U.S. patent application number 16/168275 was filed with the patent office on 2019-04-11 for antibody against peptide encoded by exon-21 of periostin and pharmaceutical composition for preventing or treating inflammation-associated diseases containing the same.
This patent application is currently assigned to OSAKA UNIVERSITY. The applicant listed for this patent is OSAKA UNIVERSITY. Invention is credited to Junya AZUMA, Naruto KATSURAGI, Ryuichi MORISHITA, Fumihiro SANADA, Yoshiaki TANIYAMA.
Application Number | 20190106484 16/168275 |
Document ID | / |
Family ID | 51491417 |
Filed Date | 2019-04-11 |
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United States Patent
Application |
20190106484 |
Kind Code |
A1 |
TANIYAMA; Yoshiaki ; et
al. |
April 11, 2019 |
ANTIBODY AGAINST PEPTIDE ENCODED BY EXON-21 OF PERIOSTIN AND
PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING
INFLAMMATION-ASSOCIATED DISEASES CONTAINING THE SAME
Abstract
The present invention provides a drug for preventing or treating
inflammation-associated diseases in which a periostin isoform
having cell adhesion activity is involved, and also provides an
inhibitor of a periostin isoform having cell adhesion activity.
Inventors: |
TANIYAMA; Yoshiaki; (Osaka,
JP) ; MORISHITA; Ryuichi; (Osaka, JP) ; AZUMA;
Junya; (Osaka, JP) ; SANADA; Fumihiro; (Osaka,
JP) ; KATSURAGI; Naruto; (Hyogo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OSAKA UNIVERSITY |
Osaka |
|
JP |
|
|
Assignee: |
OSAKA UNIVERSITY
Osaka
JP
|
Family ID: |
51491417 |
Appl. No.: |
16/168275 |
Filed: |
October 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14772122 |
Dec 1, 2015 |
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PCT/JP2014/055861 |
Mar 6, 2014 |
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16168275 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/68 20130101;
C07K 2317/34 20130101; C07K 14/47 20130101; A61P 35/00 20180101;
C07K 2317/76 20130101; A61P 9/08 20180101; C07K 2317/14 20130101;
A61P 29/00 20180101; C07K 2317/73 20130101; A61K 2039/505 20130101;
C07K 16/18 20130101; G01N 2333/47 20130101; C12N 5/12 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 14/47 20060101 C07K014/47; C12N 5/12 20060101
C12N005/12; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2013 |
JP |
2013-047097 |
Claims
1-16. (canceled)
17. An antibody binding to one or more peptides selected from the
group consisting of a peptide consisting of an amino acid sequence
of SEQ ID NO: 6, a peptide consisting of an amino acid sequence of
SEQ ID NO: 17 and a peptide consisting of an amino acid sequence of
SEQ ID NO: 18.
18. The antibody according to claim 17, which specifically binds to
one or more peptides selected from the group consisting of a
peptide consisting of an amino acid sequence of SEQ ID NO: 19, a
peptide consisting of an amino acid sequence of SEQ ID NