase or peroxidase is
added to the plate. After incubation, the plate is washed, and the
DSG3 protein can be detected after addition of a substrate for the
avidin-conjugated enzyme, using the substrate's enzymatic change or
such as an indicator.
[0197] In the present invention, another embodiment of the method
for detecting the DSG3 protein includes a method that uses one or
more types of primary antibodies that specifically recognize the
DSG3 protein, and one or more types of secondary antibodies that
specifically recognize the primary antibodies.
[0198] For example, after immobilizing an anti-DSG3 antibody to a
support such as a plate, the plate is blocked with bovine serum
albumin (BSA), gelatin, albumin, or such to prevent non-specific
binding of proteins to the support. Then, after adding a test
sample to the plate, it is incubated to allow the DSG3 protein to
bind to the anti-DSG3 antibody bound to the plate. Thereafter, the
plate is washed with a washing solution so that the DSG3 proteins
bound to the support by non-specific binding, and not by specific
binding to the anti-DSG3 antibody, are removed from the plate. A
different type of anti-DSG3 antibody from the antibody bound to the
support binds to the DSG3 protein, and then a secondary antibody
that can only bind to the anti-DSG3 antibody that binds to the DSG3
protein but not to the support, is made to react with the
DSG3-protein/anti-DSG3-antibody complexes. An example is a method
that detects the DSG3 protein in a test sample by qualitatively or
quantitatively detecting the secondary antibody that binds as a
result of the above-mentioned operation. In this case, the
secondary antibody can be more suitably labeled with a labeling
substance.
[0199] In the present invention, another embodiment of the methods
for detecting the DSG3 protein is, for example, a detection method
that uses aggregation reaction. In this method, the DSG3 protein
can be detected using a carrier onto which an anti-DSG3 antibody is
adsorbed. Any carrier may be used for adsorbing the antibody, so
long as it is insoluble and stable, and does not cause non-specific
reactions. For example, latex particles, bentonite, collodion,
kaolin, or immobilized sheep erythrocytes can be used, but the use
of latex particles is preferred. Latex particles that can be used
are, for example, polystyrene latex particles, styrene-butadiene
copolymer latex particles, or polyvinyl toluene latex particles,
but the use of polystyrene latex particles is preferred. Sensitized
particles are mixed with a sample, and this is stirred for a given
period of time. Since the degree of particle aggregation becomes
larger as the concentration of DSG3 protein in the sample
increases, the DSG3 protein can be detected by assessing the degree
of aggregation with the naked eye. Furthermore, the DSG3 protein
can also be detected by measuring the increase in turbidity caused
by aggregation using a spectrophotometer or such.
[0200] In the present invention, another embodiment of the methods
for detecting the DSG3 protein includes, for example, a method that
uses a biosensor utilizing the surface plasmon resonance
phenomenon. The use of a biosensor utilizing the surface plasmon
resonance phenomenon enables real-time observation of
protein-protein interactions as surface plasmon resonance signals
without the need of protein labeling. For example, by using a
biosensor such as BIAcore (Biacore), binding between the DSG3
protein and an anti-DSG3 antibody can be detected. Specifically, a
test sample is contacted with a sensor chip onto which an anti-DSG3
antibody is immobilized, and the DSG3 protein that binds to the
anti-DSG3 antibody can be detected as a change in resonance
signals.
[0201] Anti-DSG3 antibodies can be labeled by general methods
using, in addition to the labels mentioned above, positron-emitting
nuclides such as 18F, 55Co, 64Cu, 66Ga, 68Ga, 76Br, 89Zr, and 124I
(Acta. Oncol. 32, 825-830, 1993). By using PET (positron emission
tomography scanner), which is an instrument for non-invasively
obtaining data on the in vivo behavior of drugs, after
administering an anti-DSG3 antibody labeled with an above-mentioned
positron-emitting nuclide to humans or animals, radiation emitted
by the radioactive nuclide is measured from outside the body and
then converted into a quantitative image by computed tomography
methods. By using PET as described above, antigenic molecules that
are highly expressed in a particular cancer can be detected without
collecting samples from patients. In addition to the
above-mentioned nuclides, anti-DSG3 antibodies can be radiolabeled
with short-lived RI using positron-emitting nuclides such as 11C,
13N, 15O, 18F, and 45Ti.
[0202] At present, the use of a medical cyclotron for production of
short-lived nuclides using the above-mentioned nuclides, techniques
for producing short-lived RI-labeled compounds, and such, are
currently under research and development, and anti-DSG3 antibodies
can be labeled using such techniques. By administering an anti-DSG3
antibody to patients after labeling it with the above-mentioned
positron-emitting nuclides, the labeled anti-DSG3 antibody that
recognizes the DSG3 protein present in the living body gathers at
primary foci and metastatic foci according to the specificity of
the anti-DSG3 antibody at each site of the pathological tissue.
Therefore, the presence of primary foci and metastatic foci can be
diagnosed by detecting their radioactivity. For use in such
diagnostic purpose, emission activity values of 25-4000 keV gamma
particles or positrons can be used appropriately. Furthermore,
therapeutic effects can be expected by selecting a suitable nuclide
and administering it in large quantities. In this case, emission of
70-700 keV gamma particles or positrons can be suitably used.
[0203] In another embodiment of the methods of the present
invention, the expression of DSG3 mRNA is detected. In the present
invention, detection includes quantitative and qualitative
detections. Examples of qualitative detection include simple
measurement for the presence or absence of DSG3 mRNA, measurement
to see whether or not the DSG3 mRNA is present above a certain
amount, and measurement that compares the amount of DSG3 mRNA to
that of other samples (for example, a control sample). On the other
hand, quantitative detection includes, for example, measurement of
the DSG3 mRNA concentration and measurement of the amount of DSG3
mRNA.
[0204] Test samples are not particularly limited so long as they
are samples that may contain DSG3 mRNA, and are preferably samples
collected from the body of organisms such as mammals, and more
preferably samples collected from humans. Specific examples of the
test samples include blood, interstitial fluid, plasma,
extravascular fluid, cerebrospinal fluid, synovial fluid, pleural
fluid, serum, lymphatic fluid, saliva, and urine, but the test
samples are preferably blood, serum, or plasma. Test samples of the
present invention also include samples obtained from test samples,
such as cell culture solutions and specimens of immobilized tissues
or cells collected from the body of an organism.
[0205] The cancers that are diagnosed are not particularly limited
and may be any cancer, and specific examples include lung cancer,
colon cancer, esophageal cancer, stomach cancer, pancreatic cancer,
skin cancer, and uterine cancer. Lung cancer is preferable and
non-small cell lung cancer is particularly preferable.
[0206] Subjects in the present invention may be animal species that
genetically carry a DSG3 protein, and many non-human mammals such
as monkeys, cattle, sheep, mice, dogs, cats, and hamsters are known
as such animal species. Subjects that are suitably used are, in
particular, humans, but are not limited thereto.
[0207] Specific embodiments of the detection method are described
below, but the methods of the present invention are not limited to
those methods. First, a sample is prepared from a subject. Next,
DSG3 mRNAs included in the sample are detected. In the present
invention, it is also acceptable to detect cDNAs synthesized from
mRNAs. In the present invention, when the DSG3 mRNA or
DSG3-encoding cDNA is detected in a test sample, if a greater
amount of DSG3 mRNA or DSG3-encoding cDNA is detected in the test
sample than in a negative control or a healthy individual, it can
be determined that the subject has cancer or has a high risk of
being affected by cancer in the future.
[0208] Examples of such methods include methods known to those
skilled in the art such as the Northern blotting method, RT-PCR
method, and DNA array method.
[0209] The detection methods of the present invention described
above can be automated using various automatic testing devices, and
large quantities of sample can be examined at a time.
[0210] A further objective of the present invention is to provide
diagnostic agents or kits for detecting the DSG3 protein in a test
sample for cancer diagnosis. The diagnostic agents or kits contain
at least an anti-DSG3 antibody. When the diagnostic agents or kits
are based on an EIA method such as the ELISA method, a carrier for
immobilizing the antibody may be included, or a carrier may be
bound to the antibody in advance. If the diagnostic agents or kits
are based on an aggregation method that uses a carrier such as
latex, they may include an antibody-adsorbed carrier.
[0211] A further objective of the present invention is to provide
diagnostic agents or kits for detecting DSG3 mRNA or DSG3-encoding
cDNA in a test sample for cancer diagnosis. The diagnostic agents
or kits contain at least a DSG3-encoding DNA (a DNA consisting of
the nucleotide sequence of SEQ ID NO: 39) or an oligonucleotide
comprising at least 15 nucleotides that are complementary to its
complementary strand.
[0212] Herein, the term "complementary strand" refers to the other
strand with respect to one of the strands of a double-stranded
nucleic acid consisting of A:T (U in the case of RNA) and G:C base
pairs. In addition, "complementary" refers not only to cases of
completely complementary sequences within a region of at least 15
consecutive nucleotides, but also to cases of at least 70%,
preferably at least 80%, more preferably 90%, and even more
preferably 95% homology or higher in a nucleotide sequence.
Homology may be determined using an algorithm described herein.
[0213] The oligonucleotides of the present invention can be used as
probes or primers for detecting or amplifying DSG3-endcoding DNA,
and probes or primers for detecting the expression of these DNAs.
Furthermore, the oligonucleotides of the present invention can be
used in the form of a DNA array substrate.
[0214] When such oligonucleotides are used as primers, their
lengths are normally 15 by to 100 bp, and preferably 17 by to 30
bp. The primers are not particularly limited as long as at least a
portion of the DSG3-encoding DNA, or a complementary strand
thereof, can be amplified. Furthermore, when they are used as
primers, their 3'-end regions can be made to be complementary, and
restriction enzyme recognition sequences or tags can be added to
their 5' ends.
[0215] When using these oligonucleotides as probes, the probes are
not particularly limited, as long as they specifically hybridize to
at least a portion of the DSG3-encoding DNA, or to a complementary
strand thereof. The probes may be synthetic oligonucleotides, and
are normally at least 15 by or longer.
[0216] When the oligonucleotides of the present invention are used
as probes, it is preferable to use the labeled ones. Examples of
labeling methods include labeling methods that use T4
polynucleotide kinase to phosphorylate the 5' ends of
oligonucleotides with .sup.32P, and methods that incorporate a
substrate nucleotide labeled with an isotope such as .sup.32P, a
fluorescent dye, biotin or the like, by using a DNA polymerase such
as Klenow enzyme, and a random hexamer oligonucleotide or such as a
primer (random priming methods and so on).
[0217] The oligonucleotides of the present invention can be
produced using, for example, a commercially available
oligonucleotide synthesizer. The probes may be produced as
double-stranded DNA fragments obtained by restriction enzyme
treatment or the like.
[0218] In the diagnostic agents or kits mentioned above, sterilized
water, physiological saline, vegetable oil, surfactants, lipids,
solubilizers, buffers, protein stabilizers (BSA, gelatin, or such),
preservatives, blocking solutions, reaction solution,
reaction-stopping solution, reagents for treating samples, and such
may be combined as necessary, in addition to the oligonucleotides
and antibodies, which are the active ingredients.
[0219] The diagnostic methods of the present invention can be
performed both in vitro and in vivo, but preferably preformed in
vitro.
[0220] In a preferred embodiment of the present invention, an
example of the methods for diagnosing cancer is a method comprising
the following steps of:
(a) providing a sample collected from a subject; and (b) detecting
for DSG3 proteins contained in the sample of (a).
[0221] Moreover, in a preferred embodiment of the present
invention, an example of the methods for diagnosing cancer is a
method comprising the following steps:
(a) providing a sample collected from a subject; and (b) detecting
for DSG3 genes contained in the sample of (a).
Production of Anti-DSG3 Antibodies
[0222] The anti-DSG3 antibodies used in the present invention may
be derived from any origin, and may be of any type (monoclonal or
polyclonal), and in any form, as long as they specifically bind to
a DSG3 protein. Specifically, known antibodies such as animal
antibodies (for example, mouse antibodies, rat antibodies, and
camel antibodies), human antibodies, chimeric antibodies, and
humanized antibodies can be used. The antibodies may be polyclonal
antibodies, and are preferably monoclonal antibodies.
[0223] Anti-DSG3 antibodies to be used in the present invention can
be obtained as polyclonal or monoclonal antibodies using known
techniques. In particular, monoclonal antibodies derived from a
mammal are preferable as the anti-DSG3 antibody to be used in the
present invention. Examples of monoclonal antibodies derived from a
mammal include antibodies produced by hybridoma, and antibodies
produced by a host transformed by genetic engineering techniques
with an expression vector containing an antibody gene.
[0224] A monoclonal antibody-producing hybridoma can be prepared
essentially using known techniques as follows. Specifically,
immunization is performed using the DSG3 protein as a sensitizing
antigen according to a general immunization method to obtain
immunocytes, which are then fused to known parent cells by a
general cell fusion method. Then, hybridoma that produce an
anti-DSG3 antibody can be selected by screening for monoclonal
antibody-producing cells using a general screening method.
[0225] Specifically, monoclonal antibodies are prepared as follows.
First, the DSG3 gene having the nucleotide sequence disclosed in
GenBank Accession No. NM.sub.--001944 (SEQ ID NO: 39) is expressed,
and the DSG3 protein is obtained and used as the sensitizing
antigen for obtaining the antibody. Specifically, the gene sequence
encoding DSG3 is inserted into a known expression vector, and it is
used to transform an appropriate host cell. Then, the human DSG3
protein of interest can be purified by a known method from the host
cell or its culture supernatant. Alternatively, a purified
naturally occurring DSG3 protein can be used in the same
manner.
[0226] The purified DSG3 protein can be used as a sensitizing
antigen for immunization of mammals. A partial peptide of DSG3 can
also be used as the sensitizing antigen. In that case, the partial
peptide can be obtained from the amino acid sequence of human DSG3
by chemical synthesis, they can also be obtained by incorporating a
part of the DSG3 gene into an expression vector and expressing it.
Alternatively, the partial peptide can be obtained by degrading the
DSG3 protein with a protease, and there are no limitations on the
region or size of the partial DSG3 peptides used.
[0227] The mammal to be immunized with the sensitizing antigen is
not particularly limited, but is preferably selected in
consideration of the compatibility with parent cells to be used for
cell fusion. For example, rodents such as mice, rats, and hamsters,
rabbits, or monkeys are generally used.
[0228] The above-described animals can be immunized with a
sensitizing antigen according to a known method. For example, as a
general method, immunization can be performed by injecting a mammal
intraperitoneally or subcutaneously with a sensitizing antigen.
Specifically, the sensitizing antigen is diluted at an appropriate
dilution with PBS (Phosphate-Buffered Saline), physiological
saline, or the like; mixed with a standard adjuvant such as a
Freund's complete adjuvant as desired; emulsified; and then
administered to mammals several times every four to 21 days.
Furthermore, an appropriate carrier can be used when the
sensitizing antigen is used for immunization. Particularly when a
partial peptide with a small molecular weight is used as a
sensitizing antigen, the sensitizing antigen peptide is desirably
bound to a carrier protein such as albumin or keyhole limpet
hemocyanin, and then used for immunization.
[0229] Mammals are immunized as described, and when an increase in
the amount of desired antibody in the serum is confirmed,
immunocytes are collected from the mammals and subjected to cell
fusion. A particularly preferred immunocyte is a splenocyte.
[0230] A mammalian myeloma cell is used as a cell to be fused with
the above-mentioned immunocyte. A variety of known cell lines can
be suitably used as the myeloma cell, and examples include: P3
(P3x63Ag8.653) (J. Immunol. (1979) 123, 1548-1550); P3x63Ag8U.1
(Current Topics in Microbiology and Immunology (1978) 81, 1-7);
NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6,
511-519); MPC-11 (Margulies. D. H. et al., Cell (1976) 8, 405-415);
SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270); FO (de St.
Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21); 5194
(Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323); and 8210
(Galfre, G et al., Nature (1979) 277, 131-133).
[0231] Cell fusion of the above-mentioned immunocytes with myeloma
cells is essentially performed according to a known method, for
example, the method of Kohler and Milstein et al. (Kohler. G and
Milstein, C., Methods Enzymol. (1981) 73, 3-46).
[0232] More specifically, the above-mentioned cell fusion can be
performed in a standard nutritional culture medium in the presence
of, for example, a cell-fusion accelerator. A cell-fusion
accelerator is, for example, polyethylene glycol (PEG), Sendai
virus (HVJ), or the like. If desired, an auxiliary agent such as
dimethylsulfoxide can be added to further enhance fusion
efficiency.
[0233] The ratio of immunocytes to myeloma cells used can be
established at one's discretion. For example, the number of
immunocytes is preferably set to one to ten times of that of
myeloma cells. As a medium to be used for the above-mentioned cell
fusion, for example, RPMI1640 medium and MEM medium, which are
appropriate for the growth of the above-mentioned myeloma cell
line, or other standard media that are used for this type of cell
culture can be used. Moreover, a serum supplement solution such as
fetal calf serum (FCS) can be suitably added and used in
combination.
[0234] Cell fusion is performed by thoroughly mixing predetermined
amounts of the above-mentioned immunocytes and myeloma cells in the
above-mentioned medium, adding and mixing with a PEG solution of
generally 30 to 60% (w/v) concentration that has been pre-heated to
approximately 37.degree. C. and has, for example, an average
molecular weight of approximately 1000 to 6000, so as to form the
desired fused cells (hybridomas). Subsequently, the agent for cell
fusion or the like which is unfavorable for the growth of
hybridomas can be removed by successively adding an appropriate
medium such as those listed above, removing the supernatant by
centrifugation, and repeating these steps.
[0235] Hybridomas obtained in this manner can be selected by
culturing the hybridomas in a standard selection medium such as HAT
medium (a medium containing hypoxanthine aminopterin, and
thymidine). The above-mentioned HAT medium can be used to continue
the culturing for a sufficient period of time to kill the cells
other than the hybridoma of interest (non-fused cells) (typically,
a sufficient period of time is several days to several weeks).
Subsequently, hybridomas that produce the antibody of interest can
be screened and monocloned by carrying out a standard limiting
dilution method.
[0236] Alternatively, a DSG3-recognizing antibody can be prepared
using the method described in International Patent Publication No.
WO 03/104453.
[0237] Screening and monocloning an antibody of interest can be
suitably performed by a screening method based on known
antigen-antibody reaction. For example, the antigen is bound to a
carrier such as polystyrene beads or the like, or a commercially
available 96-well microtiter plate, followed by reaction with the
culture supernatant of the hybridomas. After the carrier is washed,
it is reacted with an enzyme-labeled secondary antibody or the like
to determine whether or not the antibody of interest that reacts
with the sensitizing antigen is contained in the culture
supernatant. Hybridomas producing the desired antibodies that have
a binding ability to the antigen can be cloned by the limiting
dilution method or the like. Antigens used for immunization as well
as an operably similar DSG3 protein can be used suitably in this
case.
[0238] In addition to the above-mentioned method where hybridoma
are obtained by immunizing non-human animals with the antigen,
desired human antibodies having the activity to bind to a DSG3
protein can also be obtained by sensitizing human lymphocytes with
the DSG3 protein in vitro, and fusing the sensitized lymphocytes
with human-derived myeloma cells that have infinite division
potential (see Japanese Patent Publication Kokoku Publication No.
(JP-B) H01-59878 (examined, approved Japanese patent application
published for opposition)). Alternatively, desired human antibodies
can also be obtained by administering a DSG3 protein that serves as
an antigen to a transgenic animal having a complete human antibody
gene repertoire to obtain anti-DSG3 antibody-producing cells,
immortalizing these cells, and isolating human antibodies against
the DSG3 protein from the immortalized cells (see International
Patent Publication Nos. WO 94/25585, WO 93/12227, WO 92/03918, and
WO 94/02602).
[0239] The monoclonal antibody-producing hybridoma produced in this
manner can be passaged and cultured in a standard medium, or can be
stored for a long period in liquid nitrogen.
[0240] To obtain monoclonal antibodies from hybridoma, a method for
obtaining monoclonal antibodies as a culture supernatant after
culturing the hybridoma according to a standard method, a method
for obtaining monoclonal antibodies as an ascites after
administering and growing the hybridoma in a compatible mammal, or
the like can be suitably carried out. The former method is suitable
for obtaining highly purified antibodies, while the latter method
is suitable for mass production of antibodies.
[0241] In the present invention, a recombinant antibody is produced
from recombinant cells generated by genetic engineering techniques
that involve cloning the antibody gene from hybridoma,
incorporating the gene into an appropriate vector, and introducing
the vector into a host, and can be used as a monoclonal antibody
(see for example, Vandamme, A. M. et al., Eur. J. Biochem. (1990)
192, 767-775). Specifically, the gene can be obtained from
hybridoma cells producing an anti-DSG3 antibody by isolating mRNA
that encodes the variable region (V region) of the anti-DSG3
antibody. That is, total RNA can be prepared from the hybridoma
cells by a known method such as the guanidine ultracentrifugation
method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299)
or AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162,
156-159), and then, the mRNA of interest can be prepared using an
mRNA purification kit (GE Healthcare Bio-Sciences) or the like. In
addition, mRNA can also be directly prepared from hybridoma using
QuickPrep mRNA Purification Kit (GE Healthcare Bio-Sciences).
[0242] cDNA of the antibody V region can be synthesized from the
obtained mRNA using reverse transcriptase. cDNA can be synthesized
using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit
(SEIKAGAKU CORPORATION) or the like. To synthesize and amplify
cDNA, for example, 5'-Ampli FINDER RACE Kit (Clontech) and the
5'-RACE method using PCR (Frohman, M. A. et al., Proc. Natl. Acad.
Sci. USA (1988) 85, 8998-9002; Belyaysky, A. et al., Nucleic Acids
Res. (1989) 17, 2919-2932) can also be used favorably, and in the
process of such cDNA synthesis, appropriate restriction enzyme
sites, which will be described later, can be introduced into both
ends of the cDNA.
[0243] The cDNA fragment of interest is purified from the obtained
PCR product, and then ligated to a vector DNA. The recombinant
vector is prepared in this manner and introduced into Escherichia
coli or the like, and after colonies are selected, the desired
recombinant vector can be prepared from the E. coli that formed the
colonies. Whether or not the recombinant vector has the cDNA
nucleotide sequence of interest can be confirmed by a known method,
such as the dideoxynucleotide chain termination method. Once cDNA
encoding the V region of the anti-DSG3 antibody of interest is
obtained, this cDNA is digested by enzymes that recognize the
restriction enzyme sites inserted to both ends of this cDNA. The
cDNA encoding the anti-DSG3 antibody V region, which has been
digested as described above, is incorporated by ligation into an
expression vector that contains a desired antibody constant region
(C region), so that the DNA encoding this C region can be fused in
frame with the cDNA when digested with the same combination of
enzymes.
[0244] A preferred method for producing the anti-DSG3 antibody used
in the present invention is a method that incorporates the antibody
gene into an expression vector so that the gene is expressed under
the regulation of an expression control region, for example, an
enhancer or a promoter. Next, by suitably transforming a host cell
with this expression vector, recombinant cells that express the
anti-DSG3 antibody-encoding DNA can be obtained.
[0245] An antibody gene can be expressed by incorporating a DNA
encoding the antibody heavy chain (H-chain) and a DNA encoding the
antibody light chain (L-chain) separately into expression vectors,
and then simultaneously transforming a host cell with the vectors;
or by incorporating a DNA encoding the H-chain and the L-chain into
a single expression vector, and then transforming a host cell with
the vector (see International Patent Publication No. WO
94/11523).
[0246] Appropriate combinations of suitable hosts and expression
vectors can be used for isolating an antibody gene and introducing
the gene into an appropriate host to produce the antibody. When
using eukaryotic cells as a host, animal cells, plant cells, and
fungal cells can be used. Known animal cells include (1) mammalian
cells such as CHO, COS, myeloma, baby hamster kidney (BHK), HeLa,
and Vero cells; (2) amphibian cells such as Xenopus oocytes; and
(3) insect cells such as sf9, sf21, and Tn5. Known plant cells
include cells derived from the Nicotiana genus such as Nicotiana
tabacum, from which callus can be cultured. Known fungal cells
include yeasts such as the Saccharomyces genus, for example,
Saccharomyces cerevisiae, and filamentous fungi such as the
Aspergillus genus, for example, Aspergillus niger. Production
systems that utilize bacterial cells can be suitably used when
using prokaryotic cells. Known bacterial cells include E. coli and
Bacillus subtilis. By introducing expression vectors comprising the
antibody genes of interest into these cells by transformation, and
then culturing the transformed cells in vitro, the desired
antibodies can be obtained from the transformed cell culture.
[0247] In addition to the above host cells, transgenic animals can
also be used suitably to produce a recombinant antibody. For
example, the antibody gene can be inserted in frame into a gene
that encodes a protein inherently produced in milk, for example,
goat .beta.-casein to construct a fused gene. A DNA fragment
containing the fused gene, which has been inserted with the
antibody gene, is injected into a goat embryo, and then this
injected embryo is introduced into a female goat. Desired
antibodies can be obtained from milk produced by the transgenic
goat born from the goat that received the embryo or progeny
thereof. To increase the volume of milk containing the desired
antibody produced by the transgenic goat, hormones can be used on
the transgenic goat as necessary (Ebert, K. M. et al.,
Bio/Technology (1994) 12, 699-702).
[0248] Animal-derived antibody C regions can be used for the C
regions of a recombinant antibody of the present invention. For
example, C.gamma.1, C.gamma.2a, C.gamma.2b, C.gamma.3, C.mu.,
C.delta., C.alpha.1, C.alpha.2, and C.epsilon. can be used for the
mouse antibody H-chain C-region, and C.kappa. and C.lamda. can be
used for the L-chain C-region. In addition to mouse antibodies,
antibodies of animals such as rats, rabbits, goat, sheep, camels,
and monkeys can be used as animal antibodies. Their sequences are
known. Furthermore, the C region can be modified to improve the
stability of the antibodies or their production.
[0249] In the present invention, genetically recombinant antibodies
that are artificially modified for the purpose of reducing
xenoantigenicity against humans, or the like can be used. Examples
of such include chimeric antibodies and humanized antibodies. These
modified antibodies can be produced using known methods. A chimeric
antibody is an antibody comprising the antibody heavy chain and
light chain variable regions of a nonhuman mammal such as a mouse,
and the antibody heavy chain and light chain constant regions of a
human. The DNA encoding a mouse antibody variable region is ligated
to the DNA encoding a human antibody constant region, and this is
incorporated into an expression vector to produce a recombinant
vector expressing the DNA. The recombinant cells that have been
transformed with the vector are cultured, and the incorporated DNA
is expressed to obtain the chimeric antibody produced in the
culture.
[0250] A human antibody C region can be used for the C regions of
the chimeric antibody and humanized antibody, and for example,
C.gamma.1, C.gamma.2, C.gamma.3, C.gamma.4, C.mu., C.delta.,
C.alpha.1, C.alpha.2, and C.epsilon. can be used for the H chain,
and C.kappa. and C.lamda. can be used for the L-chain. Their
sequences are known. Furthermore, the human antibody C region can
be modified to improve the stability of the antibody or its
production.
[0251] A chimeric antibody consists of the V region of an antibody
derived from a non-human animal, and a C region derived from a
human antibody. On the other hand, a humanized antibody consists of
the complementarity determining region (CDR) of an antibody derived
from a non-human animal, and the framework region (FR) and C region
derived from a human antibody. Since the antigenicity of a
humanized antibody in human body is reduced, a humanized antibody
is useful as an active ingredient for therapeutic agents of the
present invention.
[0252] A humanized antibody, which is also called a reshaped human
antibody, is obtained by transplanting, in place of a human
antibody CDR, the CDR of a non-human animal antibody such as a
mouse antibody, and common genetic recombination techniques for
such preparation are also known. Specifically, a DNA sequence is
designed for ligating a mouse antibody CDR in frame with a human
antibody FR, and is synthesized by PCR using several
oligonucleotides designed to contain overlapping portions at their
ends as primers. An integration vector can be produced by inserting
the DNA obtained as described above and a DNA that encodes a human
antibody C region into an expression vector so that they will
ligate in frame. After incorporating this integration vector into a
host to establish recombinant cells, the recombinant cells are
cultured, and the DNA encoding the humanized antibody is expressed
to produce the humanized antibody in the cell culture (see,
European Patent Application No. EP 239,400, and International
Patent Application No. WO 96/02576).
[0253] By qualitatively or quantitatively measuring and evaluating
the activity of the humanized antibody produced as described above
to bind to antigens, human antibody FRs that would make the CDRs
form a favorable antigen-binding site when ligated through the CDRs
can be suitably selected. As necessary, amino acids in an FR may be
substituted such that the CDRs of a reshaped human antibody forms
an appropriate antigen-binding site. The above-mentioned amino acid
substitution can be introduced by appropriately using the PCR
method used when fusing mouse CDR with human FR, and mutant FR
sequences having the desired characteristics can be selected by
measuring and evaluating the activity of the amino acid-substituted
mutant antibody to bind to the antigen by the above-mentioned
method (Sato, K. et al., Cancer Res. 1993, 53, 851-856).
[0254] Methods for obtaining human antibodies are also known. For
example, desired human antibodies with antigen-binding activity can
be obtained by sensitizing human lymphocytes with a desired antigen
or cells expressing a desired antigen in vitro; and fusing the
sensitized lymphocytes with human myeloma cells such as U266 (see
JP-B H01-59878). Alternatively, a desired human antibody can be
obtained by using a desired antigen to immunize a transgenic animal
that comprises the entire repertoire of human antibody genes (see
International Patent Application Nos. WO 93/12227, WO 92/03918, WO
94/02602, WO 94/25585, WO 96/34096, and WO 96/33735). Furthermore,
techniques to obtain human antibodies by panning a human antibody
library are also known. For example, the V region of a human
antibody is expressed as a single chain antibody (scFv) on the
phage surface using a phage display method, and phages that bind to
the antigen can be selected. By analyzing the genes of selected
phages, the DNA sequences encoding the V regions of human
antibodies that bind to the antigen can be determined. After
determining the DNA sequences of scFvs that bind to the antigen,
the V region sequence is fused in frame with the desired human
antibody C region sequence, and this is inserted into a suitable
expression vector to produce an expression vector. This expression
vector can be introduced into suitable expression cells such as
those described above, and the human antibody-encoding gene can be
expressed to obtain the human antibodies. Such methods are well
known and one can refer to International Patent Application Nos. WO
92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO
95/01438, and WO 95/15388.
[0255] The antibody used in the present invention is not limited to
bivalent antibodies represented by IgG, but includes monovalent
antibodies and multivalent antibodies represented by IgM, so long
as it binds to the DSG3 protein. The multivalent antibody of the
present invention includes a multivalent antibody that has the same
antigen binding sites, and a multivalent antibody that has
partially or completely different antigen binding sites.
[0256] The antibody used in the present invention is not limited to
the whole antibody molecule, but includes minibodies and modified
products thereof, so long as they bind to the DSG3 protein.
[0257] A minibody comprises antibody fragments lacking a portion of
a whole antibody (for example, whole IgG), and is not particularly
limited so long as it has antigen-binding ability. There are no
particular limitations on the antibody fragments of the present
invention, so long as they are portions of a whole antibody, but
they preferably contain a heavy chain variable region (VH) and/or a
light chain variable region (VL). The amino acid sequence of VH or
VL may have substitutions, deletions, additions, and/or insertions.
Furthermore, so long as it has antigen-binding ability, part of VH
and/or VL can be deleted. The variable region may be chimerized or
humanized. Specific examples of the antibody fragments include Fab,
Fab', F(ab').sub.2, and Fv. Specific examples of minibodies include
Fab, Fab', F(ab')2, Fv, scFv (single chain Fv), diabody, and
sc(Fv)2 (single chain (Fv)2). Multimers of these antibodies (for
example, dimers, trimers, tetramers, and polymers) are also
included in the minibodies of the present invention.
[0258] Antibody fragments can be produced by treating an antibody
with an enzyme, such as papain or pepsin. Alternatively, genes
encoding these antibody fragments can be constructed, introduced
into expression vectors, and expressed in appropriate host cells
(see, for example, Co, M. S. et al., J. Immunol. (1994) 152,
2968-2976; Better, M. and Horwitz, A. H., Methods in Enzymology
(1989) 178, 476-496; Plueckthun, A. and Skerra, A., Methods in
Enzymology (1989) 178, 476-496; Lamoyi, E., Methods in Enzymology
(1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology
(1989) 121, 663-669; and Bird, R. E. et al., TIBTECH (1991) 9,
132-137).
[0259] A diabody refers to a bivalent antibody fragment constructed
by gene fusion (Hollinger P. et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993); EP 404,097; WO 93/11161; and such). A diabody is
a dimer composed of two polypeptide chains, and generally, the
polypeptide chains are individually linked by a linker of, for
example, five residues or so, which is short enough to prevent
binding between VL and VH in the same chain. VL and VH that are
encoded by the same polypeptide chain have a short linker between
them, and form a dimer because they cannot form a single chain
variable region fragment. Therefore, diabodies have two antigen
binding sites.
[0260] scFv can be obtained by ligating the H-chain V region and
L-chain V region of an antibody. In this scFv, the H-chain V region
and L-chain V region are ligated via a linker, preferably a peptide
linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A., 1988,
85, 5879-5883). The H-chain V region and L-chain V region of the
scFv and may be derived from any of the antibodies described
herein. The peptide linker for ligating the V regions is not
particularly limited, but for example, any single-chain peptide
consisting of 3 to 25 residues or so, or a peptide linker described
below can be used. PCR methods such as those described above can be
used for ligating the V regions. An scFv-encoding DNA can be
amplified by a PCR method using as a template, either a whole DNA
or a partial DNA encoding a desired amino acid sequence selected
from a DNA sequence encoding the H chain or the H-chain V region of
the above-mentioned antibody, and a DNA sequence encoding the L
chain or the L-chain V region of the above-mentioned antibody; and
using a primer pair having sequences corresponding to the sequences
of the two ends. Next, a DNA comprising the desired sequence can be
obtained by performing a PCR reaction using the combination of a
DNA encoding the peptide linker portion, and a primer pair having
sequences designed so that both ends of the DNA will be ligated to
the H chain and L chain. Once the scFv-encoding DNA is constructed,
expression vectors containing the DNA, and recombinant cells
transformed by these expression vectors can be obtained according
to conventional methods. Furthermore, the scFvs can be obtained by
culturing the resulting recombinant cells and expressing the
scFv-encoding DNA.
[0261] sc(Fv)2 is a minibody prepared by ligating two VHs and two
VLs with linkers or such to form a single chain (Hudson et al., J.
Immunol. Methods 1999; 231: 177-189). sc(Fv)2 can be produced, for
example, by joining scFvs with a linker.
[0262] Moreover, antibodies in which two VHs and two VLs are
arranged in the order of VH, VL, VH, and VL
([VH]-linker-[VL]-linker-[VH]-linker-[VL]), starting from the
N-terminal side of a single chain polypeptide, are preferred.
[0263] The order of the two VHs and the two VLs is not particularly
limited to the above-mentioned arrangement, and they may be placed
in any order. Examples include the following arrangements:
[VL]-linker-[VH]-linker-[VH]-linker-[VL]
[VH]-linker-[VL]-linker-[VL]-linker-[VH]
[VH]-linker-[VH]-linker-[VL]-linker-[VL]
[VL]-linker-[VL]-linker-[VH]-linker-[VH]
[VL]-linker-[VH]-linker-[VL]-linker-[VH]
[0264] Any arbitrary peptide linker can be introduced by genetic
engineering, and synthetic linkers (see, for example, those
disclosed in Protein Engineering, 9(3), 299-305, 1996) or such can
be used as linkers for linking the antibody variable regions, but
in the present invention, peptide linkers are preferable. The
length of the peptide linkers is not particularly limited, and can
be suitably selected by those skilled in the art according to the
purpose; however, the length is generally 1 to 100 amino acids,
preferably 3 to 50 amino acids, more preferably 5 to 30 amino
acids, and particularly preferably 12 to 18 amino acids (for
example, 15 amino acids).
[0265] Examples of the peptide linkers include:
TABLE-US-00001 Ser Gly-Ser Gly-Gly-Ser Ser-Gly-Gly Gly-Gly-Gly-Ser
(SEQ ID NO: 72) Ser-Gly-Gly-Gly (SEQ ID NO: 73) Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 74) Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 75)
Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 76) Ser-Gly-Gly-Gly-Gly-Gly
(SEQ ID NO: 77) Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 78)
Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 79) (Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 74))n (Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 75))n
in which n is an integer of 1 or larger. The length and sequence of
the peptide linkers can be selected appropriately by those skilled
in the art according to the purpose.
[0266] Therefore, a particularly preferred embodiment of sc(Fv)2 in
the present invention is, for example, the following sc(Fv)2:
[VH]-peptide linker (15 amino acids)-[VL]-peptide linker (15 amino
acids)-[VH]-peptide linker (15 amino acids)-[VL]
[0267] Synthetic chemical linkers (chemical crosslinking agents)
which include crosslinking agents routinely used to crosslink
peptides and are, for example, N-hydroxy succinimide (NHS),
disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate
(BS.sup.3), dithiobis(succinimidyl propionate) (DSP),
dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol
bis(succinimidyl succinate) (EGS), ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST),
bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).
These crosslinking agents are commercially available.
[0268] Usually, three linkers are required to link four antibody
variable regions. The linkers to be used may all be of the same
type or different types. In the present invention, a preferred
minibody is a diabody or an sc(Fv)2. Such minibody can be obtained
by treating an antibody with an enzyme, such as papain or pepsin,
to generate antibody fragments, or by constructing DNAs that encode
these antibody fragments, introducing them into expression vectors,
and then expressing them in appropriate host cells (see, for
example, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976;
Better, M. and Horwitz, A. H., Methods Enzymol. (1989) 178,
476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178,
497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;
Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; and
Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,
132-137).
[0269] The antibodies of the present invention can be exemplified
by the antibodies of (1) to (62) below, but are not limited
thereto. The antibodies of (1) to (62) include, for example,
full-length antibodies, minibodies, animal antibodies, chimeric
antibodies, humanized antibodies, and human antibodies:
[0270] (1) an antibody that comprises an H chain having the amino
acid sequence of SEQ ID NO: 2 (sequence of the DF151 antibody
H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 4
(sequence of the DF151 antibody H chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 6 (sequence of the DF151 antibody
H-chain CDR3) as CDR3;
[0271] (2) an antibody that comprises the H chain of (1) having the
amino acid sequence of SEQ ID NO: 8 (sequence of the DF151 antibody
CH) as CH(H-chain constant region);
[0272] (3) an antibody that comprises the H chain of (1) having the
amino acid sequence of SEQ ID NO: 10 (sequence of the CH of the
mouse-human chimeric DF151 antibody) as CH;
[0273] (4) an antibody that comprises an L chain having the amino
acid sequence of SEQ ID NO: 12 (sequence of the DF151 antibody
L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 14
(sequence of the DF151 antibody L-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 16 (sequence of the DF151
antibody L-chain CDR3) as CDR3;
[0274] (5) an antibody that comprises the L chain of (4) having the
amino acid sequence of SEQ ID NO: 18 (sequence of the DF151
antibody CL) as CL (L-chain constant region);
[0275] (6) an antibody that comprises the L chain of (4) having the
amino acid sequence of SEQ ID NO: 20 (sequence of the mouse-human
chimeric DF151 antibody CL) as CL;
[0276] (7) an antibody that comprises the H chain of (1) and the L
chain of (4);
[0277] (8) an antibody that comprises the H chain of (2) and the L
chain of (5);
[0278] (9) an antibody that comprises the H chain of (3) and the L
chain of (6);
[0279] (10) an antibody that comprises an H chain having the amino
acid sequence of SEQ ID NO: 22 (sequence of the DF364 antibody
H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 24
(sequence of the DF364 antibody H-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 26 (sequence of the DF364
antibody H-chain CDR3) as CDR3;
[0280] (11) an antibody that comprises the H chain of (10) having
the amino acid sequence of SEQ ID NO: 28 (sequence of the DF364
antibody CH) as CH;
[0281] (12) an antibody that comprises the H chain of (10) having
the amino acid sequence of SEQ ID NO: 10 (sequence of the
mouse-human chimeric DF364 antibody CH) as CH;
[0282] (13) an antibody that comprises an L chain having the amino
acid sequence of SEQ ID NO: 30 (sequence of the DF364 antibody
L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 32
(sequence of the DF364 antibody L-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 34 (sequence of the DF364
antibody L-chain CDR3) as CDR3;
[0283] (14) an antibody that comprises the L chain of (13) having
the amino acid sequence of SEQ ID NO: 36 (sequence of the DF364
antibody CL) as CL;
[0284] (15) an antibody that comprises the L chain of (13) having
the amino acid sequence of SEQ ID NO: 20 (sequence of the
mouse-human chimeric DF364 antibody CL) as CL;
[0285] (16) an antibody that comprises the H chain of (10) and the
L chain of (13);
[0286] (17) an antibody that comprises the H chain of (11) and the
L chain of (14);
[0287] (18) an antibody that comprises the H chain of (12) and the
L chain of (15);
[0288] (19) an antibody that comprises the H chain of (1) and the L
chain of (13);
[0289] (20) an antibody that comprises the H chain of (2) and the L
chain of (14);
[0290] (21) an antibody that comprises the H chain of (3) and the L
chain of (15);
[0291] (22) an antibody that comprises the H chain of (10) and the
L chain of (4);
[0292] (23) an antibody that comprises the H chain of (11) and the
L chain of (5);
[0293] (24) an antibody that comprises the H chain of (12) and the
L chain of (6);
[0294] (25) an antibody that comprises an H chain having the amino
acid sequence of SEQ ID NO: 81 (sequence of the DF366 antibody
H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 83
(sequence of the DF366 antibody H-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 85 (sequence of the DF366
antibody H-chain CDR3) as CDR3;
[0295] (26) an antibody that comprises the H chain of (25) having
the amino acid sequence of SEQ ID NO: 28 (sequence of the DF366
antibody CH) as CH;
[0296] (27) an antibody that comprises the H chain of (25) having
the amino acid sequence of SEQ ID NO: 10 (sequence of the
mouse-human chimeric DF366 antibody CH) as CH;
[0297] (28) an antibody that comprises an L chain having the amino
acid sequence of SEQ ID NO: 87 (sequence of the DF366 antibody
L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 89
(sequence of the DF366 antibody L-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 91 (sequence of the DF366
antibody L-chain CDR3) as CDR3;
[0298] (29) an antibody that comprises the L chain of (28) having
the amino acid sequence of SEQ ID NO: 36 (sequence of the CL of the
DF366 antibody) as CL;
[0299] (30) an antibody that comprises the L chain of (28) having
the amino acid sequence of SEQ ID NO: 20 (sequence of the CL of the
mouse-human chimeric DF366 antibody) as CL;
[0300] (31) an antibody that comprises the H chain of (25) and the
L chain of (28);
[0301] (32) an antibody that comprises the H chain of (26) and the
L chain of (29);
[0302] (33) an antibody that comprises the H chain of (27) and the
L chain of (30);
[0303] (34) an antibody that comprises the H chain of (1) and the L
chain of (28);
[0304] (35) an antibody that comprises the H chain of (2) and the L
chain of (29);
[0305] (36) an antibody that comprises the H chain of (3) and the L
chain of (30);
[0306] (37) an antibody that comprises the H chain of (10) and the
L chain of (28);
[0307] (38) an antibody that comprises the H chain of (11) and the
L chain of (29);
[0308] (39) an antibody that comprises the H chain of (12) and the
L chain of (30);
[0309] (40) an antibody that comprises the H chain of (25) and the
L chain of (4);
[0310] (41) an antibody that comprises the H chain of (26) and the
L chain of (5);
[0311] (42) an antibody that comprises the H chain of (27) and the
L chain of (6);
[0312] (43) an antibody that comprises the H chain of (25) and the
L chain of (13);
[0313] (44) an antibody that comprises the H chain of (26) and the
L chain of (14);
[0314] (45) an antibody that comprises the H chain of (27) and the
L chain of (15);
[0315] (46) an antibody that comprises the H chain of (1) having
the amino acid sequence of SEQ ID NO: 108 (sequence of the CH of a
mouse IgG2a antibody) as CH;
[0316] (47) an antibody that comprises the L chain of (4) having
the amino acid sequence of SEQ ID NO: 112 (sequence of the CL of a
mouse IgG2a antibody) as CL;
[0317] (48) an antibody that comprises the H chain of (10) having
the amino acid sequence of SEQ ID NO: 108 (sequence of the CH of a
mouse IgG2a antibody) as CH;
[0318] (49) an antibody that comprises the L chain of (13) having
the amino acid sequence of SEQ ID NO: 112 (sequence of the CL of a
mouse IgG2a antibody) as CL;
[0319] (50) an antibody that comprises the H chain of (25) having
the amino acid sequence of SEQ ID NO: 108 (sequence of the CH of a
mouse IgG2a antibody) as CH;
[0320] (51) an antibody that comprises the L chain of (28) having
the amino acid sequence of SEQ ID NO: 112 (sequence of the CL of a
mouse IgG2a antibody) as CL;
[0321] (52) an antibody that comprises the H chain of (46) and the
L chain of (47);
[0322] (53) an antibody that comprises the H chain of (48) and the
L chain of (49);
[0323] (54) an antibody that comprises the H chain of (50) and the
L chain of (51);
[0324] (55) an antibody that comprises the H chain of (46) and the
L chain of (49);
[0325] (56) an antibody that comprises the H chain of (48) and the
L chain of (51);
[0326] (57) an antibody that comprises the H chain of (50) and the
L chain of (47);
[0327] (58) an antibody that comprises the H chain of (46) and the
L chain of (51);
[0328] (59) an antibody that comprises the H chain of (48) and the
L chain of (47);
[0329] (60) an antibody that comprises the H chain of (50) and the
L chain of (49);
[0330] (61) an antibody having one or more amino acid
substitutions, deletions, additions, and/or insertions in the
antibody of any one of (1) to (60) and having an activity
equivalent to that of the antibody of any one of (1) to (60);
and
[0331] (62) an antibody that binds to the same epitope as the DSG3
protein epitope to which the antibody of any one of (1) to (60)
binds.
[0332] An example of VH in the above-mentioned "H chain having the
amino acid sequence of SEQ ID NO: 2 (sequence of the DF151 antibody
H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID NO: 4
(sequence of the DF151 antibody H-chain CDR2) as CDR2, and the
amino acid sequence of SEQ ID NO: 6 (sequence of the DF151 antibody
H-chain CDR3) as CDR3" of (1) includes a VH having the amino acid
sequence of SEQ ID NO: 46 (sequence of the DF151 antibody VH).
[0333] An example of VL in the above-mentioned "L chain having the
amino acid sequence of SEQ ID NO: 12 (sequence of the DF151
antibody L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID
NO: 14 (sequence of the DF151 antibody L-chain CDR2) as CDR2, and
the amino acid sequence of SEQ ID NO: 16 (sequence of the DF151
antibody L-chain CDR3) as CDR3" of (4) includes a VL having the
amino acid sequence of SEQ ID NO: 48 (sequence of the DF151
antibody VL).
[0334] An example of VH in the above-mentioned "H chain having the
amino acid sequence of SEQ ID NO: 22 (sequence of the DF364
antibody H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID
NO: 24 (sequence of the DF364 antibody H-chain CDR2) as CDR2, and
the amino acid sequence of SEQ ID NO: 26 (sequence of the DF364
antibody H-chain CDR3) as CDR3" of (10) includes a VH having the
amino acid sequence of SEQ ID NO: 50 (sequence of the DF364
antibody VH).
[0335] An example of VL in the above-mentioned "L chain having the
amino acid sequence of SEQ ID NO: 30 (sequence of the DF364
antibody L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID
NO: 32 (sequence of the DF364 antibody L-chain CDR2) as CDR2, and
the amino acid sequence of SEQ ID NO: 34 (sequence of the DF364
antibody L-chain CDR3) as CDR3" of (13) includes a VL having the
amino acid sequence of SEQ ID NO: 52 (sequence of the DF364
antibody VL).
[0336] An example of VH in the above-mentioned "H chain having the
amino acid sequence of SEQ ID NO: 81 (sequence of the DF366
antibody H-chain CDR1) as CDR1, the amino acid sequence of SEQ ID
NO: 83 (sequence of the DF366 antibody H-chain CDR2) as CDR2, and
the amino acid sequence of SEQ ID NO: 85 (sequence of the DF366
antibody H-chain CDR3) as CDR3" of (25) includes a VH having the
amino acid sequence of SEQ ID NO: 93 (sequence of the DF366
antibody VH).
[0337] An example of VL in the above-mentioned "L chain having the
amino acid sequence of SEQ ID NO: 87 (sequence of the DF366
antibody L-chain CDR1) as CDR1, the amino acid sequence of SEQ ID
NO: 89 (sequence of the DF366 antibody L-chain CDR2) as CDR2, and
the amino acid sequence of SEQ ID NO: 91 (sequence of the DF366
antibody L-chain CDR3) as CDR3" of (28) includes a VL having the
amino acid sequence of SEQ ID NO: 95 (sequence of the DF366
antibody VL).
[0338] A preferred embodiment of the above-mentioned antibody of
(61) is an antibody in which CDR has not been modified. As an
example, a preferred embodiment of "an antibody having one or more
amino acid substitutions, deletions, additions, and/or insertions
in the antibody of (1) and having an activity equivalent to that of
the antibody of (1)" among the above-mentioned antibody of (61) is
"an antibody having an activity equivalent to that of the antibody
of (1) and having one or more amino acid substitutions, deletions,
additions, and/or insertions in the antibody of (1), and also
comprising an H chain having the amino acid sequence of SEQ ID NO:
2 as CDR1, the amino acid sequence of SEQ ID NO: 4 as CDR2, and the
amino acid sequence of SEQ ID NO: 6 as CDR3". Preferred embodiments
of other antibodies included in the above-mentioned antibody of
(61) can be expressed in a similar manner.
[0339] Methods of introducing mutations into polypeptides are well
known to those skilled in the art as methods for preparing
polypeptides that are functionally equivalent to a certain
polypeptide. For example, those skilled in the art can prepare an
antibody functionally equivalent to an antibody of the present
invention by introducing appropriate mutations into the antibody
using site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995)
Gene 152, 271-275; Zoller, M J, and Smith, M. (1983) Methods
Enzymol. 100, 468-500; Kramer, W. et al. (1984) Nucleic Acids Res.
12, 9441-9456; Kramer W, and Fritz H J (1987) Methods. Enzymol.
154, 350-367; Kunkel, T A (1985) Proc. Natl. Acad. Sci. USA. 82,
488-492; Kunkel (1988) Methods Enzymol. 85, 2763-2766) and such.
Amino acid mutations may also occur naturally. In this way, the
antibodies of the present invention also comprise antibodies
comprising amino acid sequences with one or more amino acid
mutations in the amino acid sequences of the antibodies of the
present invention, and which are functionally equivalent to the
antibodies of the present invention. The number of amino acids that
are mutated in such mutants is generally considered to be 50 amino
acids or less, preferably 30 amino acids or less, and more
preferably 10 amino acids or less (for example, 5 amino acids or
less).
[0340] It is desirable that the amino acid residues are mutated
into amino acids in which the properties of the amino acid side
chains are conserved. Examples of amino acid side chain properties
include: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),
hydrophilic amino acids (R, D, N, C, E, Q, G H, K, S, and T), amino
acids comprising the following side chains: aliphatic side chains
(G, A, V, L, I, and P); hydroxyl-containing side chains (S, T, and
Y); sulfur-containing side chains (C and M); carboxylic acid- and
amide-containing side chains (D, N, E, and Q); basic side chains
(R, K, and H); or aromatic ring-containing side chains (H, F, Y,
and W) (amino acids are represented by one-letter codes in
parentheses).
[0341] Polypeptides comprising a modified amino acid sequence, in
which one or more amino acid residues in a certain amino acid
sequence is deleted, added, and/or substituted with other amino
acids, are known to retain their original biological activities
(Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984) 81,
5662-5666; Zoller, M. J. & Smith, M. Nucleic Acids Research
(1982) 10, 6487-6500; Wang, A. et al., Science 224, 1431-1433;
Dalbadie-McFarland, G et al., Proc. Natl. Acad. Sci. USA (1982) 79,
6409-6413).
[0342] Antibodies that bind to the same epitope as the anti-DSG3
antibodies disclosed in the present invention are also provided.
Such antibodies can be obtained, for example, by the following
method.
[0343] To determine if a test antibody can compete for binding to
the same epitope bound by the anti-DSG3 antibodies disclosed in the
invention of this application, a cross-blocking assay, for example,
a competitive ELISA assay can be performed. For example, in a
competitive ELISA assay, DSG3 protein-coated wells of a microtiter
plate are pre-incubated with or without a candidate competing
antibody, and then a biotin-labeled anti-DSG3 antibody of the
present invention is added. The amount of labeled anti-DSG3
antibody bound to the DSG3 protein in the wells can be measured
using avidin-peroxidase conjugate and an appropriate substrate. The
antibody can be labeled, for example, with a radioactive label or
fluorescent label, or some other detectable and measurable label.
The amount of labeled anti-DSG3 antibody bound to the DSG3 protein
is indirectly correlated to the binding ability of the candidate
competing antibody (test antibody) that competes for binding to the
same epitope. That is, the greater the affinity of the test
antibody for the same epitope, the lower the binding activity of
the labeled anti-DSG3 antibody to the DSG3 protein-coated wells. A
candidate competing antibody is considered to be an antibody that
binds substantially to the same epitope or that competes for
binding to the same epitope as an anti-DSG3 antibody of the present
invention if the candidate competing antibody can block binding of
the DSG3 antibody by at least 20%, preferably by at least 20% to
50%, and even more preferably, by at least 50%, as compared to the
binding activity obtained in a control experiment performed in the
absence of the candidate competing antibody.
[0344] Antibodies that bind to the same epitope as the anti-DSG3
antibodies include, for example, the above-mentioned antibody of
(62), but are not limited thereto.
[0345] As described above, the above-mentioned antibodies of (1) to
(62) include not only monovalent antibodies but also multivalent
antibodies with two or more valencies. Multivalent antibodies of
the present invention include multivalent antibodies whose antigen
binding sites are all the same and multivalent antibodies whose
antigen binding sites are partially or completely different.
[0346] The following antibodies are examples of multivalent
antibodies that have different antigen binding sites, but the
antibodies of the present invention are not limited thereto:
[0347] an antibody comprising at least two H chain and L chain
pairs (hereinafter referred to as HL pairs) selected from the HL
pairs of (7), (16), (19), (22), (31), (34), (37), (40), and
(43);
[0348] an antibody comprising at least two HL pairs selected from
the HL pairs of (8), (17), (20), (23), (32), (35), (38), (41), and
(44);
[0349] an antibody comprising at least two HL pairs selected from
the HL pairs of (9), (18), (21), (24), (33), (36), (39), (42), and
(45); and
[0350] an antibody comprising at least two HL pairs selected from
the HL pairs of (52) to (60).
[0351] Antibodies bound to various types of molecules such as
polyethylene glycol (PEG) can also be used as modified antibodies.
Moreover, chemotherapeutic agents, toxic peptides, or radioactive
chemical substances can be bound to the antibodies. Such modified
antibodies (hereinafter referred to as antibody conjugates) can be
obtained by subjecting the obtained antibodies to chemical
modification. Methods for modifying antibodies are already
established in this field. Furthermore, as described below, such
antibodies can also be obtained in the molecular form of a
bispecific antibody designed using genetic engineering techniques
to recognize not only DSG3 proteins, but also chemotherapeutic
agents, toxic peptides, radioactive chemical compounds, or such.
These antibodies are included in the "antibodies" of the present
invention.
[0352] Low-molecular-weight chemotherapeutic agents such as
azaribine, anastrozole, azacytidine, bleomycin, bortezomib,
bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin,
carmustine, celebrex, chlorambucil, cisplatin, irinotecan,
carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine,
docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,
dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin
glucuronide, epirubicin, ethinyl estradiol, estramustine,
etoposide, etoposide glucuronide, floxuridine, fludarabine,
flutamide, fluorouracil, fluoxymesterone, gemcitabine,
hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,
leucovorin, lomustine, mechlorethamine, medroxyprogesterone
acetate, megestrol acetate, melphalan, mercaptopurine,
methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane,
phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin,
semustine streptozocin, tamoxifen, taxanes, taxol, testosterone
propionate, thalidomide, thioguanine, thiotepa, teniposide,
topotecan, uracil mustard, vinblastine, vinorelbine, and
vincristine can be suitably used as chemotherapeutic agents
(including prodrugs that are converted to such chemotherapeutic
agents nonenzymatically or enzymatically in vivo) that are bound to
anti-DSG3 antibodies to bring about cytotoxic activity. Moreover,
toxic peptides such as ricin, abrin, ribonuclease, onconase, DNase
I, Staphylococcal enterotoxin-A, pokeweed antiviral protein,
gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas
endotoxin, L-asparaginase, and PEG L-Asparaginase can also be
suitably used. In another embodiment, one or two or more of the
low-molecular-weight chemotherapeutic agents can be suitably used
in combination and one or two or more of the toxic peptides. For
the bond between an anti-DSG3 antibody and the above-mentioned
low-molecular-weight chemotherapeutic agent, a covalent bond or
non-covalent bond can be suitably selected, and methods for
preparing chemotherapeutic agent-bound antibodies are known.
[0353] Furthermore, for binding with proteinaceous pharmaceutical
agents or toxins, gene recombination techniques can be used to
construct a recombinant vector in which a DNA encoding the
above-mentioned toxic peptide and a DNA encoding an anti-DSG3
antibody are fused in frame and inserted into an expression vector.
This vector is introduced into suitable host cells, and transformed
cells are obtained and cultured. Recombinant proteins can be
prepared by expressing the incorporated DNA.
[0354] Furthermore, the antibody used in the present invention may
be a bispecific antibody. The bispecific antibody may have
antigen-binding sites that recognize different epitopes on a DSG3
molecule. Alternatively, one antigen-binding site may recognize
DSG3 and the other antigen-binding site may recognize a cytotoxic
substance such as a chemotherapeutic agent, toxic peptide, or
radioactive chemical substance. This enables the cytotoxic
substance to directly act on cells expressing DSG3, thereby
specifically damaging tumor cells and suppressing tumor cell
proliferation. Alternatively, one may prepare a bispecific antibody
in which the other antigen-binding site recognizes an antigen that
is similar to but different from DSG3, and specifically expressed
on the surface of the target cancer cells. Bispecific antibodies
can be produced by linking the HL pairs from two types of
antibodies, or by fusing hybridomas producing different monoclonal
antibodies to prepare bispecific antibody-producing fused cells.
Bispecific antibodies can also be prepared by genetic engineering
techniques.
[0355] Antibody genes constructed described above can be obtained
through expression by known methods. In the case of mammalian
cells, the antibody genes can be expressed by operably linking an
effective, commonly used promoter, the antibody gene to be
expressed, and a polyA signal on its 3' downstream side. An example
of the promoter/enhancer is human cytomegalovirus immediate early
promoter/enhancer.
[0356] Examples of other promoters/enhancers that can be used for
expression of an antibody to be used in the present invention
include viral promoters/enhancers from retrovirus, polyoma virus,
adenovirus, or simian virus 40 (SV40), and mammalian cell-derived
promoters/enhancers such as human elongation factor 1.alpha.
(HEF1.alpha.).
[0357] When an SV40 promoter/enhancer is used, gene expression can
be readily carried out by the method of Mulligan et al. (Nature
(1979) 277, 108), and when an HEF1.alpha. promoter/enhancer is
used, gene expression can be readily carried out by the method of
Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322).
[0358] In the case of E. coli, an effective, commonly used
promoter, a signal sequence for antibody secretion, and the
antibody gene to be expressed are functionally linked to express
the gene. Examples of a promoter include the lacZ promoter and the
araB promoter. When the lacZ promoter is used, the gene can be
expressed by the method of Ward et al. (Nature (1098) 341, 544-546;
FASEB J. (1992) 6, 2422-2427), and when the araB promoter is used,
the gene can be expressed by the method of Better et al. (Science
(1988) 240, 1041-1043).
[0359] With regard to the signal sequence for antibody secretion,
the pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987)
169, 4379) may be used for production in the periplasm of E. coli.
After the antibody produced in the periplasm is isolated, the
antibody structure is refolded by using a protein denaturant like
guanidine hydrochloride or urea so that the antibody will have the
desired binding activity.
[0360] The replication origin inserted into the expression vector
includes, for example, those derived from SV40, polyoma virus,
adenovirus, or bovine papilloma virus (BPV). In order to amplify
the gene copy number in the host cell system, the expression vector
can have, for example, the aminoglycoside transferase (APH) gene,
thymidine kinase (TK) gene, E. coli xanthine guanine
phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase
(dhfr) gene inserted as a selection marker.
[0361] Any expression system, for example, a eukaryotic cell system
or a prokaryotic cell system, can be used to produce antibodies
used in the present invention. Examples of eukaryotic cells include
animal cells such as established mammalian cell system, insect cell
system, and filamentous fungus cells and yeast cells. Examples of
prokaryotic cells include bacterial cells such as E. coli cells.
Antibodies used in the present invention are preferably expressed
in mammalian cells such as CHO, COS, myeloma, BHK, Vero, or HeLa
cells.
[0362] Next, the transformed host cell is then cultured in vitro or
in vivo to induce production of the antibody of interest. The host
cells are cultured according to known methods. For example, DMEM,
MEM, RPMI 1640, or IMDM can be used as the culture medium. A serum
supplement solution such as fetal calf serum (FCS) can also be used
in combination.
[0363] Antibodies expressed and produced as described above can be
purified by using a single known method or a suitable combination
of known methods generally used for purifying proteins. Antibodies
can be separated and purified by, for example, appropriately
selecting and combining affinity columns such as protein A column,
chromatography column, filtration, ultrafiltration, salt
precipitation, dialysis, and such (Antibodies A Laboratory Manual.
Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
[0364] Known means can be used to measure the antigen-binding
activity of the antibodies (Antibodies A Laboratory Manual. Ed
Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). For
example, an enzyme linked immunosorbent assay (ELISA), an enzyme
immunoassay (EIA), a radioimmunoassay (RIA), or a fluoroimmunoassay
can be used.
[0365] The antibodies used in the present invention may be
antibodies with a modified sugar chain. It is known that the
cytotoxic activity of an antibody can be increased by modifying its
sugar chain. Antibodies having modified sugar chains are, for
example, antibodies with modified glycosylation (for example, WO
99/54342), antibodies deficient in fucose which is added to sugar
chains (for example, WO 00/61739 and WO 02/31140), antibodies
having a sugar chain with bisecting GlcNAc (for example, WO
02/79255).
[0366] The antibodies used in the present invention are preferably
antibodies having cytotoxic activity.
[0367] In the present invention, the cytotoxic activity includes,
for example, antibody-dependent cell-mediated cytotoxicity (ADCC)
activity and complement-dependent cytotoxicity (CDC) activity. In
the present invention, CDC activity means cytotoxic activity caused
by the complement system. ADCC activity refers to the activity of
damaging a target cell when a specific antibody attaches to its
cell surface antigen, and an Fc.gamma. receptor-carrying cell
(immune cell, or such) binds to the Fc portion of the antigen via
the Fc.gamma. receptor damages the target cell.
[0368] An anti-DSG3 antibody can be tested to see whether it has
ADCC activity or CDC activity using known methods (for example,
Current Protocols in Immunology, Chapter 7. Immunologic studies in
humans, Editor, John E. Coligan et al., John Wiley & Sons,
Inc., (1993) and the like).
[0369] First, specifically, effector cells, complement solution,
and target cells are prepared.
[0370] (1) Preparation of Effector Cells
[0371] Spleen is removed from a CBA/N mouse or the like, and spleen
cells are isolated in RPMI1640 medium (manufactured by Invitrogen).
After washing in the same medium containing 10% fetal bovine serum
(FBS, manufactured by HyClone), the cell concentration is adjusted
to 5.times.10.sup.6/mL to prepare the effector cells.
[0372] (2) Preparation of Complement Solution
[0373] Baby Rabbit Complement (manufactured by CEDARLANE) is
diluted 10-fold in a culture medium (manufactured by Invitrogen)
containing 10% FBS to prepare a complement solution.
[0374] (3) Preparation of Target Cells
[0375] The target cells can be radioactively labeled by incubating
cells expressing the DSG3 protein (cells transformed with a gene
encoding the DSG3 protein, lung cancer cells, colon cancer cells,
esophageal cancer cells, gastric cancer cells, pancreatic cancer
cells, skin cancer cells, uterine cancer cells, or the like) with
0.2 mCi of sodium chromate-.sup.51Cr (manufactured by GE Healthcare
Bio-Sciences) in a DMEM medium containing 10% FBS for one hour at
37.degree. C. After radioactive labeling, cells are washed three
times in RPMI1640 medium containing 10% FBS, and the target cells
can be prepared by adjusting the cell concentration to
2.times.10.sup.5/mL.
[0376] ADCC activity or CDC activity can be measured by the method
described below. In the case of ADCC activity measurement, the
target cell and anti-DSG3 antibody (50 .mu.L each) are added to a
96-well U-bottom plate (manufactured by Becton Dickinson), and
reacted for 15 minutes on ice. Thereafter, 100 .mu.L of effector
cells are added and incubated in a carbon dioxide incubator for
four hours. The final concentration of the antibody is adjusted to
0 or 10 .mu.g/mL. After culturing, 100 .mu.L of the supernatant is
collected, and the radioactivity is measured with a gamma counter
(COBRAII AUTO-GAMMA, MODEL D5005, manufactured by Packard
Instrument Company). The cytotoxic activity (%) can be calculated
using the obtained values according to the equation:
(A-C)/(B-C).times.100, wherein A represents the radioactivity (cpm)
in each sample, B represents the radioactivity (cpm) in a sample
where 1% NP-40 (manufactured by Nacalai Tesque) has been added, and
C represents the radioactivity (cpm) of a sample containing the
target cells only.
[0377] Meanwhile, in the case of CDC activity measurement, 50 .mu.L
of target cell and 50 .mu.L of an anti-DSG3 antibody are added to a
96-well flat-bottomed plate (manufactured by Becton Dickinson), and
reacted for 15 minutes on ice. Thereafter, 100 .mu.L of complement
solution is added, and incubated in a carbon dioxide incubator for
four hours. The final concentration of the antibody is adjusted to
0 or 3 .mu.g/mL. After incubation, 100 .mu.L of supernatant is
collected, and the radioactivity is measured with a gamma counter.
The cytotoxic activity can be calculated in the same way as in the
ADCC activity determination.
[0378] On the other hand, in the case of measuring the cytotoxic
activity of an antibody conjugate, 50 .mu.L of target cell and 50
.mu.L of an anti-DSG3 antibody conjugate are added to a 96-well
flat-bottomed plate (manufactured by Becton Dickinson), and reacted
for 15 minutes on ice. Thereafter, this is incubated in a carbon
dioxide incubator for one to four hours. The final concentration of
the antibody is adjusted to 0 or 3 .mu.g/mL. After culturing, 100
.mu.L of supernatant is collected, and the radioactivity is
measured with a gamma counter. The cytotoxic activity can be
calculated in the same way as in the ADCC activity
determination.
[0379] An antibody of the present invention having cytotoxic
activity is more preferably an antibody that does not have
cell-dissociating activity. An antibody that does not have
cell-dissociating activity can be suitably selected and obtained by
measuring the cell-dissociating activity that inhibits cell
adhesion of keratinocytes even in a test tube. The method of
measuring cell-dissociating activity can be carried out in a test
tube, for example, by the method described in J. Invest. Dermatol.,
124, 939-946, 2005. Furthermore, as a method for observing this
cellular activity in vivo, the activity can be evaluated as the
activity to induce PV lesions, which are phenotypes of in vivo
cell-dissociating activity. The PV-lesion-inducing activity can be
evaluated by the method described in J. Immunology 170, 2170-2178,
2003.
[0380] The cells whose proliferation is suppressed by the anti-DSG3
antibody are not particularly limited as long as they express a
DSG3 protein, but are preferably cancer cells, and more preferably,
lung cancer cells, colon cancer cells, esophageal cancer cells,
gastric cancer cells, pancreatic cancer cells, skin cancer cells,
or uterine cancer cells. More preferably, they are from
non-small-cell lung cancer. Therefore, the anti-DSG3 antibody can
be used for the purpose of treating or preventing diseases
attributed to cell proliferation, for instance, lung cancer, colon
cancer, esophageal cancer, stomach cancer, pancreatic cancer, skin
cancer, or uterine cancer, more preferably non-small-cell lung
cancer, and even more preferably lung squamous cell carcinoma,
adenocarcinoma, adenosquamous carcinoma, or large cell
carcinoma.
[0381] The present invention also provides polynucleotides encoding
the antibodies of the present invention, and polynucleotides that
hybridize under stringent conditions to these polynucleotides and
encode antibodies having an activity equivalent to that of the
antibodies of the present invention. The present invention also
provides vectors containing these polynucleotides and transformants
(including transformed cells) containing such vectors. The
polynucleotides of the present invention are polymers comprising
multiple nucleotides or base pairs of deoxyribonucleic acids (DNA)
or ribonucleic acids (RNA), and are not particularly limited, as
long as they encode the antibodies of the present invention. They
may also contain non-natural nucleotides. The polynucleotides of
the present invention can be used to express antibodies using
genetic engineering techniques. Furthermore, they can be used as
probes in the screening of antibodies that are functionally
equivalent to the antibodies of the present invention.
Specifically, a DNA that hybridizes under stringent conditions to
the polynucleotide encoding an antibody of the present invention,
and encodes an antibody having an activity equivalent to that of
the antibody of the present invention, can be obtained by
techniques such as hybridization and gene amplification technique
(for example, PCR), using the polynucleotide encoding an antibody
of the present invention, or a portion thereof, as a probe. Such
DNAs are included in the polynucleotides of the present invention.
Hybridization techniques are well known to those skilled in the art
(Sambrook, J. et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold
Spring Harbor Lab. press, 1989). Conditions for hybridization
include, for example, those with low stringency. Examples of
conditions of low stringency include post-hybridization washing
under conditions of 0.1.times.SSC and 0.1% SDS at 42.degree. C.,
and preferably under conditions of 0.1.times.SSC and 0.1% SDS at
50.degree. C. More preferable hybridization conditions include
those of high stringency. Highly stringent conditions include, for
example, conditions of 5.times.SSC and 0.1% SDS at 65.degree. C.
Under these conditions, the higher the temperature, polynucleotides
having high homology would be obtained efficiently. However,
several factors such as temperature and salt concentration can
influence hybridization stringency, and those skilled in the art
can suitably select these factors to realize similar
stringencies.
[0382] An antibody that is encoded by a polynucleotide obtained by
these hybridization and gene amplification techniques, and which is
functionally equivalent to antibodies of the present invention,
usually has a high homology to the amino acid sequences of these
antibodies. The antibodies of the present invention also include
antibodies that are functionally equivalent to and have high amino
acid sequence homology to the antibodies of the present invention.
The term "high homology" generally refers to amino acid identity of
at least 50% or higher, preferably 75% or higher, more preferably
85% or higher, still more preferably 95% or higher. Polypeptide
homology can be determined by the algorithm described in literature
(Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983)
80, 726-730).
Pharmaceutical Compositions
[0383] In another aspect, the present invention features
pharmaceutical compositions comprising an antibody that binds to a
DSG3 protein as an active ingredient. In addition, the present
invention features a cell proliferation inhibitor, in particular an
anticancer agent, comprising an antibody that binds to a DSG3
protein as an active ingredient. Cell proliferation inhibitors and
anticancer agents of the present invention are preferably
administered to a subject affected by cancer, or to a subject who
is likely to be affected by cancer. Subjects in the present
invention are animal species that genetically carry a DSG3 protein
and are affected by cancer or likely to be affected by cancer, and
include, for example, mammals such as humans, monkeys, cattle,
sheep, mice, dogs, cats, and hamsters, but are not limited
thereto.
[0384] In the present invention, a cell proliferation inhibitor
comprising as an active ingredient an antibody that binds to a DSG3
protein can also be described as a method for suppressing cell
proliferation which comprises the step of administering an antibody
that binds to a DSG3 protein to a subject, or as use of an antibody
that binds to a DSG3 protein in the production of a cell
proliferation inhibitor.
[0385] Furthermore, in the present invention, an anticancer agent
comprising as an active ingredient an antibody that binds to a DSG3
protein can also be described as a method for preventing or
treating cancer which comprises the step of administering an
antibody that binds to a DSG3 protein to a subject, or as use of an
antibody that binds to a DSG3 protein in the production of an
anticancer agent.
[0386] In the present invention, the phrase "comprising an antibody
that binds to DSG3 as an active ingredient" means comprising an
anti-DSG3 antibody as the main active substance, and does not limit
the content percentage of the anti-DSG3 antibody.
[0387] The antibody included in the pharmaceutical composition of
the present invention (for example, cell proliferation inhibitor
and anticancer agent; same hereinafter) is not particularly limited
so long as it binds to a DSG3 protein, and examples include
antibodies described herein.
[0388] The pharmaceutical compositions of the present invention can
be administered orally or parenterally. Particularly preferably,
the method of administration is parenteral administration, and
specifically, the method of administration is, for example,
administration by injection, transnasal administration,
transpulmonary administration, or transdermal administration.
Examples of administration by injection include systemic and local
administrations of a pharmaceutical composition of the present
invention by intravenous injection, intramuscular injection,
intraperitoneal injection, subcutaneous injection, or such. A
suitable administration method may be selected according to the age
of the patient and symptoms. The dosage may be selected, for
example, within the range of 0.0001 mg to 1000 mg per kg body
weight in each administration. Alternatively, for example, the
dosage for each patient may be selected within the range of 0.001
to 100,000 mg/body. However, the pharmaceutical composition of the
present invention is not limited to these doses.
[0389] The pharmaceutical compositions of the present invention can
be formulated according to conventional methods (for example,
Remington's Pharmaceutical Science, latest edition, Mark Publishing
Company, Easton, U.S.A), and may also contain pharmaceutically
acceptable carriers and additives. Examples include, but are not
limited to, surfactants, excipients, coloring agents, perfumes,
preservatives, stabilizers, buffers, suspending agents,
isotonization agents, binders, disintegrants, lubricants, fluidity
promoting agents, and flavoring agents; and other commonly used
carriers can be suitably used. Specific examples include light
anhydrous silicic acid, lactose, crystalline cellulose, mannitol,
starch, carmellose calcium, carmellose sodium, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, polyvinylacetal
diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain
fatty acid triglyceride, polyoxyethylene hardened castor oil 60,
saccharose, carboxymethyl cellulose, corn starch, inorganic salt,
and such.
[0390] In addition, the present invention provides methods for
inducing damages in a DSG3-expressing cell and methods for
inhibiting cell growth by contacting a DSG3-expressing cell with a
DSG3 protein-binding antibody. The DSG3 protein-binding antibody is
the same as the above-described antibody that binds to a DSG3
protein, which is to be contained in the cell growth inhibitor of
the present invention. The cell that is bound by the anti-DSG3
antibody is not particularly limited as long as the cell is
expressing DSG3, and is preferably a cancer cell, more preferably a
lung cancer cell, a colon cancer cell, an esophageal cancer cell, a
stomach cancer cell, a pancreatic cancer cell, a skin cancer cell,
or a uterine cancer cell, and more preferably a non-small-cell lung
cancer.
[0391] In the present invention "contacting" is accomplished, for
example, by adding an antibody to a culture solution of
DSG3-expressing cells cultured in a test tube. In this case, the
antibody can be added in the form of, for example, a solution or a
solid obtained by freeze-drying or the like. When adding the
antibody as an aqueous solution, the aqueous solution used may
purely contain only the antibody, or the solution may include, for
example, the above-mentioned surfactants, excipients, coloring
agents, perfumes, preservatives, stabilizers, buffers, suspending
agents, isotonization agents, binders, disintegrants, lubricants,
fluidity promoting agents, or flavoring agents. The concentration
for addition is not particularly limited, but the final
concentration in the culture that may be suitably used is
preferably in the range of 1 pg/mL to 1 g/mL, more preferably 1
ng/mL to 1 mg/mL, and even more preferably 1 .mu.g/mL to 1
mg/mL.
[0392] Furthermore, in another embodiment, "contacting" in the
present invention is carried out by administration to a non-human
animal to which a DSG3-expressing cell has been transplanted into
the body, or to an animal carrying cancer cells endogenously
expressing DSG3. The method of administration may be oral or
parenteral administration. Particularly preferably, the method of
administration is parenteral administration, and specifically, the
method of administration is, for example, administration by
injection, transnasal administration, transpulmonary
administration, or transdermal administration. Examples of
administration by injection include systemic and local
administrations of pharmaceutical compositions, cell proliferation
inhibitors and anticancer agents of the present invention by
intravenous injection, intramuscular injection, intraperitoneal
injection, subcutaneous injection, or such. A suitable
administration method may be selected according to the age of the
test animal and symptoms. When administering as an aqueous
solution, the aqueous solution used may purely contain only the
antibody, or the solution may include, for example, the
above-mentioned surfactants, excipients, coloring agents, perfumes,
preservatives, stabilizers, buffers, suspending agents,
isotonization agents, binders, disintegrants, lubricants, fluidity
promoting agents, or flavoring agents. The dosage may be selected,
for example, within the range of 0.0001 mg to 1000 mg per kg body
weight in each administration. Alternatively, for example, the
dosage for each patient may be selected within the range of 0.001
to 100,000 mg/body. However, the antibody dose of the present
invention is not limited to these doses.
[0393] The following method is suitably used as a method for
evaluating or measuring cell damage induced by contacting
DSG3-expressing cells with an anti-DSG3 antibody. Examples of a
method for evaluating or measuring the cytotoxic activity in a test
tube include methods for measuring the above-mentioned
antibody-dependent cell-mediated cytotoxicity (ADCC) activity,
complement-dependent cytotoxicity (CDC) activity, and such. Whether
or not an anti-DSG3 antibody has ADCC activity or CDC activity can
be measured by known methods (for example, Current protocols in
Immunology, Chapter 7. Immunologic studies in humans, Editor, John
E. Coligan et al., John Wiley & Sons, Inc., (1993) and the
like). For activity measurements, a binding antibody having the
same isotype as anti-DSG3 antibody but not having any cell-damaging
activity can be used as a control antibody in the same manner as
the anti-DSG3 antibody, and it can be determined that the activity
is present when the anti-DSG3 antibody shows a stronger cytotoxic
activity than the control antibody.
[0394] The isotype of an antibody is defined by the sequence of its
H chain constant region in the antibody amino acid sequence, and is
determined as a result of class switching that arises from genetic
recombinations in chromosomes which occur during maturation of
antibody-producing B-cells. Difference in isotype is reflected in
the difference of physiological and pathological functions of
antibodies, and for example, the strength of cytotoxic activity is
known to be influenced by antibody isotype in addition to the
expression level of the antigen. Therefore, when measuring the
above-described cell damaging activity, an antibody of the same
isotype as the test antibody is preferably used as the control.
[0395] A method for evaluating or measuring cell damaging activity
in vivo is, for example, intradermally or subcutaneously
transplanting DSG3-expressing cancer cells to a non-human test
animal, and then intravenously or intraperitoneally administering a
test antibody daily or at the interval of few days, starting from
the day of transplantation or the following day. Cytotoxicity can
be defined by daily measurement of tumor size. In a similar manner
to the evaluation in a test tube, cytotoxicity can be determined by
administering a control antibody having the same isotype, and
observing that the tumor size in the anti-DSG3
antibody-administered group is significantly smaller than the tumor
size in the control antibody-administered group. When using a mouse
as the non-human test animal, it is suitable to use a nude (nu/nu)
mouse whose thymus has been made genetically defective so that its
T lymphocyte function is lost. The use of such a mouse can
eliminate the participation of T lymphocytes in the test animals
when evaluating or measuring the cytotoxicity of the administered
antibody.
[0396] The following method can be used suitably as a method for
evaluating or measuring the inhibitory effect of an anti-DSG3
antibody on proliferation of DSG3-expressing cells through contact.
A method for measuring the incorporation of [.sup.3H]-labeled
thymidine added to the medium by living cells as an indicator for
DNA replication ability is used as a method for evaluating or
measuring the cell proliferation inhibitory activity in a test
tube. As a more convenient method, a dye exclusion method that
measures under a microscope the ability of a cell to exclude a dye
such as trypan blue to outside, or the MTT method is used. The
latter makes use of the ability of living cells to convert MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide),
which is a tetrazolium salt to a blue formazan product. More
specifically, a test antibody is added to the culture solution of a
test cell, and after a certain period of time passes, the MTT
solution is added to the culture solution, and this is left to
stand for a certain time for MTT to be incorporated into the cell.
As a result, MTT which is a yellow compound is converted to a blue
compound by the action of succinate dehydrogenase in the
mitochondria of the cell. After dissolving this blue product for
coloration, absorbance is measured and used as an indicator for the
number of viable cells. Besides MTT, reagents such as MTS, XTT,
WST-1, and WST-8 are commercially available (Nacalai Tesque, and
such) and can be suitably used. For activity measurements, a
binding antibody having the same isotype as the anti-DSG3 antibody
but not having any cell proliferation inhibitory activity can be
used as a control antibody in the same manner as the anti-DSG3
antibody, and it can be determined that the activity is present
when the anti-DSG3 antibody has a stronger cell proliferation
inhibitory activity than the control antibody.
[0397] For a method that evaluates or measures cell proliferation
inhibiting activity in vivo, the same method as the one described
above for evaluating or measuring cytotoxicity in vivo can be
suitably used.
[0398] All prior art references cited herein are incorporated by
reference into this description.
EXAMPLES
[0399] Herein below, the present invention will be specifically
described with reference to the Examples, but it is not to be
construed as being limited thereto.
Example 1
DSG3 mRNA Expression Analysis in Various Types of Cancers
[0400] Gene chip was used to perform DSG3 gene expression analysis.
To search for a gene whose expression is enhanced in cancer cells,
various RNAs and total RNAs prepared from various extracted tissues
by conventional methods using ISOGEN (manufactured by Nippon Gene)
shown in Tables 1 and 2 were used. More specifically, gene
expression analysis was carried out using 10 .mu.g each of total
RNAs, and subjecting them to GeneChip U-133A (manufactured by
Affymetrix) according to the Expression Analysis Technical Manual
(manufactured by Affymetrix). When analyzing lung adenocarcinoma
and hepatocellular carcinoma, a total of 10 .mu.g was obtained by
combining total RNAs of twelve lung adenocarcinoma cases and three
hepatocellular carcinoma cases to perform the analysis (Table
1).
TABLE-US-00002 TABLE 1 Tissue Source Whole brain Clontech 64020-1
Lung Clinical sample, 1 case Trachea Clontech 64091-1 Heart Ambion
7966 Kidney Ambion 7976 Liver Clinical sample (Surgery) Pancreas
Ambion 7954 Stomach Clinical sample (Surgery) Small intestine
Ambion 7984 Large intestine Ambion 7986 Bone marrow Clontech
64106-1 Peripheral blood mononuclear cell Clinical sample, 1 case
Testis Clontech 64027-1 Prostate Ambion 7988 Ovary Ambion 7974 Skin
Stratagene 735031 Lung cancer (adenocarcinoma) Clinical sample, 12
cases Lung cancer (squamous cell carcinoma) Clinical sample, 1 case
Lung cancer (squamous cell carcinoma) Clinical sample, 1 case Lung
cancer (squamous cell carcinoma) Clinical sample, 1 case Lung
cancer (squamous cell carcinoma) Clinical sample, 1 case Lung
cancer (squamous cell carcinoma) Clinical sample, 1 case Liver
cancer (moderately differentiated) Clinical sample, 3 cases Liver
cancer (well differentiated) Clinical sample, 3 cases Colon cancer
Clinical sample, 1 case Colon cancer Clinical sample, 1 case Colon
cancer Clinical sample, 1 case
Tissues Used for DSG3 Gene Expression Analysis
TABLE-US-00003 [0401] TABLE 2 Type of cancer Cell line Medium Serum
(%) Brain tumor U251 DMEM 10 Breast cancer MCF7 RPMI1640 10
Esophageal TE2 RPMI1640 10 cancer Stomach cancer AGS RPMI1640 10
GT3 DMEM 10 KatoIII RPMI1640:DMEM = 1:1 10 MKN45 RPMI1640 10 MKN74
RPMI1640 10 2M DMEM 10 2MD3 DMEM 10 Colon cancer CACO2 DMEM 20 DLD1
RPMI1640 10 hCT116 McCoy5A 10 LOVO HamF12:DMEM = 1:1 10 SW480
RPMI1640 10 Liver cancer Alexander DMEM 10 HepG2 DMEM 10 HLE DMEM
10 HuH6 DMEM 10 HuH7 DMEM 10 Pancreatic Capan1 DMEM 20 cancer KLM1
RPMI1640 10 Panc1 RPMI1640 10 Paca2 RPMI1640 10 PK-1 RPMI1640 10
Kidney cancer Caki2 RPMI1640 10 Lung cancer A549 DMEM 10 Lu130
RPMI1640 10 H1359 RPMI1640 10 H157 RPMI1640 10 H1648 HamF12:DMEM =
1:1 10 H2009 HamF12:DMEM = 1:1 10 H23 RPMI1640 10 H2347 RPMI1640 10
H522 RPMI1640 10 Cervical Hela DMEM 10 cancer
Cancer Cell Lines and Culturing Conditions Used for the DSG3 Gene
Expression Analysis
[0402] Search for genes whose expression is enhanced in cancer
tissues or cancer cells was performed by setting the mean value of
the expression scores for all genes to 100, and comparing the
relative expression levels of each gene. As a result, while
expression of the DSG3 mRNA (probe ID: 205595_at HG-U133A) in
normal tissues was limited to the skin, it was enhanced in lung
cancer (lung squamous cell carcinoma) and colon cancer tissues, and
in TE2 (esophageal cancer), 2M (stomach cancer), and PK-1
(pancreatic cancer) cancer cell lines (FIGS. 1 and 2).
[0403] From the above, it became apparent that while expression of
the DSG3 gene (probe ID: 205595_at HG-U133A) is very low in normal
tissues other than the skin, expression of the DSG3 gene is
enhanced in a wide variety of cancers including lung cancer, colon
cancer, esophageal cancer, stomach cancer, and pancreatic cancer.
These results suggested that there is a high possibility that
development of the above-mentioned cancers can be diagnosed using
the DSG3 expression as an indicator.
Example 2
Immunohistological Staining of DSG3 in Lung Squamous Cell
Carcinoma
[0404] Since transcription of the DSG3 gene is enhanced in cancer
cells, in particular, lung squamous cell carcinoma cells,
immunohistological staining analysis was performed to confirm
expression of the DSG3 protein.
[0405] Each sample was prepared as a fixed paraffin embedded
preparation, and a section sliced to a thickness of 4 .mu.m was
mounted on a slide glass and then left at 37.degree. C. for about
16 hours to dry sufficiently. The section was deparaffinized by
soaking three times in 100% xylene for five minutes each, and then
hydrophilized by soaking three times in 100% ethanol for five
minutes each and further soaking in 70% ethanol for five minutes.
Then, after washing three times in a 50 mM TBS buffer solution for
five minutes, the antigen in the section was activated by treating
the section with a citrate buffer (10 mM, pH 7.0) at 120.degree. C.
for ten minutes. The section in which the antigen had been
activated was washed three times in a TBS buffer for five minutes
each, and then treated for one hour at room temperature in a TBS
buffer containing an anti-DSG3 antibody (5G11) (Zymed) diluted to a
final concentration of 50 .mu.g/mL. To inactivate the endogenous
peroxidase, the anti-DSG3 antibody-bound section was treated with
0.3% hydrogen peroxide for 15 minutes at room temperature. After
washing three times with a TBS buffer solution, the above-mentioned
section was treated with the secondary antibody, ENVISION+kit/HRP
(DAKO), for one hour at room temperature. After washing three times
with the TBS buffer solution for five minutes each, DAB
(3,3'-diaminobenzamide tetrahydrochloride) was added as a coloring
substrate to stain the section. Hematoxylin was used as a staining
agent for counter staining of the nucleus.
[0406] As a result, of the five cases of tissue sections from
cancer patients affected by lung squamous cell carcinoma, all five
cases showed a positive reaction in which the section is stained by
the anti-DSG3 antibody (5G11) (FIG. 3). Since a lung
cancer-specific staining image was obtained, it became apparent
that in lung cancer, the DSG3 expression is enhanced at the protein
level as well. This indicated that development of lung cancer can
be detected using an anti-DSG3 antibody.
Example 3
Preparation of Anti-DSG3 Antibody
[0407] 3-1) Cloning of a Full-Length cDNA Encoding Human DSG3
[0408] A full-length cDNA encoding human DSG3 was obtained by PCR
amplification using Human Small Intestine Marathon-Ready cDNA
(CLONTECH) as a template. Specifically, 50 .mu.L of a reaction
solution containing 2 .mu.L of cDNA, 1 .mu.L of sense primer (SEQ
ID NO: 37), 1 .mu.L of antisense primer (SEQ ID NO: 38), 5 .mu.L of
10.times.KOD-Plus buffer, 5 .mu.L of 2 mM dNTPs, 2 .mu.L of 25 mM
MgSO.sub.4, and 1 .mu.L of KOD-Plus was subjected to a PCR reaction
performed by five cycles of a reaction cycle consisting of
reactions at 94.degree. C. for 15 seconds and 70.degree. C. for two
minutes, five cycles of a reaction cycle consisting of reactions at
94.degree. C. for 15 seconds and 68.degree. C. for two minutes, and
28 cycles of a reaction cycle consisting of reactions at 94.degree.
C. for 15 seconds and 66.degree. C. for two minutes. The amplified
product obtained by the above-mentioned PCR reaction was inserted
into pGEM-T easy using a pGEM-T Easy Vector System I (Promega).
This was sequenced using an ABI3730 DNA sequencer to confirm that
the human DSG3-encoding cDNA sequence was successfully cloned. The
sequence represented by SEQ ID NO: 39 shows the nucleotide sequence
of the human DSG3 gene, and the sequence represented by SEQ ID NO:
40 shows the amino acid sequence of the human DSG3 protein.
3-2) Establishment of Cells Showing Constant Expression of
Full-Length Human DSG3
[0409] The full-length human DSG3 cDNA was cloned into a vector
(pMCN) for expression in mammalian cells (pMCN/hDSG3). pMCN enables
induced expression under the control of a mouse CMV promoter
(ACCESSION No. U68299), and is a vector into which a neomycin
resistance gene has been incorporated. A CHO cell line that shows
constant expression of full-length human DSG3 was established by
introducing pMCN/hDSG3 into the CHO DG44 cell strain (Invitrogen)
by electroporation, and subjecting them to selection with 500
.mu.g/mL of Geneticin. Similarly, an A549 cell line that shows
constant expression of full-length human DSG3 was established by
introducing pMCN/hDSG3 into A549 cells (human lung epithelial
cancer cell line) that do not show DSG3 expression, and selection
with 1000 .mu.g/mL of Geneticin.
3-3) Preparation of Soluble Human DSG3/Mouse IgG2a Fc-Fusion
Protein
[0410] Soluble human DSG3/mouse IgG2a Fc-fusion protein
(hereinafter, shDSG3_mIgG2aFc) was prepared as an immunizing
antigen for anti-DSG3 antibody production. shDSG3_mIgG2aFc was
prepared by linking the extracellular domain of human DSG3
(Met1-Leu616) with the mouse IgG2a constant region through the CpoI
recognition sequence in the hinge region, and cloned into the pMCDN
vector prepared by incorporating the DHFR gene to the pMCN
expression vector (pMCDN/shDSG3_mIgG2aFc). The sequence represented
by SEQ ID NO: 41 shows the nucleotide sequence of the
shDSG3_mIgG2aFc gene, and the sequence represented by SEQ ID NO: 42
shows the amino acid sequence of shDSG3_mIgG2aFc. A CHO cell line
that shows constant expression of shDSG3_mIgG2aFc was established
by introducing pMCDN/shDSG3_mIgG2aFc into DG44 cells by
electroporation, and selection with 500 .mu.g/mL of Geneticin.
Next, shDSG3_mIgG2aFc was purified from culture supernatant of the
established shDSG3 mIgG2aFc-expressing CHO cell line. The culture
supernatant was applied to a Hi Trap Protein G HP (GE Healthcare
Bio-Sciences) column, and after washing with a binding buffer (20
mM sodium phosphate (pH 7.0)), elution was carried out using an
elution buffer (0.1 M glycine-HCl (pH 2.7)). The eluate was
immediately neutralized by elution into a tube containing a
neutralization buffer (1 M Tris-HCl (pH 9.0)). This eluate was
subjected to gel filtration using Superdex 200 HR 10/30 (GE
Healthcare Bio-Sciences) so that the solvent of the solution
containing the desired antibody is replaced by a PBS buffer. The
purified protein was quantified using a DC protein assay kit
(BIO-RAD) and converted into a concentration using bovine IgG
included in the kit as standard preparation.
3-4) Preparation of Anti-DSG3 Antibody
[0411] Balb/c mice or MRL/MpJUmmCrj-lpr/lpr mice (hereinafter
MRL/lpr mice, purchased from Charles River Japan) were used as the
animals for immunization. Immunization was initiated at the
7.sup.th week or 8th week. For the first immunization, an antigen
was prepared using a PBS buffer so as to include 100 .mu.g of
shDSG3_mIgG2aFc for each mouse, emulsified using Freund's complete
adjuvant (Beckton Dickinson), and administered subcutaneously. Two
weeks later, an antigen was prepared using a PBS buffer so as to
include 50 .mu.g for each mouse, emulsified using Freund's
incomplete adjuvant (Beckton Dickinson), and administered
subcutaneously. Subsequently, boosting immunization was performed
at 1-week intervals for two to four times, and for the final
immunization, the antigen was diluted in PBS at 50 .mu.g/mouse, and
then administered into the tail vein. Four days after the final
immunization, spleen cells were extirpated and mixed with mouse
myeloma cells P3-X63Ag8U1 (P3U1, purchased from ATCC) at 2:1 ratio,
and cell fusion was carried out by gradual addition of PEG 1500
(Roche Diagnostics). Next, RPMI1640 medium (Invitrogen) was added
carefully to dilute PEG 1500, and then PEG 1500 was removed by
centrifuging and removing the supernatant. The group of fused cells
suspended in RPMI1640 containing 10% FBS was seeded into a 96-well
culture plate at 100 .mu.L/well. The following day, RPMI1640
containing 10% FBS, 1.times.HAT media supplement (SIGMA), and
0.5.times.BM-Condimed H1 Hybridoma cloning supplement (Roche
Diagnostics) (hereinafter referred to as HAT medium) was added at
100 .mu.L/well. On the second or third day, half of the culture
solution was replaced with HAT medium, and the culture supernatant
from the seventh day was used in the screening in which binding
activity to the DSG3 molecule was used as an indicator. The
screening was performed by flow cytometric analysis which detects
binding to CHO cells that show constant expression of full-length
human DSG3. Positive clones obtained by this analysis were
monocloned by the limiting dilution method. Specifically, DF120,
DF122, DF148, DF151, DF153, DF168, DF331, DF364, and DF366 were
established as antibodies that specifically bind to DSG3.
[0412] In a similar manner to the case with shDSG3 mIgG2aFc, the
monoclonal antibodies were purified using a Hi Trap Protein G HP
column, from the culture supernatant of hybridomas cultured in HAT
medium that uses FBS (Ultra low IgG) (Invitrogen) as the serum. The
eluted fractions were subjected to solvent replacement with PBS
using a PD-10 column (GE Healthcare Bio-Sciences), and then stored
at 4.degree. C. The purified antibodies were quantified using a DC
protein assay kit (BIO-RAD) and converted into concentration using
bovine IgG included in the kit as the standard preparation.
3-5) Evaluation of Binding Activity by Flow Cytometry
[0413] Flow cytometry was used to evaluate the binding of the
obtained antibodies to CHO cells that show constant expression of
full-length human DSG3. The cells were suspended in FACS Buffer (1%
FBS/PBS) at 5.times.10.sup.5 cells/mL and dispensed into
Multiscreen-HV Filter Plates (Millipore), and the supernatant was
removed from this cell suspension solution by centrifugation. After
adding to the supernatant-free cells an FACS buffer containing
anti-DSG3 monoclonal antibodies which have been diluted to a
suitable concentration (3 .mu.g/mL) in the FACS buffer, this was
left to stand for 30 minutes on ice to let the cells react with the
monoclonal antibodies. After removing the supernatant from this
reaction solution by centrifugation, the cells were washed once
with FACS buffer. Next, by suspending the cells in a FACS buffer
containing FITC-labeled anti-mouse IgG antibody as the secondary
antibody, the cells were reacted with the secondary antibody for 30
minutes on ice. After the reaction was completed, the supernatant
was removed from the cells by centrifugation. The cells were
suspended in 100 .mu.L of FACS buffer, and then subjected to flow
cytometric analysis. FACS Calibur (Becton Dickinson) was used as
the flow cytometer. The living cell population was gated to a
histogram of forward scatter and side scatter. As shown in FIG. 4,
3 .mu.g/mL of anti-DSG3 monoclonal antibodies (DF120, DF122, DF148,
DF151, DF153, DF168, DF331, DF364, and DF366) bound strongly to the
DSG3-expressing CHO cells and did not bind to the parent CHO cells,
indicating that the anti-DSG3 monoclonal antibodies specifically
bind to DSG3 present on the cell membrane.
Example 4
Measurement of Cytotoxic Activities of the Anti-DSG3 Antibodies
4-1) Measurement of Complement-Dependent Cytotoxicity (CDC)
Activities of the Anti-DSG3 Antibodies
[0414] The CHO cell line showing constant expression of full-length
human DSG3 (DSG3-CHO, described in Example 3-2)) was used as the
target cell. CHO-S-SFM II medium (Invitrogen) containing 500
.mu.g/mL Geneticin (Invitrogen), HT supplement (Invitrogen), and
penicillin/streptomycin (Invitrogen) (hereinafter referred to as
"medium") was used to culture the DSG3-CHO cell line. The cell
pellet obtained by centrifuging 5.times.10.sup.5 DSG3-CHO cell line
cells (1000 rpm) for five minutes at 4.degree. C. was suspended in
approximately 200 .mu.L of medium containing 3.7 MBq of Chromium-51
(GE Healthcare Bio-Sciences), and then cultured in a 5% carbon
dioxide incubator for one hour at 37.degree. C. These cells were
washed three times with the medium, then adjusted to cell density
of 1.times.10.sup.5 cells/mL in the medium, and then dispensed into
a 96-well flat-bottomed plate at 100 .mu.L/well. Next, the
anti-DSG3 antibodies and a control mouse IgG2a antibody (BD
Biosciences Pharmingen) diluted with the medium were individually
added at 50 .mu.L/well. The final concentration of the antibodies
was adjusted to 10 .mu.g/mL. Next, baby rabbit complement
(Cederlane) diluted 5-fold in the medium was added at 50
.mu.L/well, and then the plate was left to stand in a 5% carbon
dioxide incubator for 1.5 hours at 37.degree. C. Thereafter, this
was centrifuged (1000 rpm) for five minutes at 4.degree. C., 1004
of the supernatant was collected from each well of the plate, and
the radioactivity was measured using a gamma counter (1480 WIZARD
3'', Wallac). The specific chromium release rate was determined
based on the following equation:
Specific chromium release rate (%)=(A-C).times.100/(B-C)
where A represents the radioactivity (cpm) in each well, B
represents the mean value of radioactivity (cpm) in wells where 100
.mu.L of the cells and 100 .mu.L of 2% Nonidet P-40 solution
(Nacalai Tesque) have been added, and C represents the mean value
of radioactivity (cpm) in wells where 100 .mu.L of the cells and
100 .mu.L of the medium have been added. The measurements were
conducted in duplicate, and the mean value and standard deviation
were calculated for the specific chromium release rate.
[0415] All of the anti-DSG3 antibodies used in the experiment were
confirmed to have CDC activity (FIG. 5). On the other hand, the
control mouse IgG2a antibody did not show CDC activity at the same
concentration.
[0416] Next, human epidermoid carcinoma cell line A431 (purchased
from ATCC), human lung epithelial cancer cell line A549 (purchased
from ATCC), and an A549 cell line showing constant expression of
full-length human DSG3 (DSG3-A549, described in Example 3-2)) were
used as target cells to examine whether the antibodies have CDC
activity. A431 and DSG3-A549 express DSG3 on the cell membrane.
Dulbecco's Modified Eagle Medium (Invitrogen) (hereinafter referred
to as DMEM medium) containing 10% fetal bovine serum (Invitrogen)
and penicillin/streptomycin was used to culture A431 and A549. DMEM
medium containing 1 mg/mL Geneticin was used to culture the
DSG3-A549 cell line. A431, A549, and DSG3-A549 cells were
individually added to a 96-well flat-bottomed plate at
2.times.10.sup.3 cells/well (A549 and DSG3-A549) or
4.times.10.sup.3 cells/well (A431), and cultured in a 5% carbon
dioxide incubator for three days at 37.degree. C. After culturing,
Chromium-51 was added at a final concentration of 1.85 MBq/mL, and
culturing was continued for another hour. Each well was washed with
300 .mu.L of DMEM medium, and then 1004 of DMEM medium was added.
Next, specific chromium release rates were determined by adding an
anti-DSG3 antibody and baby rabbit complement under conditions
similar to those used for the examination using DSG3-CHO cell
line.
[0417] Anti-DSG3 antibody DF151 induced concentration-dependent CDC
activity against DSG3-expressing A431 and DSG3-A549 cell lines, but
did not show CDC activity against A549 cell line which does not
express DSG3 (FIG. 6). These results showed that the anti-DSG3
antibody exhibits antigen-specific CDC activity.
4-2) Measurement of Antibody-Dependent Cellular Cytotoxicity (ADCC)
Activity of Anti-DSG3 Antibodies
[0418] DSG3-A549 cell line and A431 cell line were used for ADCC
activity measurements. Similar to the case of CDC activity
measurements, the above-mentioned cells were cultured in a 96-well
flat-bottomed plate and then reacted with Chromium-51. Thereafter,
each well was washed with RPMI1640 medium (Invitrogen) containing
10% fetal bovine serum and penicillin/streptomycin (hereinafter
referred to as RPMI medium), and then 100 .mu.L of RPMI medium was
added. Next, 50 .mu.L each of an anti-DSG3 antibody and the control
mouse IgG2a antibody diluted in RPMI medium was added to each well.
The final concentration of the antibody was adjusted to 10 .mu.g/mL
(bone marrow-derived effector cells) or 1 .mu.g/mL (spleen-derived
effector cells). Next, 50 .mu.L of an effector cell solution
(1.times.10.sup.7 cells/mL), which will be described later, was
added to each well, and then the plate was left to stand in a 5%
carbon dioxide incubator for four hours at 37.degree. C. Specific
chromium release rate was determined from the measured
radioactivity of each well in this plate. Cells obtained by
culturing the spleen cells of a Balb/c mouse (Charles River Japan)
in RPMI medium containing 50 ng/mL recombinant human interleukin-2
(Peprotech) for five days or cells obtained by culturing the bone
marrow cells of the same mouse in RPMI medium containing 50 ng/mL
of recombinant human interleukin-2 and 10 ng/mL of recombinant
mouse GM-CSF (Peprotech) for six days were used as effector
cells.
[0419] Anti-DSG3 antibodies DF151, DF364, and DF366 induced ADCC
against DSG3-A549 and A431 cell lines (FIG. 7). The above-mentioned
results showed that anti-DSG3 antibodies induce cell damage in DSG3
protein-expressing cells through ADCC activity.
Example 5
Determination of the Anti-DSG3 Antibody Variable-Region Gene
Sequences
[0420] Antibody variable region genes were cloned from hybridomas
that produce monoclonal antibodies DF151, DF364, and DF366, which
are antibodies showing ADCC activity and CDC activity in
DSG3-expressing cells, and their sequences were determined.
Antibody genes encoding monoclonal antibodies DF151, DF364, and
DF366 were amplified by the RT-PCR method using total RNAs
extracted from the anti-DSG3 antibody-producing hybridomas. Total
RNA was extracted from 1.times.10.sup.7 hybridoma cells using the
RNeasy Plant Mini Kit (QIAGEN). Using 1 .mu.g of total RNA, the
5'-end gene fragment was amplified by the SMART RACE cDNA
Amplification Kit (CLONTECH), using synthetic oligonucleotide
MHC-IgG2b (SEQ ID NO: 43) complementary to the mouse IgG2b constant
region sequence, synthetic oligonucleotide MHC-IgG1 (SEQ ID NO:
100) complementary to the mouse IgG1 constant region sequence, or
synthetic oligonucleotide kappa (SEQ ID NO: 44) complementary to
the mouse .kappa. chain constant region nucleotide sequence. The
reverse transcription reaction was performed for one hour and
thirty minutes at 42.degree. C. The PCR reaction was performed in
50 .mu.L of PCR reaction solution containing 5 .mu.L of 10.times.
Advantage 2 PCR Buffer, 5 .mu.L of 10.times. Universal Primer A
Mix, 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP), 1 .mu.L of
Advantage 2 Polymerase Mix (the above were manufactured by
CLONTECH), 2.5 .mu.L of reverse transcription reaction product, and
10 pmol of synthetic oligonucleotide MHC-IgG2b, MHC-IgG1, or kappa.
The PCR reaction was performed under the reaction conditions of
reaction at an initial temperature of 94.degree. C. for 30 seconds,
followed by five cycles of a reaction cycle consisting of reactions
at 94.degree. C. for 5 seconds and 72.degree. C. for three minutes,
five cycles of a reaction cycle consisting of reactions at
94.degree. C. for 5 seconds, 70.degree. C. for 10 seconds, and
72.degree. C. for three minutes, and 25 cycles of a reaction cycle
consisting of reactions at 94.degree. C. for five seconds,
68.degree. C. for ten seconds, and 72.degree. C. for three minutes.
Finally, the reaction product was heated at 72.degree. C. for seven
minutes. Each PCR product was purified from agarose gel using the
QIAquick Gel Extraction Kit (manufactured by QIAGEN), then cloned
into pGEM-T Easy vector (manufactured by Promega), and the
nucleotide sequence of the clone was determined.
[0421] For the H chain of DF151, the nucleotide sequence and amino
acid sequence of CDR1 are shown in SEQ ID NO: 1 and SEQ ID NO: 2,
respectively, the nucleotide sequence and amino acid sequence of
CDR2 are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and
the nucleotide sequence and amino acid sequence of CDR3 are shown
in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. For the L chain of
DF151, the nucleotide sequence and amino acid sequence of CDR1 are
shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, the
nucleotide sequence and amino acid sequence of CDR2 are shown in
SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the nucleotide
sequence and amino acid sequence of CDR3 are shown in SEQ ID NO: 15
and SEQ ID NO: 16, respectively.
[0422] For the H chain of DF364, the nucleotide sequence and amino
acid sequence of CDR1 are shown in SEQ ID NO: 21 and SEQ ID NO: 22,
respectively, the nucleotide sequence and amino acid sequence of
CDR2 are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively,
and the nucleotide sequence and amino acid sequence of CDR3 are
shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively. For the L
chain of DF364, the nucleotide sequence and amino acid sequence of
CDR1 are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively,
the nucleotide sequence and amino acid sequence of CDR2 are shown
in SEQ ID NO: 31 and SEQ ID NO: 32, respectively, and the
nucleotide sequence and amino acid sequence of CDR3 are shown in
SEQ ID NO: 33 and SEQ ID NO: 34, respectively.
[0423] For the H chain of DF366, the nucleotide sequence and amino
acid sequence of CDR1 are shown in SEQ ID NO: 80 and SEQ ID NO: 81,
respectively, the nucleotide sequence and amino acid sequence of
CDR2 are shown in SEQ ID NO: 82 and SEQ ID NO: 83, respectively,
and the nucleotide sequence and amino acid sequence of CDR3 are
shown in SEQ ID NO: 84 and SEQ ID NO: 85, respectively. For the L
chain of DF366, the nucleotide sequence and amino acid sequence of
CDR1 are shown in SEQ ID NO: 86 and SEQ ID NO: 87, respectively,
the nucleotide sequence and amino acid sequence of CDR2 are shown
in SEQ ID NO: 88 and SEQ ID NO: 89, respectively, and the
nucleotide sequence and amino acid sequence of CDR3 are shown in
SEQ ID NO: 90 and SEQ ID NO: 91, respectively.
[0424] For DF151, the nucleotide sequence and the amino acid
sequence of the H-chain variable region are shown in SEQ ID NO: 45
and SEQ ID NO: 46, respectively, and the nucleotide sequence and
the amino acid sequence of the L-chain variable region are shown in
SEQ ID NO: 47 and SEQ ID NO: 48, respectively. For DF364, the
nucleotide sequence and the amino acid sequence of the H-chain
variable region are shown in SEQ ID NO: 49 and SEQ ID NO: 50,
respectively, and the nucleotide sequence and the amino acid
sequence of the L-chain variable region are shown in SEQ ID NO: 51
and SEQ ID NO: 52, respectively. For DF366, the nucleotide sequence
and the amino acid sequence of the H-chain variable region are
shown in SEQ ID NO: 92 and SEQ ID NO: 93, respectively, and the
nucleotide sequence and the amino acid sequence of the L-chain
variable region are shown in SEQ ID NO: 94 and SEQ ID NO: 95,
respectively.
Example 6
Determination of Full-Length Gene Sequences of Anti-DSG3
Antibodies
[0425] When the variable region gene sequences of DF151, DF364, and
DF366 were determined, the gene sequences of the constant regions
adjacent to the variable regions were also determined. By searching
for genes that have the same sequences as these sequences using the
Basic Local Alignment Search Tool (BLAST) of the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/),
the nucleotide sequences of the entire constant regions can be
obtained. The full-length nucleotide sequence can be determined by
linking the obtained nucleotide sequence of the constant region to
the variable region nucleotide sequence. In this manner, the mouse
IgG2b nucleotide sequence (DDBJ Accession No. BC025447), mouse
kappa light chain nucleotide sequence (DDBJ Accession No.
AY704179), and mouse IgG1 nucleotide sequence (DDBJ Accession No.
BCO57688) can be obtained from the nucleotide sequence of the
H-chain constant region of DF151 (SEQ ID NO: 53), the nucleotide
sequence of the L-chain constant region of DF151, DF364, and DF366
(SEQ ID NO: 54), and the nucleotide sequence of the H-chain
constant region of DF364 and DF366 (SEQ ID NO: 55),
respectively.
[0426] The isotypes of DF151 (mouse IgG2b.kappa.), DF364 (mouse
IgG1.kappa.), and DF366 (mouse IgG1.kappa.) were determined in
advance using the IsoStrip Mouse Monoclonal Antibody Isotyping Kit
(ROCHE). The predicted nucleotide sequence and amino acid sequence
of the full-length DF151H chain are shown in SEQ ID NO: 56 and SEQ
ID NO: 57, respectively, and the predicted nucleotide sequence and
amino acid sequence of the full-length DF151 L chain are shown in
SEQ ID NO: 58 and SEQ ID NO: 59, respectively. The predicted
nucleotide sequence and amino acid sequence of the full-length
DF364H chain are shown in SEQ ID NO: 60 and SEQ ID NO: 61,
respectively, and the predicted nucleotide sequence and amino acid
sequence of the full-length DF364 L chain are shown in SEQ ID NO:
62 and SEQ ID NO: 63, respectively. The predicted nucleotide
sequence and amino acid sequence of the full-length DF366H chain
are shown in SEQ ID NO: 101 and SEQ ID NO: 102, respectively, and
the predicted nucleotide sequence and amino acid sequence of the
full-length DF366 L chain are shown in SEQ ID NO: 103 and SEQ ID
NO: 104, respectively. For DF151, the nucleotide sequence and the
amino acid sequence of the H-chain constant region are shown in SEQ
ID NO: 7 and SEQ ID NO: 8, respectively, and the nucleotide
sequence and the amino acid sequence of the L-chain constant region
are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. For
DF364 and DF366, the nucleotide sequence and the amino acid
sequence of the H-chain constant region are shown in SEQ ID NO: 27
and SEQ ID NO: 28, respectively, and the nucleotide sequence and
the amino acid sequence of the L-chain constant region are shown in
SEQ ID NO: 35 and SEQ ID NO: 36, respectively.
Example 7
Production of Anti-DSG3 Mouse-Human Chimeric Antibodies
[0427] The H-chain and L-chain variable region sequences of each
antibody were ligated in frame with human H-chain and L-chain
constant region sequences. PCR was performed using a synthetic
oligonucleotide having a sequence complementary to a Kozak sequence
and an EcoRI site at the 5' end of a nucleotide sequence encoding
the H-chain variable region, and a synthetic oligonucleotide
complementary to the 3' end nucleotide sequence which has a NheI
site inserted. PCR was performed using a synthetic oligonucleotide
having a sequence complementary to a Kozak sequence and a BamHI
site at the 5' end of a nucleotide sequence encoding the L-chain
variable region, and a synthetic oligonucleotide complementary to
the 3' end nucleotide sequence which has a BsiWI site inserted. The
obtained PCR products were cloned into antibody expression plasmid
pMCDN_G1k. pMCDN_G1k has the human IgG1 constant region (the
nucleotide sequence is shown in SEQ ID NO: 9 and the amino acid
sequence is shown in SEQ ID NO: 10) cloned into the pMCDN vector,
and has a structure in which the mouse H-chain variable region and
the human H-chain (.kappa. chain) constant region are linked by a
NheI site. Furthermore, another expression unit comprising a mouse
CMV promoter, and a human .kappa. constant region (the nucleotide
sequence is shown in SEQ ID NO: 19, and the amino acid sequence is
shown in SEQ ID NO: 20) are inserted, and it has a structure in
which the mouse L-chain variable region and human L chain (.kappa.
chain) constant region are linked by a BsiWI site. This plasmid
expresses the neomycin resistance gene, DHFR gene, and anti-DSG3
mouse-human chimeric antibody gene in animal cells.
[0428] pMCDN_G1k_DF151, pMCDN_G1k_DF364, and pMCDN_G1k_DF366
prepared as described above were introduced into DG44 cells by
electroporation. Geneticin selection (500 .mu.g/mL) established CHO
cells that show constant expression of DF151 mouse-human chimeric
antibody (hereinafter referred to as DF151c), DF364 mouse-human
chimeric antibody (hereinafter referred to as DF364c), and DF366
mouse-human chimeric antibody (hereinafter referred to as DF366c).
Next, the anti-DSG3 mouse-human chimeric antibodies were purified
from the culture supernatants of the CHO cells using a Hi Trap
rProtein A column (GE Healthcare Bio-Sciences). The purified
antibodies were subjected to buffer replacement with PBS buffer
using PD-10 columns (GE Healthcare Bio-Sciences), quantified by DC
Protein Assay, and then stored at 4.degree. C. The purified
anti-DSG3 mouse-human chimeric antibodies were subjected to flow
cytometric analysis to confirm that they bind specifically to DSG3
in the same way as the mouse antibodies. The nucleotide sequence
and amino acid sequence of the full-length DF151c H chain are shown
in SEQ ID NO: 64 and SEQ ID NO: 65, respectively, and the
nucleotide sequence and amino acid sequence of the full-length
DF151c L chain are shown in SEQ ID NO: 66 and SEQ ID NO: 67,
respectively. The nucleotide sequence and amino acid sequence of
the full-length DF364c H chain are shown in SEQ ID NO: 68 and SEQ
ID NO: 69, respectively, and the nucleotide sequence and amino acid
sequence of the full-length DF364c L chain are shown in SEQ ID NO:
70 and SEQ ID NO: 71, respectively. The nucleotide sequence and
amino acid sequence of the full-length DF366c H chain are shown in
SEQ ID NO: 96 and SEQ ID NO: 97, respectively, and the nucleotide
sequence and amino acid sequence of the full-length DF366c L chain
are shown in SEQ ID NO: 98 and SEQ ID NO: 99, respectively.
Example 8
Production of Low-Fucose Anti-DSG3 Mouse-Human Chimeric
Antibodies
[0429] The method of modifying the sugar chain of an antibody is a
known method for enhancing the ADCC activity of an antibody. For
example, improvement of ADCC activity by modified antibody
glycosylation is described in WO 99/54342. Furthermore, WO 00/61739
describes the adjustment of ADCC activity by the presence or
absence of fucose on an antibody sugar chain. WO 02/31140 describes
the use of a YB2/0 cell line to prepare an antibody comprising a
sugar chain that does not have .alpha.-1,6-core fucose. Whether the
ADCC improvement techniques described above enhance the activity of
the anti-DSG3 antibodies was examined. First, as host cells, the
YB2/0 cell line (purchased from ATCC) was cultured in RPMI1640
medium containing 10% FBS. An anti-DSG3 mouse-human chimeric
antibody expression vector prepared in Example 7 was introduced
into the YB2/0 cell line by the electroporation method under
conditions of 1.4 kV and 25 .mu.F. By Geneticin selection (500
.mu.g/mL), YB2/0 cell lines that show constant expression of
low-fucose DF151 mouse-human chimeric antibody (hereinafter
referred to as YB-DF151c), low-fucose DF364 mouse-human chimeric
antibody (hereinafter referred to as YB-DF364c), and low-fucose
DF366 mouse-human chimeric antibody (hereinafter referred to as
YB-DF366c) were established. Next, the low-fucose anti-DSG3
mouse-human chimeric antibodies were purified from the culture
supernatant using a Hi Trap rProtein A column. Purified antibodies
were subjected to buffer exchange with PBS buffer using a PD-10
column, quantified by DC Protein Assay, and then stored at
4.degree. C. The purified low-fucose anti-DSG3 mouse-human chimeric
antibodies were subjected to flow cytometric analysis to confirm
that they bind specifically to DSG3 in the same way as the
anti-DSG3 mouse-human chimeric antibodies.
Example 9
Measurement of CDC Activity and ADCC Activity of Anti-DSG3
Mouse-Human Chimeric Antibodies and Low-Fucose Anti-DSG3
Mouse-Human Chimeric Antibodies
[0430] 9-1) Establishment of Cell Lines that Show Constant
Expression of Full-Length Human DSG3
[0431] The full-length human DSG3 cDNA was cloned into a vector
(pMCDN) for expression in mammalian cells (pMCDN/hDSG3). The pMCDN
vector, into which a neomycin resistance gene and a DHFR gene are
incorporated, enables induced expression under the control of a
mouse CMV promoter (ACCESSION No. U68299). A Ba/F3 cell line
(DSG3-Ba/F3) that shows constant expression of full-length human
DSG3 was established by introducing pMCDN/hDSG3 into Ba/F3 cells
(purchased from RIKEN BioResource Center) by electroporation, and
subjecting them to selection with 500 .mu.g/mL of Geneticin
(Invitrogen). DSG3-Ba/F3 cells were cultured using RPMI1640 medium
(Invitrogen) containing 500 .mu.g/mL Geneticin,
penicillin/streptomycin (Invitrogen), recombinant mouse
interleukin-3 (R&D Systems), and 10% fetal bovine serum
(Invitrogen).
9-2) Establishment of Cells Showing Constant Expression of
Full-Length Human CD16
[0432] Full-length human CD16 (RefSeq ID, NM.sub.--000569) was
cloned into pMCDN, then introduced into NK-92 cells (purchased from
ATCC) by electroporation and then subjected to Geneticin selection
(500 .mu.g/mL) to establish a NK-92 cell line (CD16-NK92) that
shows constant expression of full-length human CD16. The CD16-NK92
cell line was cultured using Minimum Essential Medium Alpha Medium
with L-glutamine, without ribonucleosides, deoxyribonucleosides
(Invitrogen) containing 500 .mu.g/mL Geneticin,
penicillin/streptomycin, 0.2 mM inositol (Sigma), 0.1 mM
2-mercaptoethanol (Invitrogen), 0.02 mM folic acid (Sigma), 100
U/mL recombinant human interleukin-2 (Peprotech), 12.5% horse serum
(Invitrogen), and 12.5% fetal bovine serum.
9-3) Measurement of CDC Activity of Anti-DSG3 Mouse-Human Chimeric
Antibodies
[0433] A suspension of 5.times.10.sup.5 DSG3-Ba/F3 cells was
centrifuged (1000 rpm for five minutes at 4.degree. C.), the
resulting cell pellet was suspended in approximately 200 .mu.L of
RPMI1640 medium containing 10% fetal bovine serum,
penicillin/streptomycin, and 3.7 MBq of Chromium-51 (GE Healthcare
Bio-Sciences) (hereinafter referred to as medium), and the cells
were cultured in a 5% carbon dioxide incubator for one hour at
37.degree. C. These cells were washed three times in the medium,
then adjusted to 2.times.10.sup.5 cells/mL, and then added to a
96-well round-bottomed plate at 50 .mu.L/well. Next, DF151c,
DF364c, DF366c, and a control human IgG antibody (Zymed) were
individually added at 50 pt/well. Final concentration of the
antibodies was adjusted to 10 .mu.g/mL. Next, baby rabbit
complement (Cederlane) diluted 5-fold in the medium was added at
100 .mu.L each. The plate was left to stand in a 5% carbon dioxide
incubator at 37.degree. C. for four hours. After the culturing, the
plate was centrifuged (1000 rpm for five minutes at 4.degree. C.),
and 100 .mu.L of the supernatant was used for the radioactivity
measurement on a gamma counter (1480 WIZARD 3'', Wallac). The
specific chromium release rate was determined according to the
following equation:
Specific chromium release rate (%)=(A-C).times.100/(B-C)
where A represents the radioactivity (cpm) in each well, B
represents the mean value of radioactivity (cpm) in wells where 50
.mu.L of the cells and 150 .mu.L of 2% Nonidet P-40 solution
(Nacalai Tesque) have been added, and C represents the mean value
of radioactivity (cpm) in wells where 50 .mu.L of the cells and 150
.mu.L of the medium have been added. The assay was conducted in
duplicate, and the mean value and standard deviation were
calculated for the specific chromium release rate. DF151c, DF364c,
and DF366c were shown to have CDC activity (FIG. 8).
9-4) Measurement of ADCC Activity of Anti-DSG3 Mouse-Human Chimeric
Antibodies and Low-Fucose Anti-DSG3 Mouse-Human Chimeric
Antibodies
[0434] DSG3-Ba/F3 cells were labeled with Chromium-51, and then
added to a 96-well round-bottomed plate at 50 .mu.L/well. Next,
DF364c, DF366c, YB-DF364c, YB-DF366c, and a control human IgG
antibody were individually added at 50 .mu.L/well. Final
concentrations of the antibodies were adjusted by four 10-fold
serial dilutions starting from 1 .mu.g/mL. Subsequently, CD16-NK92
cells at 2.times.10.sup.5 cells/mL were added at 100 .mu.L/well.
The plate was left to stand in a 5% carbon dioxide incubator at
37.degree. C. for four hours, and then the specific chromium
release rate was determined using the same method as that of
8-3).
[0435] All antibodies showed ADCC activity in an antibody
concentration-dependent manner (FIG. 9). In particular, low-fucose
antibodies YB-DF364c and YB-DF366c showed strong ADCC activity.
Example 10
Immunohistological Staining of DSG3 in Lung Cancer, Skin Cancer,
and Uterine Cancer
[0436] The DSG3 expression was enhanced at the protein level in
lung squamous cell carcinoma (see Example 2). Therefore,
immunohistological staining analyses were newly performed to
confirm the DSG3 protein expression in skin cancer, uterine cancer,
and lung adenocarcinoma which is a lung cancer that affects a large
number of people. First, 4% paraformaldehyde (PFA) or
periodate-lysine-paraformaldehyde (PLP)-fixed AMeX embedded
paraffin block and 10% neutral buffer formaldehyde (NBF)-fixed
paraffin-embedded block were prepared from each sample, and
3-.mu.m-thin sections were prepared. After deparaffinization, these
sections were stained immunohistochemically as described below
using the Ventana HX Discovery System (Ventana Medical Systems,
Inc., Arizona, USA). Each preparation was washed with water after
deparaffinization, and reacted with 3.0% hydrogen peroxide solution
(Inhibitor D) for four minutes at room temperature to eliminate
endogenous peroxidase. This was washed, and with addition of
protein blocker to eliminate non-specific reactions, this was
reacted for 30 minutes at room temperature. After washing, a mouse
anti-human Desmoglein 3 antibody (Clone 5G11, ZYMED Laboratories
Inc., California, USA) was added as a primary antibody, and then
reacted for one hour at room temperature. After washing, a
secondary antibody (Ventana Universal Secondary Antibody, Ventana
Medical Systems) was added and reacted for 30 minutes at room
temperature. After washing, reaction with Blocker D was carried out
for two minutes at room temperature to remove non-specific
reactions, and then streptavidin horseradish peroxidase (SA-HRP,
Ventana Medical Systems) was added and reacted at 37.degree. C. for
16 minutes. After washing, a mixture of diaminobenzidine (DAB map
solution, Ventana Medical Systems) and hydrogen peroxide solution
(DAB map solution, Ventana Medical Systems) was added and reacted
for eight minutes at 37.degree. C. for substrate color development.
Next, the color was intensified using a copper sulfate solution
(Ventana Medical Systems). After washing, this was subjected to
nuclear staining with hematoxylin, dehydration, penetration, and
inclusion.
[0437] As a result, the DSG3 expression was confirmed in two out of
three cases in lung squamous cell carcinoma, one out of nine cases
in lung adenocarcinoma, two out of two cases in skin squamous cell
carcinoma, one out of one case in skin basal cell carcinoma, and
one out of one case in uterine squamous cell carcinoma (Table
3).
TABLE-US-00004 TABLE 3 Lung.sup.a Skin Uterus SCC.sup.b
Adenocarcinoma SCC BCC SCC 2.sup.c 3 3-M 1 2 2 2-M 3 3 3 3-M 3-M 1
3 -- 2 Desmoglein-3 1.sup.d 2 3 4 5 6 7 8 9 10 11 12 14 15 16 17
Intensity 3-4.sup.e 2-4 0 0 0 0 0 2-4 0 0 0 0 2-4 1-4 1-4 2-4
Frequency 4.sup.f 4 -- -- -- -- -- 2 -- -- -- -- 4 3 3 4
Abbreviations: BCC, basal cell carcinoma; M, metastatic cancer;
SCC, squamous cell carcinoma .sup.atissue site of cancer .sup.bPL
tissue type .sup.cgrade of cancer (1, well-differentiated; 2,
moderately-differentiated; 3, poorly-differentiated) .sup.dcase
number .sup.e1, faint; 2, weak; 3, moderate; 4, strong .sup.f1,
rare (less than 10%); 2, occasional (10% and above, less than 50%);
3, frequent (50% and above, less than 90%); 4, constant (90% and
above)
Example 11
Evaluation of Antitumor Activity of Anti-DSG3 Antibodies
11-1) Production of Mouse IgG2a Chimeric DF366 Antibody
(DF366m)
[0438] The nucleotide sequence of the H-chain variable region gene
of DF366 antibody was ligated in frame to the nucleotide sequence
of H-chain constant region gene of mouse IgG2a. First, PCR was
performed using a primer (SEQ ID NO: 105) having the 5' end
nucleotide sequence of the H-chain variable region gene, a Kozak
sequence, and an EcoRI restriction enzyme sequence, and an
antisense primer (SEQ ID NO: 106) having a c residue attached to a
sequence complementary to the 3'-end nucleotide sequence. The
obtained amplified product was treated with the EcoRI restriction
enzyme, and then incorporated into the EcoRI-NruI site of a mouse
IgG2a chimeric H-chain expression plasmid (pMCD/G2a) to construct a
mouse IgG2a chimeric DF366 antibody H-chain expression vector
(pMCD/G2a-DF366). pMCD/G2a has the H-chain constant region gene of
mouse IgG2a (nucleotide sequence: SEQ ID NO: 107; amino acid
sequence: SEQ ID NO: 108) cloned into a pMCD plasmid for expression
in mammalian cells, and the NruI restriction enzyme sequence of the
H-chain constant region is ligated to the H-chain variable region.
The pMCD vector, into which a DHFR gene has been incorporated,
enables induced expression under the control of a mouse CMV
promoter (ACCESSION No. U68299).
[0439] The nucleotide sequence of the L-chain variable region gene
of DF366 antibody was ligated in frame to the nucleotide sequence
of the L-chain (.kappa. chain) constant region gene of mouse IgG2a.
First, PCR was performed using a primer (SEQ ID NO: 109) having the
5'-end nucleotide sequence of the L-chain variable region gene, a
Kozak sequence, and the EcoRI restriction enzyme sequence, and an
antisense primer (SEQ ID NO: 110) having gcccg residues attached to
a sequence complementary to the 3'-end nucleotide sequence. The
amplified product obtained was treated with EcoRI restriction
enzyme, and then incorporated into the EcoRI-NruI site of a mouse
IgG2a chimeric L-chain (.kappa. chain) expression plasmid (pMCN/k)
to construct a mouse IgG2a chimeric DF366 antibody L-chain
expression vector (pMCN/k-DF366). pMCN/k has the L-chain (.kappa.
chain) constant region gene of mouse IgG2a (nucleotide sequence:
SEQ ID NO: 111; amino acid sequence: SEQ ID NO: 112) cloned into
the plasmid pMCN, and the NruI restriction enzyme sequence of the
L-chain (.kappa. chain) constant region is ligated to the L-chain
variable region.
[0440] The plasmid pMCD/G2a-DF366 and the plasmid pMCN/k-DF366 were
introduced into DG44 cells by electroporation. CHO cells
(DF366m-DG44) showing constant expression of the mouse IgG2a
chimeric DF366 antibody (DF366m) were established by Geneticin
selection (500 .mu.g/mL) and nucleic acid (HT supplement)-free
medium. Subsequently, the DF366m antibody was purified from the
culture supernatant of DF366m-DG44 using a Hi Trap Protein G HP
column. The solvent was substituted with PBS using a PD-10 column.
The concentration of the purified DF366m antibody was quantified
using a DC Protein Assay kit. The DF366m antibody was subjected to
flow cytometric analysis to confirm that it specifically binds to
DSG3 in the same way as the DF366 antibody (described in Example
3-5). The nucleotide sequence of the full-length DF366m antibody
H-chain gene and the corresponding amino acid sequence are shown in
SEQ ID NO: 113 and SEQ ID NO: 114, respectively, and the nucleotide
sequence of the full-length DF366m antibody L-chain gene and the
corresponding amino acid sequence are shown in SEQ ID NO: 115 and
SEQ ID NO: 116, respectively.
11-2) Production of Low-Fucose Mouse IgG2a Chimeric DF366 Antibody
(Low Fucose DF366m)
[0441] The plasmid pMCD/G2a-DF366 and the plasmid pMCN/k-DF366 were
introduced into fucose transporter knockout CHO cells (FTPKO-DXB11
cells, International Patent Publication Nos. WO 2006/067913 and WO
2006/067847) by electroporation. CHO cells (DF366m-DXB11) showing
constant expression of the low-fucose mouse IgG2a chimeric DF366
antibody (low fucose DF366m) were established by Geneticin
selection (500 .mu.g/mL) and nucleic acid (HT supplement)-free
medium. Subsequently, the low-fucose DF366m antibody was purified
from the culture supernatant of DF366m-DXB11 using a Hi Trap
Protein G HP column. The solvent was substituted with PBS using a
PD-10 column, and the antibody concentration was quantified using a
DC Protein Assay kit.
11-3) Measurement of ADCC Activity
[0442] RPMI1640 medium (Invitrogen) containing
penicillin/streptomycin and 10% fetal bovine serum (hereinafter
referred to as RPMI medium) was used for the experiment.
1.times.10.sup.6 DSG3-Ba/F3 cells were suspended in approximately
2004 of RPMI medium containing 3.7 MBq of Chromium-51 (GE
Healthcare Bio-Sciences), and then cultured in a 5% carbon dioxide
incubator for one hour at 37.degree. C. After washing, the cell
density was adjusted to 2.times.10.sup.5 cells/mL, and then
dispensed into a 96-well U-bottom plate at 50 .mu.L/well. Next, the
antibody solution was added at 50 .mu.L/well. After incubating at
room temperature for 15 minutes, effector cells (described later)
were added at 100 .mu.L each. The plate was left to stand in a 5%
carbon dioxide incubator at 37.degree. C. for six hours.
Thereafter, 100 .mu.L of the supernatant was collected from each
well, and the radioactivity was measured with a gamma counter (1480
WIZARD 3'', Wallac). The specific chromium release rate was
calculated according to the following equation:
Specific chromium release rate (%)=(A-C).times.100/(B-C)
where A represents the radioactivity (cpm) in each well, B
represents the mean value of radioactivity (cpm) in wells where 50
.mu.L of the cells and 150 .mu.L of 2% Nonidet P-40 solution
(Nacalai Tesque) have been added, and C represents the mean value
of radioactivity (cpm) in wells where 50 .mu.L of the cells and 150
.mu.L of RPMI medium have been added. The measurements were
conducted in duplicate, and the mean value and standard deviation
were calculated for the specific chromium release rate.
[0443] Cells obtained by adding 50 ng/mL recombinant human
interleukin-2 (Peprotech) to spleen cells prepared from a C3H mouse
(Charles River Japan) (hereinafter referred to as SPL) or cells
obtained by culturing spleen cells for four days in the presence of
50 ng/mL of recombinant human interleukin-2 (hereinafter referred
to as SPL-LAK) were used as effector cells. The number of effector
cells per well was set to 5.times.10.sup.5 cells (SPL) or
2.times.10.sup.5 cells (SPL-LAK). Mouse IgG2a (Cat. No. 553453,
Becton Dickinson) and human IgG1 (Cat. No. PHP010, Serotec) were
used as negative controls.
[0444] ADCC activity was detected in DF366m and low-fucose DF366m,
but ADCC activity was hardly observed in DF366c and YB-DF366c
(FIGS. 10 and 11). Therefore, DF366m and low-fucose DF366m were
considered to show stronger medicinal effect than DF366c and
YB-DF366c in mice.
11-4) Establishment of Cell Line Showing Constant Expression of
Full-Length Human DSG3
[0445] After digesting pMCDN/hDSG3 with the PvuI restriction
enzyme, this was introduced into SK-HEP-1 cells (purchased from
ATCC) by transfection using FuGENE (Roche), and a SK-HEP-1 cell
line (hereinafter referred to as DSG3-SK) showing constant
expression of full-length human DSG3 was established by Geneticin
selection (1 mg/mL). D-MEM medium (Sigma) containing 1 mg/mL
Geneticin and 10% fetal bovine serum was used to culture the
DSG3-SK cells.
11-5) Evaluation of Antitumor Activity of DF366m and Low-Fucose
DF366m
[0446] DSG3-SK cells were adjusted to 1.times.10.sup.8 cells/mL in
a solution containing a 1:1 ratio of D-MEM medium and MATRIGEL
(Cat. No. 354234, BD Bioscience), and 100 .mu.L of this cell
solution was subcutaneously transplanted to the abdomen of a SCID
mouse (female, 9-weeks old, CLEA Japan) that had been subjected to
intraperitoneal administration of 100 .mu.L of anti-asialo GM1
antibody (Wako Pure Chemicals, after dissolving one vial using 1 mL
of distilled water for injection, 4 mL of physiological saline
solution was added) on the previous day. From the 19th day after
transplantation, DF366m and low-fucose DF366m were administered
through the tail vein once a week for four weeks. The antibodies
were prepared in PBS at 1 mg/mL (10 mg/kg administration group) or
0.2 mg/mL (2 mg/kg administration group), and administered at 10
mL/kg. PBS (vehicle) was administered similarly as a negative
control. The assay was carried out using five animals in each
group. Antitumor activity was evaluated based on tumor volume. The
tumor volume was determined based on the following equation, and
the mean value and standard deviation were calculated:
Tumor volume=major axis.times.minor axis.times.minor axis/2
[0447] Non-parametric Dunnett's multiple comparison was used for
the significant difference test, and P value less than 0.05 was
considered significant.
[0448] As a result of the examination, DF366m and low-fucose DF366m
significantly suppressed tumor growth in the 10 mg/kg
administration group as compared to the vehicle administration
group (FIG. 12). Furthermore, although not significant, low-fucose
DF366m indicated a suppressive tendency also at 2 mg/kg. From the
above, anti-DSG3 antibodies were confirmed to show antitumor
activity.
INDUSTRIAL APPLICABILITY
[0449] The DSG3 protein-specific antibodies of the present
invention can be used as a diagnostic agent not only for lung
cancer but also for colon cancer, esophageal cancer, stomach
cancer, pancreatic cancer, skin cancer, and uterine cancer.
Furthermore, by using the anti-DSG3 antibodies after labeling them
with a chemical substance or a radioisotope, the presence of lung
cancer, colon cancer, esophageal cancer, stomach cancer, pancreatic
cancer, skin cancer, or uterine cancer can be detected in vivo.
[0450] Furthermore, anti-DSG3 antibodies having cytotoxic activity
according to the present invention can be used as cytotoxic agents
or cell growth inhibitors for various types of cancer cells that
express a DSG3 protein, such as cells of lung cancer, colon cancer,
esophageal cancer, stomach cancer, pancreatic cancer, skin cancer,
or uterine cancer.
[0451] Furthermore, anti-DSG3 antibodies having cytotoxic activity
according to the present invention can be used as therapeutic
agents against various types of cancers such as lung cancer, colon
cancer, esophageal cancer, stomach cancer, pancreatic cancer, skin
cancer, or uterine cancer. In addition, anti-DSG3 antibodies of the
present invention can be used as therapeutic agents for these
cancers without inducing pemphigus conditions.
[0452] Additionally, genes encoding antibodies of the present
invention and recombinant cells transformed by these genes can be
used to produce recombinant antibodies that exhibit the
above-mentioned effects and more preferred effects.
Sequence CWU 1
1
116115DNAMus musculus 1gcctactaca tgcac 1525PRTMus musculus 2Ala
Tyr Tyr Met His1 5351DNAMus musculus 3cagattaatc ctagcactgg
tggtactacc tacaaccaga agttcaaggc c 51417PRTMus musculus 4Gln Ile
Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn Gln Lys Phe Lys1 5 10
15Ala512DNAMus musculus 5tggggtgact ct 1264PRTMus musculus 6Trp Gly
Asp Ser171011DNAMus musculus 7gccaaaacaa cacccccatc agtctatcca
ctggcccctg ggtgtggaga tacaactggt 60tcctctgtga ctctgggatg cctggtcaag
ggctacttcc ctgagtcagt gactgtgact 120tggaactctg gatccctgtc
cagcagtgtg cacaccttcc cagctctcct gcagtctgga 180ctctacacta
tgagcagctc agtgactgtc ccctccagca cctggccaag tcagaccgtc
240acctgcagcg ttgctcaccc agccagcagc accacggtgg acaaaaaact
tgagcccagc 300gggcccattt caacaatcaa cccctgtcct ccatgcaagg
agtgtcacaa atgcccagct 360cctaacctcg agggtggacc atccgtcttc
atcttccctc caaatatcaa ggatgtactc 420atgatctccc tgacacccaa
ggtcacgtgt gtggtggtgg atgtgagcga ggatgaccca 480gacgtccaga
tcagctggtt tgtgaacaac gtggaagtac acacagctca gacacaaacc
540catagagagg attacaacag tactatccgg gtggtcagtg ccctccccat
ccagcaccag 600gactggatga gtggcaagga gttcaaatgc aaggtcaaca
acaaagacct cccatcaccc 660atcgagagaa ccatctcaaa aattaaaggg
ctagtcagag ctccacaagt atacatcttg 720ccgccaccag cagagcagtt
gtccaggaaa gatgtcagtc tcacttgcct ggtcgtgggc 780ttcaaccctg
gagacatcag tgtggagtgg accagcaatg ggcatacaga ggagaactac
840aaggacaccg caccagtcct ggactctgac ggttcttact tcatatacag
caagctcgat 900ataaaaacaa gcaagtggga gaaaacagat tccttctcat
gcaacgtgag acacgagggt 960ctgaaaaatt actacctgaa gaagaccatc
tcccggtctc cgggtaaatg a 10118336PRTMus musculus 8Ala Lys Thr Thr
Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly1 5 10 15Asp Thr Thr
Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30Phe Pro
Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45Ser
Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met 50 55
60Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val65
70 75 80Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys
Lys 85 90 95Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro
Pro Cys 100 105 110Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu
Gly Gly Pro Ser 115 120 125Val Phe Ile Phe Pro Pro Asn Ile Lys Asp
Val Leu Met Ile Ser Leu 130 135 140Thr Pro Lys Val Thr Cys Val Val
Val Asp Val Ser Glu Asp Asp Pro145 150 155 160Asp Val Gln Ile Ser
Trp Phe Val Asn Asn Val Glu Val His Thr Ala 165 170 175Gln Thr Gln
Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val 180 185 190Ser
Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 195 200
205Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr
210 215 220Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr
Ile Leu225 230 235 240Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp
Val Ser Leu Thr Cys 245 250 255Leu Val Val Gly Phe Asn Pro Gly Asp
Ile Ser Val Glu Trp Thr Ser 260 265 270Asn Gly His Thr Glu Glu Asn
Tyr Lys Asp Thr Ala Pro Val Leu Asp 275 280 285Ser Asp Gly Ser Tyr
Phe Ile Tyr Ser Lys Leu Asp Ile Lys Thr Ser 290 295 300Lys Trp Glu
Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly305 310 315
320Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys
325 330 33591004DNAHomo sapiens 9gctagcacca agggcccatc ggtcttcccc
ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
240tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 300aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 360ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 420gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggatgag 720ctgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
840ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc tccgggtaaa
tgataagcgg ccgc 100410330PRTHomo sapiens 10Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 3301145DNAMus
musculus 11agagccagtg aaagtgttga atattatggc actagtttaa tgcag
451215PRTMus musculus 12Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr
Ser Leu Met Gln1 5 10 151321DNAMus musculus 13ggtgcatccg acgtagaatc
t 21147PRTMus musculus 14Gly Ala Ser Asp Val Glu Ser1 51527DNAMus
musculus 15cagcaaagta ggaaggttcc gtatacg 27169PRTMus musculus 16Gln
Gln Ser Arg Lys Val Pro Tyr Thr1 517324DNAMus musculus 17cgggctgatg
ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60ggaggtgcct
cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag
120tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac
tgatcaggac 180agcaaagaca gcacctacag catgagcagc accctcacgt
tgaccaagga cgagtatgaa 240cgacataaca gctatacctg tgaggccact
cacaagacat caacttcacc cattgtcaag 300agcttcaaca ggaatgagtg ttag
32418107PRTMus musculus 18Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Ser Glu1 5 10 15Gln Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe 20 25 30Tyr Pro Lys Asp Ile Asn Val Lys
Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu65 70 75 80Arg His Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 100 10519335DNAHomo sapiens
19cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct
60ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag
120tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac
agagcaggac 180agcaaggaca gcacctacag cctcagcagc accctgacgc
tgagcaaagc agactacgag 240aaacacaaag tctacgcctg cgaagtcacc
catcagggcc tgagctcgcc cgtcacaaag 300agcttcaaca ggggagagtg
ttgataagcg gccgc 33520107PRTHomo sapiens 20Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75
80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100
1052115DNAMus musculus 21agctactgga ttcac 15225PRTMus musculus
22Ser Tyr Trp Ile His1 52351DNAMus musculus 23tctatttatc ctggaaatag
tgatactacc tacaaccaga agttcaaggg c 512417PRTMus musculus 24Ser Ile
Tyr Pro Gly Asn Ser Asp Thr Thr Tyr Asn Gln Lys Phe Lys1 5 10
15Gly2542DNAMus musculus 25cctacttact atagttacga cgattactat
gctatggact at 422614PRTMus musculus 26Pro Thr Tyr Tyr Ser Tyr Asp
Asp Tyr Tyr Ala Met Asp Tyr1 5 1027975DNAMus musculus 27gccaaaacga
cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60tccatggtga
ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc
120tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct
gcagtctgac 180ctctacactc tgagcagctc agtgactgtc ccctccagca
cctggcccag ccagaccgtc 240acctgcaacg ttgcccaccc ggccagcagc
accaaggtgg acaagaaaat tgtgcccagg 300gattgtggtt gtaagccttg
catatgtaca gtcccagaag tatcatctgt cttcatcttc 360cccccaaagc
ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg
420gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga
tgatgtggag 480gtgcacacag ctcagacaaa accccgggag gagcagttca
acagcacttt ccgttcagtc 540agtgaacttc ccatcatgca ccaggactgg
ctcaatggca aggagttcaa atgcagggtc 600aacagtgcag ctttccctgc
ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660aaggctccac
aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc
720agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga
gtggcagtgg 780aatgggcagc cagcggagaa ctacaagaac actcagccca
tcatggacac agatggctct 840tacttcgtct acagcaagct caatgtgcag
aagagcaact gggaggcagg aaatactttc 900acctgctctg tgttacatga
gggcctgcac aaccaccata ctgagaagag cctctcccac 960tctcctggta aatga
97528324PRTMus musculus 28Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
Leu Ala Pro Gly Ser Ala1 5 10 15Ala Gln Thr Asn Ser Met Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Thr
Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60Ser Ser Ser Val Thr Val
Pro Ser Ser Thr Trp Pro Ser Gln Thr Val65 70 75 80Thr Cys Asn Val
Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95Ile Val Pro
Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110Glu
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120
125Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp
Val Glu145 150 155 160Val His Thr Ala Gln Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr 165 170 175Phe Arg Ser Val Ser Glu Leu Pro Ile
Met His Gln Asp Trp Leu Asn 180 185 190Gly Lys Glu Phe Lys Cys Arg
Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220Val Tyr Thr
Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val225 230 235
240Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn
Thr Gln 260 265 270Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr
Ser Lys Leu Asn 275 280 285Val Gln Lys Ser Asn Trp Glu Ala Gly Asn
Thr Phe Thr Cys Ser Val 290 295 300Leu His Glu Gly Leu His Asn His
His Thr Glu Lys Ser Leu Ser His305 310 315 320Ser Pro Gly
Lys2936DNAMus musculus 29agtgtcagct caagtataag ttccagcaac ttacac
363012PRTMus musculus 30Ser Val Ser Ser Ser Ile Ser Ser Ser Asn Leu
His1 5 103121DNAMus musculus 31ggcacatcca accttgcttc t 21327PRTMus
musculus 32Gly Thr Ser Asn Leu Ala Ser1 53327DNAMus musculus
33caacagtgga gtagttaccc gctcacg 27349PRTMus musculus 34Gln Gln Trp
Ser Ser Tyr Pro Leu Thr1 535324DNAMus musculus 35cgggctgatg
ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60ggaggtgcct
cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag
120tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac
tgatcaggac 180agcaaagaca gcacctacag catgagcagc accctcacgt
tgaccaagga cgagtatgaa 240cgacataaca gctatacctg tgaggccact
cacaagacat caacttcacc cattgtcaag 300agcttcaaca ggaatgagtg ttag
32436107PRTMus musculus 36Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Ser Glu1 5 10 15Gln Leu Thr Ser Gly Gly Ala Ser Val
Val Cys Phe Leu Asn Asn Phe 20 25 30Tyr Pro Lys Asp Ile Asn Val Lys
Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45Gln Asn Gly Val Leu Asn Ser
Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Met Ser Ser
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu65 70 75 80Arg His Asn Ser
Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 100 1053734DNAArtificial
SequenceSynthetic primer sequence 37taacccgggc caccatgatg
gggctcttcc ccag 343835DNAArtificial SequenceSynthetic primer
sequence 38ttagcggccg cttatcatat tagacgggag caagg 35393000DNAHomo
sapiens 39atgatggggc tcttccccag aactacaggg gctctggcca tcttcgtggt
ggtcatattg 60gttcatggag aattgcgaat agagactaaa ggtcaatatg atgaagaaga
gatgactatg 120caacaagcta aaagaaggca aaaacgtgaa tgggtgaaat
ttgccaaacc ctgcagagaa 180ggagaagata actcaaaaag aaacccaatt
gccaagatta cttcagatta ccaagcaacc 240cagaaaatca cctaccgaat
ctctggagtg ggaatcgatc agccgccttt tggaatcttt 300gttgttgaca
aaaacactgg agatattaac ataacagcta tagtcgaccg ggaggaaact
360ccaagcttcc tgatcacatg tcgggctcta aatgcccaag gactagatgt
agagaaacca 420cttatactaa cggttaaaat tttggatatt aatgataatc
ctccagtatt ttcacaacaa 480attttcatgg gtgaaattga agaaaatagt
gcctcaaact cactggtgat gatactaaat 540gccacagatg cagatgaacc
aaaccacttg aattctaaaa ttgccttcaa aattgtctct 600caggaaccag
caggcacacc catgttcctc ctaagcagaa
acactgggga agtccgtact 660ttgaccaatt ctcttgaccg agagcaagct
agcagctatc gtctggttgt gagtggtgca 720gacaaagatg gagaaggact
atcaactcaa tgtgaatgta atattaaagt gaaagatgtc 780aacgataact
tcccaatgtt tagagactct cagtattcag cacgtattga agaaaatatt
840ttaagttctg aattacttcg atttcaagta acagatttgg atgaagagta
cacagataat 900tggcttgcag tatatttctt tacctctggg aatgaaggaa
attggtttga aatacaaact 960gatcctagaa ctaatgaagg catcctgaaa
gtggtgaagg ctctagatta tgaacaacta 1020caaagcgtga aacttagtat
tgctgtcaaa aacaaagctg aatttcacca atcagttatc 1080tctcgatacc
gagttcagtc aaccccagtc acaattcagg taataaatgt aagagaagga
1140attgcattcc gtcctgcttc caagacattt actgtgcaaa aaggcataag
tagcaaaaaa 1200ttggtggatt atatcctggg aacatatcaa gccatcgatg
aggacactaa caaagctgcc 1260tcaaatgtca aatatgtcat gggacgtaac
gatggtggat acctaatgat tgattcaaaa 1320actgctgaaa tcaaatttgt
caaaaatatg aaccgagatt ctactttcat agttaacaaa 1380acaatcacag
ctgaggttct ggccatagat gaatacacgg gtaaaacttc tacaggcacg
1440gtatatgtta gagtacccga tttcaatgac aattgtccaa cagctgtcct
cgaaaaagat 1500gcagtttgca gttcttcacc ttccgtggtt gtctccgcta
gaacactgaa taatagatac 1560actggcccct atacatttgc actggaagat
caacctgtaa agttgcctgc cgtatggagt 1620atcacaaccc tcaatgctac
ctcggccctc ctcagagccc aggaacagat acctcctgga 1680gtataccaca
tctccctggt acttacagac agtcagaaca atcggtgtga gatgccacgc
1740agcttgacac tggaagtctg tcagtgtgac aacaggggca tctgtggaac
ttcttaccca 1800accacaagcc ctgggaccag gtatggcagg ccgcactcag
ggaggctggg gcctgccgcc 1860atcggcctgc tgctccttgg tctcctgctg
ctgctgttgg ccccccttct gctgttgacc 1920tgtgactgtg gggcaggttc
tactggggga gtgacaggtg gttttatccc agttcctgat 1980ggctcagaag
gaacaattca tcagtgggga attgaaggag cccatcctga agacaaggaa
2040atcacaaata tttgtgtgcc tcctgtaaca gccaatggag ccgatttcat
ggaaagttct 2100gaagtttgta caaatacgta tgccagaggc acagcggtgg
aaggcacttc aggaatggaa 2160atgaccacta agcttggagc agccactgaa
tctggaggtg ctgcaggctt tgcaacaggg 2220acagtgtcag gagctgcttc
aggattcgga gcagccactg gagttggcat ctgttcctca 2280gggcagtctg
gaaccatgag aacaaggcat tccactggag gaaccaataa ggactacgct
2340gatggggcga taagcatgaa ttttctggac tcctactttt ctcagaaagc
atttgcctgt 2400gcggaggaag acgatggcca ggaagcaaat gactgcttgt
tgatctatga taatgaaggc 2460gcagatgcca ctggttctcc tgtgggctcc
gtgggttgtt gcagttttat tgctgatgac 2520ctggatgaca gcttcttgga
ctcacttgga cccaaattta aaaaacttgc agagataagc 2580cttggtgttg
atggtgaagg caaagaagtt cagccaccct ctaaagacag cggttatggg
2640attgaatcct gtggccatcc catagaagtc cagcagacag gatttgttaa
gtgccagact 2700ttgtcaggaa gtcaaggagc ttctgctttg tccgcctctg
ggtctgtcca gccagctgtt 2760tccatccctg accctctgca gcatggtaac
tatttagtaa cggagactta ctcggcttct 2820ggttccctcg tgcaaccttc
cactgcaggc tttgatccac ttctcacaca aaatgtgata 2880gtgacagaaa
gggtgatctg tcccatttcc agtgttcctg gcaacctagc tggcccaacg
2940cagctacgag ggtcacatac tatgctctgt acagaggatc cttgctcccg
tctaatatga 300040999PRTHomo sapiens 40Met Met Gly Leu Phe Pro Arg
Thr Thr Gly Ala Leu Ala Ile Phe Val1 5 10 15Val Val Ile Leu Val His
Gly Glu Leu Arg Ile Glu Thr Lys Gly Gln 20 25 30Tyr Asp Glu Glu Glu
Met Thr Met Gln Gln Ala Lys Arg Arg Gln Lys 35 40 45Arg Glu Trp Val
Lys Phe Ala Lys Pro Cys Arg Glu Gly Glu Asp Asn 50 55 60Ser Lys Arg
Asn Pro Ile Ala Lys Ile Thr Ser Asp Tyr Gln Ala Thr65 70 75 80Gln
Lys Ile Thr Tyr Arg Ile Ser Gly Val Gly Ile Asp Gln Pro Pro 85 90
95Phe Gly Ile Phe Val Val Asp Lys Asn Thr Gly Asp Ile Asn Ile Thr
100 105 110Ala Ile Val Asp Arg Glu Glu Thr Pro Ser Phe Leu Ile Thr
Cys Arg 115 120 125Ala Leu Asn Ala Gln Gly Leu Asp Val Glu Lys Pro
Leu Ile Leu Thr 130 135 140Val Lys Ile Leu Asp Ile Asn Asp Asn Pro
Pro Val Phe Ser Gln Gln145 150 155 160Ile Phe Met Gly Glu Ile Glu
Glu Asn Ser Ala Ser Asn Ser Leu Val 165 170 175Met Ile Leu Asn Ala
Thr Asp Ala Asp Glu Pro Asn His Leu Asn Ser 180 185 190Lys Ile Ala
Phe Lys Ile Val Ser Gln Glu Pro Ala Gly Thr Pro Met 195 200 205Phe
Leu Leu Ser Arg Asn Thr Gly Glu Val Arg Thr Leu Thr Asn Ser 210 215
220Leu Asp Arg Glu Gln Ala Ser Ser Tyr Arg Leu Val Val Ser Gly
Ala225 230 235 240Asp Lys Asp Gly Glu Gly Leu Ser Thr Gln Cys Glu
Cys Asn Ile Lys 245 250 255Val Lys Asp Val Asn Asp Asn Phe Pro Met
Phe Arg Asp Ser Gln Tyr 260 265 270Ser Ala Arg Ile Glu Glu Asn Ile
Leu Ser Ser Glu Leu Leu Arg Phe 275 280 285Gln Val Thr Asp Leu Asp
Glu Glu Tyr Thr Asp Asn Trp Leu Ala Val 290 295 300Tyr Phe Phe Thr
Ser Gly Asn Glu Gly Asn Trp Phe Glu Ile Gln Thr305 310 315 320Asp
Pro Arg Thr Asn Glu Gly Ile Leu Lys Val Val Lys Ala Leu Asp 325 330
335Tyr Glu Gln Leu Gln Ser Val Lys Leu Ser Ile Ala Val Lys Asn Lys
340 345 350Ala Glu Phe His Gln Ser Val Ile Ser Arg Tyr Arg Val Gln
Ser Thr 355 360 365Pro Val Thr Ile Gln Val Ile Asn Val Arg Glu Gly
Ile Ala Phe Arg 370 375 380Pro Ala Ser Lys Thr Phe Thr Val Gln Lys
Gly Ile Ser Ser Lys Lys385 390 395 400Leu Val Asp Tyr Ile Leu Gly
Thr Tyr Gln Ala Ile Asp Glu Asp Thr 405 410 415Asn Lys Ala Ala Ser
Asn Val Lys Tyr Val Met Gly Arg Asn Asp Gly 420 425 430Gly Tyr Leu
Met Ile Asp Ser Lys Thr Ala Glu Ile Lys Phe Val Lys 435 440 445Asn
Met Asn Arg Asp Ser Thr Phe Ile Val Asn Lys Thr Ile Thr Ala 450 455
460Glu Val Leu Ala Ile Asp Glu Tyr Thr Gly Lys Thr Ser Thr Gly
Thr465 470 475 480Val Tyr Val Arg Val Pro Asp Phe Asn Asp Asn Cys
Pro Thr Ala Val 485 490 495Leu Glu Lys Asp Ala Val Cys Ser Ser Ser
Pro Ser Val Val Val Ser 500 505 510Ala Arg Thr Leu Asn Asn Arg Tyr
Thr Gly Pro Tyr Thr Phe Ala Leu 515 520 525Glu Asp Gln Pro Val Lys
Leu Pro Ala Val Trp Ser Ile Thr Thr Leu 530 535 540Asn Ala Thr Ser
Ala Leu Leu Arg Ala Gln Glu Gln Ile Pro Pro Gly545 550 555 560Val
Tyr His Ile Ser Leu Val Leu Thr Asp Ser Gln Asn Asn Arg Cys 565 570
575Glu Met Pro Arg Ser Leu Thr Leu Glu Val Cys Gln Cys Asp Asn Arg
580 585 590Gly Ile Cys Gly Thr Ser Tyr Pro Thr Thr Ser Pro Gly Thr
Arg Tyr 595 600 605Gly Arg Pro His Ser Gly Arg Leu Gly Pro Ala Ala
Ile Gly Leu Leu 610 615 620Leu Leu Gly Leu Leu Leu Leu Leu Leu Ala
Pro Leu Leu Leu Leu Thr625 630 635 640Cys Asp Cys Gly Ala Gly Ser
Thr Gly Gly Val Thr Gly Gly Phe Ile 645 650 655Pro Val Pro Asp Gly
Ser Glu Gly Thr Ile His Gln Trp Gly Ile Glu 660 665 670Gly Ala His
Pro Glu Asp Lys Glu Ile Thr Asn Ile Cys Val Pro Pro 675 680 685Val
Thr Ala Asn Gly Ala Asp Phe Met Glu Ser Ser Glu Val Cys Thr 690 695
700Asn Thr Tyr Ala Arg Gly Thr Ala Val Glu Gly Thr Ser Gly Met
Glu705 710 715 720Met Thr Thr Lys Leu Gly Ala Ala Thr Glu Ser Gly
Gly Ala Ala Gly 725 730 735Phe Ala Thr Gly Thr Val Ser Gly Ala Ala
Ser Gly Phe Gly Ala Ala 740 745 750Thr Gly Val Gly Ile Cys Ser Ser
Gly Gln Ser Gly Thr Met Arg Thr 755 760 765Arg His Ser Thr Gly Gly
Thr Asn Lys Asp Tyr Ala Asp Gly Ala Ile 770 775 780Ser Met Asn Phe
Leu Asp Ser Tyr Phe Ser Gln Lys Ala Phe Ala Cys785 790 795 800Ala
Glu Glu Asp Asp Gly Gln Glu Ala Asn Asp Cys Leu Leu Ile Tyr 805 810
815Asp Asn Glu Gly Ala Asp Ala Thr Gly Ser Pro Val Gly Ser Val Gly
820 825 830Cys Cys Ser Phe Ile Ala Asp Asp Leu Asp Asp Ser Phe Leu
Asp Ser 835 840 845Leu Gly Pro Lys Phe Lys Lys Leu Ala Glu Ile Ser
Leu Gly Val Asp 850 855 860Gly Glu Gly Lys Glu Val Gln Pro Pro Ser
Lys Asp Ser Gly Tyr Gly865 870 875 880Ile Glu Ser Cys Gly His Pro
Ile Glu Val Gln Gln Thr Gly Phe Val 885 890 895Lys Cys Gln Thr Leu
Ser Gly Ser Gln Gly Ala Ser Ala Leu Ser Ala 900 905 910Ser Gly Ser
Val Gln Pro Ala Val Ser Ile Pro Asp Pro Leu Gln His 915 920 925Gly
Asn Tyr Leu Val Thr Glu Thr Tyr Ser Ala Ser Gly Ser Leu Val 930 935
940Gln Pro Ser Thr Ala Gly Phe Asp Pro Leu Leu Thr Gln Asn Val
Ile945 950 955 960Val Thr Glu Arg Val Ile Cys Pro Ile Ser Ser Val
Pro Gly Asn Leu 965 970 975Ala Gly Pro Thr Gln Leu Arg Gly Ser His
Thr Met Leu Cys Thr Glu 980 985 990Asp Pro Cys Ser Arg Leu Ile
995412550DNAArtificial SequenceSynthetic nucleotide sequence
41atgatggggc tcttccccag aactacaggg gctctggcca tcttcgtggt ggtcatattg
60gttcatggag aattgcgaat agagactaaa ggtcaatatg atgaagaaga gatgactatg
120caacaagcta aaagaaggca aaaacgtgaa tgggtgaaat ttgccaaacc
ctgcagagaa 180ggagaagata actcaaaaag aaacccaatt gccaagatta
cttcagatta ccaagcaacc 240cagaaaatca cctaccgaat ctctggagtg
ggaatcgatc agccgccttt tggaatcttt 300gttgttgata aaaacactgg
agatattaac ataacagcta tagtcgaccg ggaggaaact 360ccaagcttcc
tgatcacatg tcgggctcta aatgcccaag gactagatgt agagaaacca
420cttatactaa cggttaaaat tttggatatt aatgataatc ctccagtatt
ttcacaacaa 480attttcatgg gtgaaattga agaaaatagt gcctcaaact
cactggtgat gatactaaat 540gccacagatg cagatgaacc aaaccacttg
aactctaaaa ttgccttcaa aattgtctct 600caggaaccag caggcacacc
catgttcctc ctaagcagaa acactgggga agtccgtact 660ttgaccaatt
ctcttgaccg agagcaagct agcagctatc gtctggttgt gagtggtgca
720gacaaagatg gagaaggact atcaactcaa tgtgaatgta atattaaagt
gaaagatgtc 780aacgataact tcccaatgtt tagagactct cagtattcag
cacgtattga agaaaatatt 840ttaagttctg aattacttcg atttcaagta
acagatttgg atgaagagta cacagataat 900tggcttgcag tatatttctt
tacctctggg aatgaaggaa attggtttga aatacaaact 960gatcctagaa
ctaatgaagg catcctgaaa gtggtgaagg ctctagatta tgaacaacta
1020caaagcgtga aacttagtat tgctgtcaaa aacaaagctg aatttcacca
atcagttatc 1080tctcgatacc gagttcagtc aaccccagtc acaattcagg
taataaatgt aagagaagga 1140attgcattcc gtcctgcttc caagacattt
actgtgcaaa aaggcataag tagcaaaaaa 1200ttggtggatt atatcctggg
aacatatcaa gccatcgatg aggacactaa caaagctgcc 1260tcaaatgtca
aatatgtcat gggacgtaac gatggtggat acctaatgat tgattcaaaa
1320actgctgaaa tcaaatttgt caaaaatatg aaccgagatt ctactttcat
agttaacaaa 1380acaatcacag ctgaggttct ggccatagat gaatacacgg
gtaaaacttc tacaggcacg 1440gtatatgtta gagtacccga tttcaatgac
aattgtccaa cagctgtcct cgaaaaagat 1500gcagtttgca gttcttcacc
ttccgtggtt gtctccgcta gaacactgaa taatagatac 1560actggcccct
atacatttgc actggaagat caacctgtaa agttgcctgc cgtatggagt
1620atcacaaccc tcaatgctac ctcggccctc ctcagagccc aggaacagat
acctcctgga 1680gtataccaca tctccctggt acttacagac agtcagaaca
atcggtgtga gatgccacgc 1740agcttgacac tggaagtctg tcagtgtgac
aacaggggca tctgtggaac ttcttaccca 1800accacaagcc ctgggaccag
gtatggcagg ccgcactcag ggaggctgga acctcgcgga 1860ccgacaatca
agccctgtcc tccatgcaaa tgcccagcac ctaacctctt gggtggacca
1920tccgtcttca tcttccctcc aaagatcaag gatgtactca tgatctccct
gagccccata 1980gtcacatgtg tggtggtgga tgtgagcgag gatgacccag
atgtccagat cagctggttt 2040gtgaacaacg tggaagtaca cacagctcag
acacaaaccc atagagagga ttacaacagt 2100actctccggg tggtcagtgc
cctccccatc cagcaccagg actggatgag tggcaaggag 2160ttcaaatgca
aggtcaacaa caaagacctc ccagcgccca tcgagagaac catctcaaaa
2220cccaaagggt cagtaagagc tccacaggta tatgtcttgc ctccaccaga
agaagagatg 2280actaagaaac aggtcactct gacctgcatg gtcacagact
tcatgcctga agacatttac 2340gtggagtgga ccaacaacgg gaaaacagag
ctaaactaca agaacactga accagtcctg 2400gactctgatg gttcttactt
catgtacagc aagctgagag tggaaaagaa gaactgggtg 2460gaaagaaata
gctactcctg ttcagtggtc cacgagggtc tgcacaatca ccacacgact
2520aagagcttct cccggactcc gggtaaatga 255042849PRTArtificial
SequenceSynthetic peptide sequence 42Met Met Gly Leu Phe Pro Arg
Thr Thr Gly Ala Leu Ala Ile Phe Val1 5 10 15Val Val Ile Leu Val His
Gly Glu Leu Arg Ile Glu Thr Lys Gly Gln 20 25 30Tyr Asp Glu Glu Glu
Met Thr Met Gln Gln Ala Lys Arg Arg Gln Lys 35 40 45Arg Glu Trp Val
Lys Phe Ala Lys Pro Cys Arg Glu Gly Glu Asp Asn 50 55 60Ser Lys Arg
Asn Pro Ile Ala Lys Ile Thr Ser Asp Tyr Gln Ala Thr65 70 75 80Gln
Lys Ile Thr Tyr Arg Ile Ser Gly Val Gly Ile Asp Gln Pro Pro 85 90
95Phe Gly Ile Phe Val Val Asp Lys Asn Thr Gly Asp Ile Asn Ile Thr
100 105 110Ala Ile Val Asp Arg Glu Glu Thr Pro Ser Phe Leu Ile Thr
Cys Arg 115 120 125Ala Leu Asn Ala Gln Gly Leu Asp Val Glu Lys Pro
Leu Ile Leu Thr 130 135 140Val Lys Ile Leu Asp Ile Asn Asp Asn Pro
Pro Val Phe Ser Gln Gln145 150 155 160Ile Phe Met Gly Glu Ile Glu
Glu Asn Ser Ala Ser Asn Ser Leu Val 165 170 175Met Ile Leu Asn Ala
Thr Asp Ala Asp Glu Pro Asn His Leu Asn Ser 180 185 190Lys Ile Ala
Phe Lys Ile Val Ser Gln Glu Pro Ala Gly Thr Pro Met 195 200 205Phe
Leu Leu Ser Arg Asn Thr Gly Glu Val Arg Thr Leu Thr Asn Ser 210 215
220Leu Asp Arg Glu Gln Ala Ser Ser Tyr Arg Leu Val Val Ser Gly
Ala225 230 235 240Asp Lys Asp Gly Glu Gly Leu Ser Thr Gln Cys Glu
Cys Asn Ile Lys 245 250 255Val Lys Asp Val Asn Asp Asn Phe Pro Met
Phe Arg Asp Ser Gln Tyr 260 265 270Ser Ala Arg Ile Glu Glu Asn Ile
Leu Ser Ser Glu Leu Leu Arg Phe 275 280 285Gln Val Thr Asp Leu Asp
Glu Glu Tyr Thr Asp Asn Trp Leu Ala Val 290 295 300Tyr Phe Phe Thr
Ser Gly Asn Glu Gly Asn Trp Phe Glu Ile Gln Thr305 310 315 320Asp
Pro Arg Thr Asn Glu Gly Ile Leu Lys Val Val Lys Ala Leu Asp 325 330
335Tyr Glu Gln Leu Gln Ser Val Lys Leu Ser Ile Ala Val Lys Asn Lys
340 345 350Ala Glu Phe His Gln Ser Val Ile Ser Arg Tyr Arg Val Gln
Ser Thr 355 360 365Pro Val Thr Ile Gln Val Ile Asn Val Arg Glu Gly
Ile Ala Phe Arg 370 375 380Pro Ala Ser Lys Thr Phe Thr Val Gln Lys
Gly Ile Ser Ser Lys Lys385 390 395 400Leu Val Asp Tyr Ile Leu Gly
Thr Tyr Gln Ala Ile Asp Glu Asp Thr 405 410 415Asn Lys Ala Ala Ser
Asn Val Lys Tyr Val Met Gly Arg Asn Asp Gly 420 425 430Gly Tyr Leu
Met Ile Asp Ser Lys Thr Ala Glu Ile Lys Phe Val Lys 435 440 445Asn
Met Asn Arg Asp Ser Thr Phe Ile Val Asn Lys Thr Ile Thr Ala 450 455
460Glu Val Leu Ala Ile Asp Glu Tyr Thr Gly Lys Thr Ser Thr Gly
Thr465 470 475 480Val Tyr Val Arg Val Pro Asp Phe Asn Asp Asn Cys
Pro Thr Ala Val 485 490 495Leu Glu Lys Asp Ala Val Cys Ser Ser Ser
Pro Ser Val Val Val Ser 500 505 510Ala Arg Thr Leu Asn Asn Arg Tyr
Thr Gly Pro Tyr Thr Phe Ala Leu 515 520 525Glu Asp Gln Pro Val Lys
Leu Pro Ala Val Trp Ser Ile Thr Thr Leu 530 535 540Asn Ala Thr Ser
Ala Leu Leu Arg Ala Gln Glu Gln Ile Pro Pro Gly545 550 555 560Val
Tyr His Ile Ser Leu Val Leu Thr Asp Ser Gln Asn Asn Arg Cys 565 570
575Glu Met Pro Arg Ser Leu Thr Leu Glu Val Cys Gln Cys Asp Asn Arg
580 585 590Gly Ile Cys Gly Thr Ser Tyr Pro Thr Thr Ser Pro Gly Thr
Arg Tyr 595 600 605Gly Arg Pro His Ser Gly Arg Leu Glu Pro Arg Gly
Pro Thr Ile Lys 610 615 620Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro
Asn Leu Leu Gly Gly Pro625 630 635 640Ser Val Phe Ile Phe Pro
Pro
Lys Ile Lys Asp Val Leu Met Ile Ser 645 650 655Leu Ser Pro Ile Val
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 660 665 670Pro Asp Val
Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr 675 680 685Ala
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 690 695
700Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
Glu705 710 715 720Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala
Pro Ile Glu Arg 725 730 735Thr Ile Ser Lys Pro Lys Gly Ser Val Arg
Ala Pro Gln Val Tyr Val 740 745 750Leu Pro Pro Pro Glu Glu Glu Met
Thr Lys Lys Gln Val Thr Leu Thr 755 760 765Cys Met Val Thr Asp Phe
Met Pro Glu Asp Ile Tyr Val Glu Trp Thr 770 775 780Asn Asn Gly Lys
Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu785 790 795 800Asp
Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys 805 810
815Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu
820 825 830Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr
Pro Gly 835 840 845Lys 4324DNAArtificial SequenceSynthetic
oligonucleotide sequence 43caggggccag tggatagact gatg
244423DNAArtificial SequenceSynthetic oligonucleotide sequence
44gctcactgga tggtgggaag atg 2345396DNAMus musculus 45atgggatgga
actggatctt tattttaatc ctgtcagtaa ctacaggtgt ccactctgag 60gtccagctgc
agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagatatcc
120tgcaaggctt ctggttactc attcactgcc tactacatgc actgggtgaa
gcaaagtcct 180gaaaagtgcc ttgagtggat tggacagatt aatcctagca
ctggtggtac tacctacaac 240cagaagttca aggccaaggc cacattgact
gtagacaaat cctccagcac agcctacatg 300cagctcaaga gcctgacatc
tgaggactct gcagtctatt attgtgcaag atggggtgac 360tcttggggcc
aaggcaccac tctcacagtc tcctca 39646132PRTMus musculus 46Met Gly Trp
Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly1 5 10 15Val His
Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro
Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40
45Thr Ala Tyr Tyr Met His Trp Val Lys Gln Ser Pro Glu Lys Cys Leu
50 55 60Glu Trp Ile Gly Gln Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr
Asn65 70 75 80Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu
Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Trp Gly Asp Ser Trp
Gly Gln Gly Thr Thr Leu 115 120 125Thr Val Ser Ser 13047393DNAMus
musculus 47atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg
ctccactggt 60gacattgtgc tcacccaatc tccagcttct ttggctgtgt ctctagggca
gagtgtcacc 120atctcctgca gagccagtga aagtgttgaa tattatggca
ctagtttaat gcagtggtac 180caacagaaac caggacagcc acccaaactc
ctcatctatg gtgcatccga cgtagaatct 240ggggtccctg ccaggtttag
tggcagtggg tctgggacag acttcagcct caacatccat 300cctgtggagg
aggatgatat tgcaatgtat ttctgtcagc aaagtaggaa ggttccgtat
360acgttcggat cggggaccaa gttggaaata aaa 39348131PRTMus musculus
48Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala 20 25 30Val Ser Leu Gly Gln Ser Val Thr Ile Ser Cys Arg Ala Ser
Glu Ser 35 40 45Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp Tyr Gln
Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser
Asp Val Glu Ser65 70 75 80Gly Val Pro Ala Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Ser 85 90 95Leu Asn Ile His Pro Val Glu Glu Asp
Asp Ile Ala Met Tyr Phe Cys 100 105 110Gln Gln Ser Arg Lys Val Pro
Tyr Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125Glu Ile Lys
13049426DNAMus musculus 49atggaatcta actggatact tccttttatt
ctgtcggtaa cttcaggggt ctactcagag 60gttcagctcc agcagtctgg gactgtgctg
gcaaggcctg gggcttcagt gaagatgtcc 120tgcaaggctt ctggctacac
ctttgccagc tactggattc actgggtaaa gcagaggcct 180ggacagggtc
tggaatggat tggctctatt tatcctggaa atagtgatac tacctacaac
240cagaagttca agggcaaggc caaactgact gtagtcacat ctgccagctc
tgcctacatg 300gagctcagca gcctgacaaa tgaggactct gcggtctatt
actgtacaga acctacttac 360tatagttacg acgattacta tgctatggac
tattggggtc aaggaacctc agtcaccgtc 420tcctca 42650142PRTMus musculus
50Met Glu Ser Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly1
5 10 15Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala
Arg 20 25 30Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
Thr Phe 35 40 45Ala Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu 50 55 60Glu Trp Ile Gly Ser Ile Tyr Pro Gly Asn Ser Asp
Thr Thr Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr
Val Val Thr Ser Ala Ser 85 90 95Ser Ala Tyr Met Glu Leu Ser Ser Leu
Thr Asn Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Thr Glu Pro Thr
Tyr Tyr Ser Tyr Asp Asp Tyr Tyr Ala 115 120 125Met Asp Tyr Trp Gly
Gln Gly Thr Ser Val Thr Val Ser Ser 130 135 14051390DNAMus musculus
51atggattttc atgtgcagat tttcagcttc atgctaatca gtgtcacagt catattgtcc
60agtggagaaa ttgtgctcac ccagtctcca gcactcatgg ctgcatctcc aggggagaag
120gtcaccatca cctgcagtgt cagctcaagt ataagttcca gcaacttaca
ctggtaccag 180cagaagtcag gaacctcccc caaaccctgg atttatggca
catccaacct tgcttctgga 240gtccctgttc gcttcagtgg cagtggatct
gggacctctt attctctcac aatcagcacc 300atggaggctg aagatgctgc
cacttattac tgtcaacagt ggagtagtta cccgctcacg 360ttcggtgctg
ggaccaagct ggagctgaaa 39052130PRTMus musculus 52Met Asp Phe His Val
Gln Ile Phe Ser Phe Met Leu Ile Ser Val Thr1 5 10 15Val Ile Leu Ser
Ser Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30Met Ala Ala
Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Val Ser 35 40 45Ser Ser
Ile Ser Ser Ser Asn Leu His Trp Tyr Gln Gln Lys Ser Gly 50 55 60Thr
Ser Pro Lys Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly65 70 75
80Val Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu
85 90 95Thr Ile Ser Thr Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
Gln 100 105 110Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Lys Leu Glu 115 120 125Leu Lys 1305332DNAMus musculus 53gccaaaacaa
cacccccatc agtctatcca ct 325430DNAMus musculus 54cgggctgatg
ctgcaccaac tgtatccatc 305533DNAMus musculus 55gccaaaacga cacccccatc
tgtctatcca ctg 33561407DNAMus musculus 56atgggatgga actggatctt
tattttaatc ctgtcagtaa ctacaggtgt ccactctgag 60gtccagctgc agcagtctgg
acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120tgcaaggctt
ctggttactc attcactgcc tactacatgc actgggtgaa gcaaagtcct
180gaaaagtgcc ttgagtggat tggacagatt aatcctagca ctggtggtac
tacctacaac 240cagaagttca aggccaaggc cacattgact gtagacaaat
cctccagcac agcctacatg 300cagctcaaga gcctgacatc tgaggactct
gcagtctatt attgtgcaag atggggtgac 360tcttggggcc aaggcaccac
tctcacagtc tcctcagcca aaacaacacc cccatcagtc 420tatccactgg
cccctgggtg tggagataca actggttcct ctgtgactct gggatgcctg
480gtcaagggct acttccctga gtcagtgact gtgacttgga actctggatc
cctgtccagc 540agtgtgcaca ccttcccagc tctcctgcag tctggactct
acactatgag cagctcagtg 600actgtcccct ccagcacctg gccaagtcag
accgtcacct gcagcgttgc tcacccagcc 660agcagcacca cggtggacaa
aaaacttgag cccagcgggc ccatttcaac aatcaacccc 720tgtcctccat
gcaaggagtg tcacaaatgc ccagctccta acctcgaggg tggaccatcc
780gtcttcatct tccctccaaa tatcaaggat gtactcatga tctccctgac
acccaaggtc 840acgtgtgtgg tggtggatgt gagcgaggat gacccagacg
tccagatcag ctggtttgtg 900aacaacgtgg aagtacacac agctcagaca
caaacccata gagaggatta caacagtact 960atccgggtgg tcagtgccct
ccccatccag caccaggact ggatgagtgg caaggagttc 1020aaatgcaagg
tcaacaacaa agacctccca tcacccatcg agagaaccat ctcaaaaatt
1080aaagggctag tcagagctcc acaagtatac atcttgccgc caccagcaga
gcagttgtcc 1140aggaaagatg tcagtctcac ttgcctggtc gtgggcttca
accctggaga catcagtgtg 1200gagtggacca gcaatgggca tacagaggag
aactacaagg acaccgcacc agtcctggac 1260tctgacggtt cttacttcat
atacagcaag ctcgatataa aaacaagcaa gtgggagaaa 1320acagattcct
tctcatgcaa cgtgagacac gagggtctga aaaattacta cctgaagaag
1380accatctccc ggtctccggg taaatga 140757468PRTMus musculus 57Met
Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly1 5 10
15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser
Phe 35 40 45Thr Ala Tyr Tyr Met His Trp Val Lys Gln Ser Pro Glu Lys
Cys Leu 50 55 60Glu Trp Ile Gly Gln Ile Asn Pro Ser Thr Gly Gly Thr
Thr Tyr Asn65 70 75 80Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr
Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Trp Gly Asp
Ser Trp Gly Gln Gly Thr Thr Leu 115 120 125Thr Val Ser Ser Ala Lys
Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala 130 135 140Pro Gly Cys Gly
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu145 150 155 160Val
Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly 165 170
175Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly
180 185 190Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr
Trp Pro 195 200 205Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala
Ser Ser Thr Thr 210 215 220Val Asp Lys Lys Leu Glu Pro Ser Gly Pro
Ile Ser Thr Ile Asn Pro225 230 235 240Cys Pro Pro Cys Lys Glu Cys
His Lys Cys Pro Ala Pro Asn Leu Glu 245 250 255Gly Gly Pro Ser Val
Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu 260 265 270Met Ile Ser
Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser 275 280 285Glu
Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu 290 295
300Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser
Thr305 310 315 320Ile Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
Asp Trp Met Ser 325 330 335Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
Lys Asp Leu Pro Ser Pro 340 345 350Ile Glu Arg Thr Ile Ser Lys Ile
Lys Gly Leu Val Arg Ala Pro Gln 355 360 365Val Tyr Ile Leu Pro Pro
Pro Ala Glu Gln Leu Ser Arg Lys Asp Val 370 375 380Ser Leu Thr Cys
Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val385 390 395 400Glu
Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala 405 410
415Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp
420 425 430Ile Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys
Asn Val 435 440 445Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys
Thr Ile Ser Arg 450 455 460Ser Pro Gly Lys46558717DNAMus musculus
58atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ctccactggt
60gacattgtgc tcacccaatc tccagcttct ttggctgtgt ctctagggca gagtgtcacc
120atctcctgca gagccagtga aagtgttgaa tattatggca ctagtttaat
gcagtggtac 180caacagaaac caggacagcc acccaaactc ctcatctatg
gtgcatccga cgtagaatct 240ggggtccctg ccaggtttag tggcagtggg
tctgggacag acttcagcct caacatccat 300cctgtggagg aggatgatat
tgcaatgtat ttctgtcagc aaagtaggaa ggttccgtat 360acgttcggat
cggggaccaa gttggaaata aaacgggctg atgctgcacc aactgtatcc
420atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt
gtgcttcttg 480aacaacttct accccaaaga catcaatgtc aagtggaaga
ttgatggcag tgaacgacaa 540aatggcgtcc tgaacagttg gactgatcag
gacagcaaag acagcaccta cagcatgagc 600agcaccctca cgttgaccaa
ggacgagtat gaacgacata acagctatac ctgtgaggcc 660actcacaaga
catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 71759238PRTMus
musculus 59Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala 20 25 30Val Ser Leu Gly Gln Ser Val Thr Ile Ser Cys Arg
Ala Ser Glu Ser 35 40 45Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp
Tyr Gln Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Asp Val Glu Ser65 70 75 80Gly Val Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Ser 85 90 95Leu Asn Ile His Pro Val Glu
Glu Asp Asp Ile Ala Met Tyr Phe Cys 100 105 110Gln Gln Ser Arg Lys
Val Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125Glu Ile Lys
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140Ser
Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu145 150
155 160Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp
Gly 165 170 175Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln Asp Ser 180 185 190Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu
Thr Leu Thr Lys Asp 195 200 205Glu Tyr Glu Arg His Asn Ser Tyr Thr
Cys Glu Ala Thr His Lys Thr 210 215 220Ser Thr Ser Pro Ile Val Lys
Ser Phe Asn Arg Asn Glu Cys225 230 235601401DNAMus musculus
60atggaatcta actggatact tccttttatt ctgtcggtaa cttcaggggt ctactcagag
60gttcagctcc agcagtctgg gactgtgctg gcaaggcctg gggcttcagt gaagatgtcc
120tgcaaggctt ctggctacac ctttgccagc tactggattc actgggtaaa
gcagaggcct 180ggacagggtc tggaatggat tggctctatt tatcctggaa
atagtgatac tacctacaac 240cagaagttca agggcaaggc caaactgact
gtagtcacat ctgccagctc tgcctacatg 300gagctcagca gcctgacaaa
tgaggactct gcggtctatt actgtacaga acctacttac 360tatagttacg
acgattacta tgctatggac tattggggtc aaggaacctc agtcaccgtc
420tcctcagcca aaacgacacc cccatctgtc tatccactgg cccctggatc
tgctgcccaa 480actaactcca tggtgaccct gggatgcctg gtcaagggct
atttccctga gccagtgaca 540gtgacctgga actctggatc cctgtccagc
ggtgtgcaca ccttcccagc tgtcctgcag 600tctgacctct acactctgag
cagctcagtg actgtcccct ccagcacctg gcccagccag 660accgtcacct
gcaacgttgc ccacccggcc agcagcacca aggtggacaa gaaaattgtg
720cccagggatt gtggttgtaa gccttgcata tgtacagtcc cagaagtatc
atctgtcttc 780atcttccccc caaagcccaa ggatgtgctc accattactc
tgactcctaa ggtcacgtgt 840gttgtggtag acatcagcaa ggatgatccc
gaggtccagt tcagctggtt tgtagatgat 900gtggaggtgc acacagctca
gacaaaaccc cgggaggagc agttcaacag cactttccgt 960tcagtcagtg
aacttcccat catgcaccag gactggctca atggcaagga gttcaaatgc
1020agggtcaaca gtgcagcttt ccctgccccc atcgagaaaa ccatctccaa
aaccaaaggc 1080agaccgaagg ctccacaggt gtacaccatt ccacctccca
aggagcagat ggccaaggat 1140aaagtcagtc tgacctgcat gataacagac
ttcttccctg aagacattac tgtggagtgg 1200cagtggaatg ggcagccagc
ggagaactac aagaacactc agcccatcat ggacacagat 1260ggctcttact
tcgtctacag caagctcaat gtgcagaaga gcaactggga ggcaggaaat
1320actttcacct gctctgtgtt acatgagggc ctgcacaacc accatactga
gaagagcctc 1380tcccactctc ctggtaaatg a 140161466PRTMus musculus
61Met Glu Ser Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly1
5 10 15Val Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala
Arg 20 25
30Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Ala Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ser Ile Tyr Pro Gly Asn Ser Asp Thr Thr
Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr Val Val
Thr Ser Ala Ser 85 90 95Ser Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn
Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Thr Glu Pro Thr Tyr Tyr
Ser Tyr Asp Asp Tyr Tyr Ala 115 120 125Met Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser Ala Lys 130 135 140Thr Thr Pro Pro Ser
Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln145 150 155 160Thr Asn
Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro 165 170
175Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val
180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
Ser Ser 195 200 205Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln
Thr Val Thr Cys 210 215 220Asn Val Ala His Pro Ala Ser Ser Thr Lys
Val Asp Lys Lys Ile Val225 230 235 240Pro Arg Asp Cys Gly Cys Lys
Pro Cys Ile Cys Thr Val Pro Glu Val 245 250 255Ser Ser Val Phe Ile
Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 260 265 270Thr Leu Thr
Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp 275 280 285Asp
Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His 290 295
300Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg305 310 315 320Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp
Leu Asn Gly Lys 325 330 335Glu Phe Lys Cys Arg Val Asn Ser Ala Ala
Phe Pro Ala Pro Ile Glu 340 345 350Lys Thr Ile Ser Lys Thr Lys Gly
Arg Pro Lys Ala Pro Gln Val Tyr 355 360 365Thr Ile Pro Pro Pro Lys
Glu Gln Met Ala Lys Asp Lys Val Ser Leu 370 375 380Thr Cys Met Ile
Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp385 390 395 400Gln
Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 405 410
415Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln
420 425 430Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
Leu His 435 440 445Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu
Ser His Ser Pro 450 455 460Gly Lys46562714DNAMus musculus
62atggattttc atgtgcagat tttcagcttc atgctaatca gtgtcacagt catattgtcc
60agtggagaaa ttgtgctcac ccagtctcca gcactcatgg ctgcatctcc aggggagaag
120gtcaccatca cctgcagtgt cagctcaagt ataagttcca gcaacttaca
ctggtaccag 180cagaagtcag gaacctcccc caaaccctgg atttatggca
catccaacct tgcttctgga 240gtccctgttc gcttcagtgg cagtggatct
gggacctctt attctctcac aatcagcacc 300atggaggctg aagatgctgc
cacttattac tgtcaacagt ggagtagtta cccgctcacg 360ttcggtgctg
ggaccaagct ggagctgaaa cgggctgatg ctgcaccaac tgtatccatc
420ttcccaccat ccagtgagca gttaacatct ggaggtgcct cagtcgtgtg
cttcttgaac 480aacttctacc ccaaagacat caatgtcaag tggaagattg
atggcagtga acgacaaaat 540ggcgtcctga acagttggac tgatcaggac
agcaaagaca gcacctacag catgagcagc 600accctcacgt tgaccaagga
cgagtatgaa cgacataaca gctatacctg tgaggccact 660cacaagacat
caacttcacc cattgtcaag agcttcaaca ggaatgagtg ttag 71463237PRTMus
musculus 63Met Asp Phe His Val Gln Ile Phe Ser Phe Met Leu Ile Ser
Val Thr1 5 10 15Val Ile Leu Ser Ser Gly Glu Ile Val Leu Thr Gln Ser
Pro Ala Leu 20 25 30Met Ala Ala Ser Pro Gly Glu Lys Val Thr Ile Thr
Cys Ser Val Ser 35 40 45Ser Ser Ile Ser Ser Ser Asn Leu His Trp Tyr
Gln Gln Lys Ser Gly 50 55 60Thr Ser Pro Lys Pro Trp Ile Tyr Gly Thr
Ser Asn Leu Ala Ser Gly65 70 75 80Val Pro Val Arg Phe Ser Gly Ser
Gly Ser Gly Thr Ser Tyr Ser Leu 85 90 95Thr Ile Ser Thr Met Glu Ala
Glu Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110Gln Trp Ser Ser Tyr
Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu 115 120 125Leu Lys Arg
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser 130 135 140Ser
Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn145 150
155 160Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly
Ser 165 170 175Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln
Asp Ser Lys 180 185 190Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr
Leu Thr Lys Asp Glu 195 200 205Tyr Glu Arg His Asn Ser Tyr Thr Cys
Glu Ala Thr His Lys Thr Ser 210 215 220Thr Ser Pro Ile Val Lys Ser
Phe Asn Arg Asn Glu Cys225 230 235641389DNAArtificial
SequenceSynthetic nucleotide sequence 64atgggatgga actggatctt
tattttaatc ctgtcagtaa ctacaggtgt ccactctgag 60gtccagctgc agcagtctgg
acctgagctg gtgaagcctg gggcttcagt gaagatatcc 120tgcaaggctt
ctggttactc attcactgcc tactacatgc actgggtgaa gcaaagtcct
180gaaaagtgcc ttgagtggat tggacagatt aatcctagca ctggtggtac
tacctacaac 240cagaagttca aggccaaggc cacattgact gtagacaaat
cctccagcac agcctacatg 300cagctcaaga gcctgacatc tgaggactct
gcagtctatt attgtgcaag atggggtgac 360tcttggggcc aaggcaccac
tctcacagtc tcctcagcta gcaccaaggg cccatcggtc 420ttccccctgg
caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg
480gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc
cctgaccagc 540ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac
tctactccct cagcagcgtg 600gtgaccgtgc cctccagcag cttgggcacc
cagacctaca tctgcaacgt gaatcacaag 660cccagcaaca ccaaggtgga
caagaaagtt gagcccaaat cttgtgacaa aactcacaca 720tgcccaccgt
gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca
780aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt
ggtggtggac 840gtgagccacg aagaccctga ggtcaagttc aactggtacg
tggacggcgt ggaggtgcat 900aatgccaaga caaagccgcg ggaggagcag
tacaacagca cgtaccgtgt ggtcagcgtc 960ctcaccgtcc tgcaccagga
ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1020aaagccctcc
cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa
1080ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca
ggtcagcctg 1140acctgcctgg tcaaaggctt ctatcccagc gacatcgccg
tggagtggga gagcaatggg 1200cagccggaga acaactacaa gaccacgcct
cccgtgctgg actccgacgg ctccttcttc 1260ctctacagca agctcaccgt
ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1320tccgtgatgc
atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg
1380ggtaaatga 138965462PRTArtificial SequenceSynthetic peptide
sequence 65Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr
Thr Gly1 5 10 15Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys 20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe 35 40 45Thr Ala Tyr Tyr Met His Trp Val Lys Gln Ser
Pro Glu Lys Cys Leu 50 55 60Glu Trp Ile Gly Gln Ile Asn Pro Ser Thr
Gly Gly Thr Thr Tyr Asn65 70 75 80Gln Lys Phe Lys Ala Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Gln Leu Lys
Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg
Trp Gly Asp Ser Trp Gly Gln Gly Thr Thr Leu 115 120 125Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 130 135 140Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu145 150
155 160Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
Gly 165 170 175Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser 180 185 190Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser Leu 195 200 205Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr 210 215 220Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr225 230 235 240Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265
270Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
275 280 285Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 290 295 300Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val305 310 315 320Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys 325 330 335Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 355 360 365Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 370 375 380Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly385 390
395 400Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp 405 410 415Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp 420 425 430Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 435 440 445Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 450 455 46066717DNAArtificial SequenceSynthetic
nucleotide sequence 66atggagacag acacactcct gctatgggtg ctgctgctct
gggttccagg ctccactggt 60gacattgtgc tcacccaatc tccagcttct ttggctgtgt
ctctagggca gagtgtcacc 120atctcctgca gagccagtga aagtgttgaa
tattatggca ctagtttaat gcagtggtac 180caacagaaac caggacagcc
acccaaactc ctcatctatg gtgcatccga cgtagaatct 240ggggtccctg
ccaggtttag tggcagtggg tctgggacag acttcagcct caacatccat
300cctgtggagg aggatgatat tgcaatgtat ttctgtcagc aaagtaggaa
ggttccgtat 360acgttcggat cggggaccaa gttggaaata aaacgtacgg
tggctgcacc atctgtcttc 420atcttcccgc catctgatga gcagttgaaa
tctggaactg cctctgttgt gtgcctgctg 480aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 540ggtaactccc
aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
600agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc
ctgcgaagtc 660acccatcagg gcctgagctc gcccgtcaca aagagcttca
acaggggaga gtgttga 71767238PRTArtificial SequenceSynthetic peptide
sequence 67Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp
Val Pro1 5 10 15Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala
Ser Leu Ala 20 25 30Val Ser Leu Gly Gln Ser Val Thr Ile Ser Cys Arg
Ala Ser Glu Ser 35 40 45Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp
Tyr Gln Gln Lys Pro 50 55 60Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly
Ala Ser Asp Val Glu Ser65 70 75 80Gly Val Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Ser 85 90 95Leu Asn Ile His Pro Val Glu
Glu Asp Asp Ile Ala Met Tyr Phe Cys 100 105 110Gln Gln Ser Arg Lys
Val Pro Tyr Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140Ser
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu145 150
155 160Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn 165 170 175Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser 180 185 190Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala 195 200 205Asp Tyr Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly 210 215 220Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys225 230 235681419DNAArtificial
SequenceSynthetic nucleotide sequence 68atggaatcta actggatact
tccttttatt ctgtcggtaa cttcaggggt ctactcagag 60gttcagctcc agcagtctgg
gactgtgctg gcaaggcctg gggcttcagt gaagatgtcc 120tgcaaggctt
ctggctacac ctttgccagc tactggattc actgggtaaa gcagaggcct
180ggacagggtc tggaatggat tggctctatt tatcctggaa atagtgatac
tacctacaac 240cagaagttca agggcaaggc caaactgact gtagtcacat
ctgccagctc tgcctacatg 300gagctcagca gcctgacaaa tgaggactct
gcggtctatt actgtacaga acctacttac 360tatagttacg acgattacta
tgctatggac tattggggtc aaggaacctc agtcaccgtc 420tcctcagcta
gcaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc
480tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga
accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca
ccttcccggc tgtcctacag 600tcctcaggac tctactccct cagcagcgtg
gtgaccgtgc cctccagcag cttgggcacc 660cagacctaca tctgcaacgt
gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 720gagcccaaat
cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg
780gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat
gatctcccgg 840acccctgagg tcacatgcgt ggtggtggac gtgagccacg
aagaccctga ggtcaagttc 900aactggtacg tggacggcgt ggaggtgcat
aatgccaaga caaagccgcg ggaggagcag 960tacaacagca cgtaccgtgt
ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1020ggcaaggagt
acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
1080atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc
cccatcccgg 1140gatgagctga ccaagaacca ggtcagcctg acctgcctgg
tcaaaggctt ctatcccagc 1200gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 1260cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 1320aggtggcagc
aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
1380tacacgcaga agagcctctc cctgtctccg ggtaaatga
141969472PRTArtificial SequenceSynthetic peptide sequence 69Met Glu
Ser Asn Trp Ile Leu Pro Phe Ile Leu Ser Val Thr Ser Gly1 5 10 15Val
Tyr Ser Glu Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg 20 25
30Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Ala Ser Tyr Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu 50 55 60Glu Trp Ile Gly Ser Ile Tyr Pro Gly Asn Ser Asp Thr Thr
Tyr Asn65 70 75 80Gln Lys Phe Lys Gly Lys Ala Lys Leu Thr Val Val
Thr Ser Ala Ser 85 90 95Ser Ala Tyr Met Glu Leu Ser Ser Leu Thr Asn
Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Thr Glu Pro Thr Tyr Tyr
Ser Tyr Asp Asp Tyr Tyr Ala 115 120 125Met Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser Ala Ser 130 135 140Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr145 150 155 160Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170
175Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser 195 200 205Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile 210 215 220Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val225 230 235 240Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295
300Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln305 310 315 320Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 325 330 335Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 340
345 350Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 355 360 365Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr 370 375 380Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser385 390 395 400Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 405 410 415Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 420 425 430Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455
460Ser Leu Ser Leu Ser Pro Gly Lys465 47070714DNAArtificial
SequenceSynthetic nucleotide sequence 70atggattttc atgtgcagat
tttcagcttc atgctaatca gtgtcacagt catattgtcc 60agtggagaaa ttgtgctcac
ccagtctcca gcactcatgg ctgcatctcc aggggagaag 120gtcaccatca
cctgcagtgt cagctcaagt ataagttcca gcaacttaca ctggtaccag
180cagaagtcag gaacctcccc caaaccctgg atttatggca catccaacct
tgcttctgga 240gtccctgttc gcttcagtgg cagtggatct gggacctctt
attctctcac aatcagcacc 300atggaggctg aagatgctgc cacttattac
tgtcaacagt ggagtagtta cccgctcacg 360ttcggtgctg ggaccaagct
ggagctgaaa cgtacggtgg ctgcaccatc tgtcttcatc 420ttcccgccat
ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat
480aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct
ccaatcgggt 540aactcccagg agagtgtcac agagcaggac agcaaggaca
gcacctacag cctcagcagc 600accctgacgc tgagcaaagc agactacgag
aaacacaaag tctacgcctg cgaagtcacc 660catcagggcc tgagctcgcc
cgtcacaaag agcttcaaca ggggagagtg ttga 71471237PRTArtificial
SequenceSynthetic peptide sequence 71Met Asp Phe His Val Gln Ile
Phe Ser Phe Met Leu Ile Ser Val Thr1 5 10 15Val Ile Leu Ser Ser Gly
Glu Ile Val Leu Thr Gln Ser Pro Ala Leu 20 25 30Met Ala Ala Ser Pro
Gly Glu Lys Val Thr Ile Thr Cys Ser Val Ser 35 40 45Ser Ser Ile Ser
Ser Ser Asn Leu His Trp Tyr Gln Gln Lys Ser Gly 50 55 60Thr Ser Pro
Lys Pro Trp Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly65 70 75 80Val
Pro Val Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 85 90
95Thr Ile Ser Thr Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
100 105 110Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys
Leu Glu 115 120 125Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser 130 135 140Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn145 150 155 160Asn Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala 165 170 175Leu Gln Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys 180 185 190Asp Ser Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 195 200 205Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 210 215
220Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
235724PRTArtificial SequenceSynthetic peptide sequence 72Gly Gly
Gly Ser1734PRTArtificial SequenceSynthetic peptide sequence 73Ser
Gly Gly Gly1745PRTArtificial SequenceAn artificially synthesized
peptide sequence 74Gly Gly Gly Gly Ser1 5755PRTArtificial
SequenceSynthetic peptide sequence 75Ser Gly Gly Gly Gly1
5766PRTArtificial SequenceSynthetic peptide sequence 76Gly Gly Gly
Gly Gly Ser1 5776PRTArtificial SequenceSynthetic peptide sequence
77Ser Gly Gly Gly Gly Gly1 5787PRTArtificial SequenceSynthetic
peptide sequence 78Gly Gly Gly Gly Gly Gly Ser1 5797PRTArtificial
SequenceSynthetic peptide sequence 79Ser Gly Gly Gly Gly Gly Gly1
58015DNAMus musculus 80gactacaaca tggac 15815PRTMus musculus 81Asp
Tyr Asn Met Asp1 58251DNAMus musculus 82tatatttatc ctaacaatgg
tggttctggc tacaaccaga agttcaagag c 518317PRTMus musculus 83Tyr Ile
Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn Gln Lys Phe Lys1 5 10
15Ser8442DNAMus musculus 84cgggatagtt actatggttt cgacatggcc
tggtttgctt ac 428514PRTMus musculus 85Arg Asp Ser Tyr Tyr Gly Phe
Asp Met Ala Trp Phe Ala Tyr1 5 108633DNAMus musculus 86cgaccaagtg
agaatattta caataattta gca 338711PRTMus musculus 87Arg Pro Ser Glu
Asn Ile Tyr Asn Asn Leu Ala1 5 108821DNAMus musculus 88gttgcaacaa
atttagcaga a 21897PRTMus musculus 89Val Ala Thr Asn Leu Ala Glu1
59027DNAMus musculus 90caacattctt atggtactcc gtggacg 27919PRTMus
musculus 91Gln His Ser Tyr Gly Thr Pro Trp Thr1 592426DNAMus
musculus 92atgggatgga actggatctt tctcttcctc ctgtcaggaa ctgcaggtgt
cctctctgag 60gtccagctgc agcagtctgg acctgaactg gtgaagcctg gggcttcagt
gaagatatcc 120tgcaaggctt ctggatacac attcactgac tacaacatgg
actgggtgaa gcagagccat 180ggagagagcc ttgagtggat tggatatatt
tatcctaaca atggtggttc tggctacaac 240cagaagttca agagcaaggc
cacattgact gtagacaagt cctccagcac agcctacatg 300gagctccaca
gcctgacatc tgaagactct gcagtctatt actgtgcaag acgggatagt
360tactatggtt tcgacatggc ctggtttgct tactggggcc aagggactct
ggtcactgtc 420tctgca 42693142PRTMus musculus 93Met Gly Trp Asn Trp
Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly1 5 10 15Val Leu Ser Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly Ala
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp
Tyr Asn Met Asp Trp Val Lys Gln Ser His Gly Glu Ser Leu 50 55 60Glu
Trp Ile Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn65 70 75
80Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala
Val 100 105 110Tyr Tyr Cys Ala Arg Arg Asp Ser Tyr Tyr Gly Phe Asp
Met Ala Trp 115 120 125Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ala 130 135 14094381DNAMus musculus 94atgagtgtgc ccactcaggt
cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60gacatccaga tgactcagtc
tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 120atcacatgtc
gaccaagtga gaatatttac aataatttag catggtatca acagaaacag
180ggaaaatctc ctcagctcct ggtctatgtt gcaacaaatt tagcagaagg
tgtgccatca 240aggttcagtg gcagtggatc aggcacacgg ttttctctga
agatcaacag cctgcagcct 300gaagattttg ggaagtatta ctgtcaacat
tcttatggta ctccgtggac gttcggtgga 360ggcaccaagc tggagatcaa a
38195127PRTMus musculus 95Met Ser Val Pro Thr Gln Val Leu Gly Leu
Leu Leu Leu Trp Leu Thr1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser 20 25 30Ala Ser Val Gly Glu Thr Val Thr
Ile Thr Cys Arg Pro Ser Glu Asn 35 40 45Ile Tyr Asn Asn Leu Ala Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60Gln Leu Leu Val Tyr Val
Ala Thr Asn Leu Ala Glu Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly
Ser Gly Ser Gly Thr Arg Phe Ser Leu Lys Ile Asn 85 90 95Ser Leu Gln
Pro Glu Asp Phe Gly Lys Tyr Tyr Cys Gln His Ser Tyr 100 105 110Gly
Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 115 120
125961416DNAArtificial SequenceSynthetic nucleotide sequence
96atgggatgga actggatctt tctcttcctc ctgtcaggaa ctgcaggtgt cctctctgag
60gtccagctgc agcagtctgg acctgaactg gtgaagcctg gggcttcagt gaagatatcc
120tgcaaggctt ctggatacac attcactgac tacaacatgg actgggtgaa
gcagagccat 180ggagagagcc ttgagtggat tggatatatt tatcctaaca
atggtggttc tggctacaac 240cagaagttca agagcaaggc cacattgact
gtagacaagt cctccagcac agcctacatg 300gagctccaca gcctgacatc
tgaagactct gcagtctatt actgtgcaag acgggatagt 360tactatggtt
tcgacatggc ctggtttgct tactggggcc aagggactct ggtcactgtc
420tctgcagcta gcaccaaggg cccatcggtc ttccccctgg caccctcctc
caagagcacc 480tctgggggca cagcggccct gggctgcctg gtcaaggact
acttccccga accggtgacg 540gtgtcgtgga actcaggcgc cctgaccagc
ggcgtgcaca ccttcccggc tgtcctacag 600tcctcaggac tctactccct
cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 660cagacctaca
tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt
720gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc
tgaactcctg 780gggggaccgt cagtcttcct cttcccccca aaacccaagg
acaccctcat gatctcccgg 840acccctgagg tcacatgcgt ggtggtggac
gtgagccacg aagaccctga ggtcaagttc 900aactggtacg tggacggcgt
ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 960tacaacagca
cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat
1020ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat
cgagaaaacc 1080atctccaaag ccaaagggca gccccgagaa ccacaggtgt
acaccctgcc cccatcccgg 1140gatgagctga ccaagaacca ggtcagcctg
acctgcctgg tcaaaggctt ctatcccagc 1200gacatcgccg tggagtggga
gagcaatggg cagccggaga acaactacaa gaccacgcct 1260cccgtgctgg
actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc
1320aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct
gcacaaccac 1380tacacgcaga agagcctctc cctgtctccg ggtaaa
141697472PRTArtificial SequenceSynthetic peptide sequence 97Met Gly
Trp Asn Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly1 5 10 15Val
Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25
30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Ser His Gly Glu Ser
Leu 50 55 60Glu Trp Ile Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Ser Gly
Tyr Asn65 70 75 80Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser
Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg Arg Asp Ser Tyr
Tyr Gly Phe Asp Met Ala Trp 115 120 125Phe Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala Ala Ser 130 135 140Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr145 150 155 160Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170
175Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser 195 200 205Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile 210 215 220Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val225 230 235 240Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala 245 250 255Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 260 265 270Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 275 280 285Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 290 295
300Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln305 310 315 320Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln 325 330 335Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala 340 345 350Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro 355 360 365Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 370 375 380Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser385 390 395 400Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 405 410
415Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
420 425 430Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe 435 440 445Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys 450 455 460Ser Leu Ser Leu Ser Pro Gly Lys465
47098702DNAArtificial SequenceSynthetic nucleotide sequence
98atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt
60gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc
120atcacatgtc gaccaagtga gaatatttac aataatttag catggtatca
acagaaacag 180ggaaaatctc ctcagctcct ggtctatgtt gcaacaaatt
tagcagaagg tgtgccatca 240aggttcagtg gcagtggatc aggcacacgg
ttttctctga agatcaacag cctgcagcct 300gaagattttg ggaagtatta
ctgtcaacat tcttatggta ctccgtggac gttcggtgga 360ggcaccaagc
tggagatcaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca
420tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa
taacttctat 480cccagagagg ccaaagtaca gtggaaggtg gataacgccc
tccaatcggg taactcccag 540gagagtgtca cagagcagga cagcaaggac
agcacctaca gcctcagcag caccctgacg 600ctgagcaaag cagactacga
gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 660ctgagctcgc
ccgtcacaaa gagcttcaac aggggagagt gt 70299234PRTArtificial
SequenceSynthetic peptide sequence 99Met Ser Val Pro Thr Gln Val
Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Gly Ala Arg Cys Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30Ala Ser Val Gly Glu
Thr Val Thr Ile Thr Cys Arg Pro Ser Glu Asn 35 40 45Ile Tyr Asn Asn
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60Gln Leu Leu
Val Tyr Val Ala Thr Asn Leu Ala Glu Gly Val Pro Ser65 70 75 80Arg
Phe Ser Gly Ser Gly Ser Gly Thr Arg Phe Ser Leu Lys Ile Asn 85 90
95Ser Leu Gln Pro Glu Asp Phe Gly Lys Tyr Tyr Cys Gln His Ser Tyr
100 105 110Gly Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 115 120 125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln 130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 195 200 205His
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215
220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 23010021DNAArtificial
SequenceSynthetic oligonucleotide 100gggccagtgg atagacagat g
211011401DNAMus musculus 101atgggatgga actggatctt tctcttcctc
ctgtcaggaa ctgcaggtgt cctctctgag 60gtccagctgc agcagtctgg acctgaactg
gtgaagcctg gggcttcagt gaagatatcc 120tgcaaggctt ctggatacac
attcactgac tacaacatgg actgggtgaa gcagagccat 180ggagagagcc
ttgagtggat tggatatatt tatcctaaca atggtggttc tggctacaac
240cagaagttca agagcaaggc cacattgact gtagacaagt cctccagcac
agcctacatg 300gagctccaca gcctgacatc tgaagactct gcagtctatt
actgtgcaag acgggatagt 360tactatggtt tcgacatggc ctggtttgct
tactggggcc aagggactct ggtcactgtc 420tctgcagcca aaacgacacc
cccatctgtc tatccactgg cccctggatc tgctgcccaa 480actaactcca
tggtgaccct gggatgcctg gtcaagggct atttccctga gccagtgaca
540gtgacctgga actctggatc cctgtccagc ggtgtgcaca ccttcccagc
tgtcctgcag 600tctgacctct acactctgag cagctcagtg actgtcccct
ccagcacctg gcccagccag 660accgtcacct gcaacgttgc ccacccggcc
agcagcacca aggtggacaa gaaaattgtg 720cccagggatt gtggttgtaa
gccttgcata tgtacagtcc cagaagtatc atctgtcttc 780atcttccccc
caaagcccaa ggatgtgctc accattactc tgactcctaa ggtcacgtgt
840gttgtggtag acatcagcaa ggatgatccc gaggtccagt tcagctggtt
tgtagatgat 900gtggaggtgc acacagctca gacaaaaccc cgggaggagc
agttcaacag cactttccgt 960tcagtcagtg aacttcccat catgcaccag
gactggctca atggcaagga gttcaaatgc 1020agggtcaaca gtgcagcttt
ccctgccccc atcgagaaaa ccatctccaa aaccaaaggc 1080agaccgaagg
ctccacaggt gtacaccatt ccacctccca aggagcagat ggccaaggat
1140aaagtcagtc tgacctgcat
gataacagac ttcttccctg aagacattac tgtggagtgg 1200cagtggaatg
ggcagccagc ggagaactac aagaacactc agcccatcat ggacacagat
1260ggctcttact tcgtctacag caagctcaat gtgcagaaga gcaactggga
ggcaggaaat 1320actttcacct gctctgtgtt acatgagggc ctgcacaacc
accatactga gaagagcctc 1380tcccactctc ctggtaaatg a 1401102466PRTMus
musculus 102Met Gly Trp Asn Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr
Ala Gly1 5 10 15Val Leu Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Val Lys 20 25 30Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Asn Met Asp Trp Val Lys Gln Ser
His Gly Glu Ser Leu 50 55 60Glu Trp Ile Gly Tyr Ile Tyr Pro Asn Asn
Gly Gly Ser Gly Tyr Asn65 70 75 80Gln Lys Phe Lys Ser Lys Ala Thr
Leu Thr Val Asp Lys Ser Ser Ser 85 90 95Thr Ala Tyr Met Glu Leu His
Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110Tyr Tyr Cys Ala Arg
Arg Asp Ser Tyr Tyr Gly Phe Asp Met Ala Trp 115 120 125Phe Ala Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys 130 135 140Thr
Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln145 150
155 160Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe
Pro 165 170 175Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
Ser Gly Val 180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
Tyr Thr Leu Ser Ser 195 200 205Ser Val Thr Val Pro Ser Ser Thr Trp
Pro Ser Gln Thr Val Thr Cys 210 215 220Asn Val Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val225 230 235 240Pro Arg Asp Cys
Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 245 250 255Ser Ser
Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 260 265
270Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp
275 280 285Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
Val His 290 295 300Thr Ala Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg305 310 315 320Ser Val Ser Glu Leu Pro Ile Met His
Gln Asp Trp Leu Asn Gly Lys 325 330 335Glu Phe Lys Cys Arg Val Asn
Ser Ala Ala Phe Pro Ala Pro Ile Glu 340 345 350Lys Thr Ile Ser Lys
Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 355 360 365Thr Ile Pro
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 370 375 380Thr
Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp385 390
395 400Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
Ile 405 410 415Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu
Asn Val Gln 420 425 430Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
Cys Ser Val Leu His 435 440 445Glu Gly Leu His Asn His His Thr Glu
Lys Ser Leu Ser His Ser Pro 450 455 460Gly Lys465103705DNAMus
musculus 103atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg
tgccagatgt 60gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga
aactgtcacc 120atcacatgtc gaccaagtga gaatatttac aataatttag
catggtatca acagaaacag 180ggaaaatctc ctcagctcct ggtctatgtt
gcaacaaatt tagcagaagg tgtgccatca 240aggttcagtg gcagtggatc
aggcacacgg ttttctctga agatcaacag cctgcagcct 300gaagattttg
ggaagtatta ctgtcaacat tcttatggta ctccgtggac gttcggtgga
360ggcaccaagc tggagatcaa acgggctgat gctgcaccaa ctgtatccat
cttcccacca 420tccagtgagc agttaacatc tggaggtgcc tcagtcgtgt
gcttcttgaa caacttctac 480cccaaagaca tcaatgtcaa gtggaagatt
gatggcagtg aacgacaaaa tggcgtcctg 540aacagttgga ctgatcagga
cagcaaagac agcacctaca gcatgagcag caccctcacg 600ttgaccaagg
acgagtatga acgacataac agctatacct gtgaggccac tcacaagaca
660tcaacttcac ccattgtcaa gagcttcaac aggaatgagt gttag
705104234PRTMus musculus 104Met Ser Val Pro Thr Gln Val Leu Gly Leu
Leu Leu Leu Trp Leu Thr1 5 10 15Gly Ala Arg Cys Asp Ile Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser 20 25 30Ala Ser Val Gly Glu Thr Val Thr
Ile Thr Cys Arg Pro Ser Glu Asn 35 40 45Ile Tyr Asn Asn Leu Ala Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60Gln Leu Leu Val Tyr Val
Ala Thr Asn Leu Ala Glu Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly
Ser Gly Ser Gly Thr Arg Phe Ser Leu Lys Ile Asn 85 90 95Ser Leu Gln
Pro Glu Asp Phe Gly Lys Tyr Tyr Cys Gln His Ser Tyr 100 105 110Gly
Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 115 120
125Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
130 135 140Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn
Phe Tyr145 150 155 160Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp
Gly Ser Glu Arg Gln 165 170 175Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Met Ser Ser Thr Leu
Thr Leu Thr Lys Asp Glu Tyr Glu Arg 195 200 205His Asn Ser Tyr Thr
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 210 215 220Ile Val Lys
Ser Phe Asn Arg Asn Glu Cys225 23010531DNAArtificial
SequenceSynthetic primer sequence 105taagaattcc accatgggat
ggaactggat c 3110621DNAArtificial SequenceSynthetic primer sequence
106cagagacagt gaccagagtc c 211071006DNAMus musculus 107tcgcgaaaac
aacagcccca tcggtctatc cactggcccc tgtgtgtgga gatacaactg 60gctcctcggt
gactctagga tgcctggtca agggttattt ccctgagcca gtgaccttga
120cctggaactc tggatccctg tccagtggtg tgcacacctt cccagctgtc
ctgcagtctg 180acctctacac cctcagcagc tcagtgactg taacctcgag
cacctggccc agccagtcca 240tcacctgcaa tgtggcccac ccggcaagca
gcaccaaggt ggacaagaaa attgagccca 300gagggcccac aatcaagccc
tgtcctccat gcaaatgccc agcacctaac ctcttgggtg 360gaccatccgt
cttcatcttc cctccaaaga tcaaggatgt actcatgatc tccctgagcc
420ccatagtcac atgtgtggtg gtggatgtga gcgaggatga cccagatgtc
cagatcagct 480ggtttgtgaa caacgtggaa gtacacacag ctcagacaca
aacccataga gaggattaca 540acagtactct ccgggtggtc agtgccctcc
ccatccagca ccaggactgg atgagtggca 600aggagttcaa atgcaaggtc
aacaacaaag acctcccagc gcccatcgag agaaccatct 660caaaacccaa
agggtcagta agagcaccac aggtatatgt cttgcctcca ccagaagaag
720agatgactaa gaaacaggtc actctgacct gcatggtcac agacttcatg
cctgaagaca 780tttacgtgga gtggaccaac aacgggaaaa cagagctaaa
ctacaagaac actgaaccag 840tcctggactc tgatggttct tacttcatgt
acagcaagct gagagtggaa aagaagaact 900gggtggaaag aaatagctac
tcctgttcag tggtccacga gggtctgcac aatcaccaca 960cgactaagag
cttctcccgg actccgggta aatgataagc ggccgc 1006108330PRTMus musculus
108Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly1
5 10 15Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr 20 25 30Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu
Ser Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
Tyr Thr Leu 50 55 60Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro
Ser Gln Ser Ile65 70 75 80Thr Cys Asn Val Ala His Pro Ala Ser Ser
Thr Lys Val Asp Lys Lys 85 90 95Ile Glu Pro Arg Gly Pro Thr Ile Lys
Pro Cys Pro Pro Cys Lys Cys 100 105 110Pro Ala Pro Asn Leu Leu Gly
Gly Pro Ser Val Phe Ile Phe Pro Pro 115 120 125Lys Ile Lys Asp Val
Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 130 135 140Val Val Val
Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp145 150 155
160Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
Ile Gln 180 185 190His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
Lys Val Asn Asn 195 200 205Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr
Ile Ser Lys Pro Lys Gly 210 215 220Ser Val Arg Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Glu Glu Glu225 230 235 240Met Thr Lys Lys Gln
Val Thr Leu Thr Cys Met Val Thr Asp Phe Met 245 250 255Pro Glu Asp
Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu 260 265 270Asn
Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe 275 280
285Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
His Thr305 310 315 320Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325
33010932DNAArtificial SequenceSynthetic primer sequence
109taagaattcc accatgagtg tgcccactca gg 3211021DNAArtificial
SequenceSynthetic primer sequence 110gcccgtttga tctccagctt g
21111333DNAMus musculus 111tcgcgatgcg gccccaactg tatccatctt
cccaccatcc agtgagcagt taacatctgg 60aggtgcctca gtcgtgtgct tcttgaacaa
cttctacccc aaagacatca atgtcaagtg 120gaagattgat ggcagtgaac
gacaaaatgg cgtcctgaac agttggactg atcaggacag 180caaagacagc
acctacagca tgagcagcac cctcacgttg accaaggacg agtatgaacg
240acataacagc tatacctgtg aggccactca caagacatca acttcaccca
ttgtcaagag 300cttcaacagg aatgagtgtt gataagcggc cgc 333112107PRTMus
musculus 112Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser
Ser Glu1 5 10 15Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu
Asn Asn Phe 20 25 30Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp
Gly Ser Glu Arg 35 40 45Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu
Thr Lys Asp Glu Tyr Glu65 70 75 80Arg His Asn Ser Tyr Thr Cys Glu
Ala Thr His Lys Thr Ser Thr Ser 85 90 95Pro Ile Val Lys Ser Phe Asn
Arg Asn Glu Cys 100 1051131430DNAArtificial SequenceSynthetic
nucleotide sequence 113atgggatgga actggatctt tctcttcctc ctgtcaggaa
ctgcaggtgt cctctctgag 60gtccagctgc agcagtctgg acctgaactg gtgaagcctg
gggcttcagt gaagatatcc 120tgcaaggctt ctggatacac attcactgac
tacaacatgg actgggtgaa gcagagccat 180ggagagagcc ttgagtggat
tggatatatt tatcctaaca atggtggttc tggctacaac 240cagaagttca
agagcaaggc cacattgact gtagacaagt cctccagcac agcctacatg
300gagctccaca gcctgacatc tgaagactct gcagtctatt actgtgcaag
acgggatagt 360tactatggtt tcgacatggc ctggtttgct tactggggcc
aagggactct ggtcactgtc 420tctgcagcga aaacaacagc cccatcggtc
tatccactgg cccctgtgtg tggagataca 480actggctcct cggtgactct
aggatgcctg gtcaagggtt atttccctga gccagtgacc 540ttgacctgga
actctggatc cctgtccagt ggtgtgcaca ccttcccagc tgtcctgcag
600tctgacctct acaccctcag cagctcagtg actgtaacct cgagcacctg
gcccagccag 660tccatcacct gcaatgtggc ccacccggca agcagcacca
aggtggacaa gaaaattgag 720cccagagggc ccacaatcaa gccctgtcct
ccatgcaaat gcccagcacc taacctcttg 780ggtggaccat ccgtcttcat
cttccctcca aagatcaagg atgtactcat gatctccctg 840agccccatag
tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc
900agctggtttg tgaacaacgt ggaagtacac acagctcaga cacaaaccca
tagagaggat 960tacaacagta ctctccgggt ggtcagtgcc ctccccatcc
agcaccagga ctggatgagt 1020ggcaaggagt tcaaatgcaa ggtcaacaac
aaagacctcc cagcgcccat cgagagaacc 1080atctcaaaac ccaaagggtc
agtaagagca ccacaggtat atgtcttgcc tccaccagaa 1140gaagagatga
ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa
1200gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa
gaacactgaa 1260ccagtcctgg actctgatgg ttcttacttc atgtacagca
agctgagagt ggaaaagaag 1320aactgggtgg aaagaaatag ctactcctgt
tcagtggtcc acgagggtct gcacaatcac 1380cacacgacta agagcttctc
ccggactccg ggtaaatgat aagcggccgc 1430114472PRTArtificial
SequenceSynthetic peptide sequence 114Met Gly Trp Asn Trp Ile Phe
Leu Phe Leu Leu Ser Gly Thr Ala Gly1 5 10 15Val Leu Ser Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Val Lys 20 25 30Pro Gly Ala Ser Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45Thr Asp Tyr Asn
Met Asp Trp Val Lys Gln Ser His Gly Glu Ser Leu 50 55 60Glu Trp Ile
Gly Tyr Ile Tyr Pro Asn Asn Gly Gly Ser Gly Tyr Asn65 70 75 80Gln
Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90
95Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110Tyr Tyr Cys Ala Arg Arg Asp Ser Tyr Tyr Gly Phe Asp Met
Ala Trp 115 120 125Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ala Ala Lys 130 135 140Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala
Pro Val Cys Gly Asp Thr145 150 155 160Thr Gly Ser Ser Val Thr Leu
Gly Cys Leu Val Lys Gly Tyr Phe Pro 165 170 175Glu Pro Val Thr Leu
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val 180 185 190His Thr Phe
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser 195 200 205Ser
Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys 210 215
220Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile
Glu225 230 235 240Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys
Lys Cys Pro Ala 245 250 255Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
Ile Phe Pro Pro Lys Ile 260 265 270Lys Asp Val Leu Met Ile Ser Leu
Ser Pro Ile Val Thr Cys Val Val 275 280 285Val Asp Val Ser Glu Asp
Asp Pro Asp Val Gln Ile Ser Trp Phe Val 290 295 300Asn Asn Val Glu
Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp305 310 315 320Tyr
Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln 325 330
335Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
340 345 350Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
Ser Val 355 360 365Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
Glu Glu Met Thr 370 375 380Lys Lys Gln Val Thr Leu Thr Cys Met Val
Thr Asp Phe Met Pro Glu385 390 395 400Asp Ile Tyr Val Glu Trp Thr
Asn Asn Gly Lys Thr Glu Leu Asn Tyr 405 410 415Lys Asn Thr Glu Pro
Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr 420 425 430Ser Lys Leu
Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr 435 440 445Ser
Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys 450 455
460Ser Phe Ser Arg Thr Pro Gly Lys465 470115716DNAArtificial
SequenceSynthetic nucleotide sequence 115atgagtgtgc ccactcaggt
cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60gacatccaga tgactcagtc
tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 120atcacatgtc
gaccaagtga gaatatttac aataatttag catggtatca acagaaacag
180ggaaaatctc ctcagctcct ggtctatgtt gcaacaaatt tagcagaagg
tgtgccatca 240aggttcagtg gcagtggatc aggcacacgg ttttctctga
agatcaacag cctgcagcct 300gaagattttg ggaagtatta ctgtcaacat
tcttatggta ctccgtggac gttcggtgga 360ggcaccaagc tggagatcaa
acgggccgat gcggccccaa ctgtatccat cttcccacca 420tccagtgagc
agttaacatc tggaggtgcc tcagtcgtgt gcttcttgaa caacttctac
480cccaaagaca tcaatgtcaa gtggaagatt gatggcagtg aacgacaaaa
tggcgtcctg 540aacagttgga ctgatcagga cagcaaagac agcacctaca
gcatgagcag caccctcacg 600ttgaccaagg acgagtatga acgacataac
agctatacct gtgaggccac tcacaagaca 660tcaacttcac ccattgtcaa
gagcttcaac aggaatgagt gttgataagc ggccgc 716116234PRTArtificial
SequenceSynthetic peptide sequence 116Met Ser Val Pro Thr Gln Val
Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Gly Ala Arg Cys Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser 20 25 30Ala Ser Val Gly Glu
Thr Val Thr Ile Thr Cys Arg Pro Ser Glu Asn 35 40 45Ile Tyr Asn Asn
Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro 50 55 60Gln Leu Leu
Val Tyr Val Ala Thr Asn Leu Ala Glu Gly Val Pro Ser65 70 75 80Arg
Phe Ser Gly Ser Gly Ser Gly Thr Arg Phe Ser Leu Lys Ile Asn 85 90
95Ser Leu Gln Pro Glu Asp Phe Gly Lys Tyr Tyr Cys Gln His Ser Tyr
100 105 110Gly Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 115 120 125Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro
Ser Ser Glu Gln 130 135 140Leu Thr Ser Gly Gly Ala Ser Val Val Cys
Phe Leu Asn Asn Phe Tyr145 150 155 160Pro Lys Asp Ile Asn Val Lys
Trp Lys Ile Asp Gly Ser Glu Arg Gln 165 170 175Asn Gly Val Leu Asn
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Met
Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 195 200 205His
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 210 215
220Ile Val Lys Ser Phe Asn Arg Asn Glu Cys225 230
* * * * *
References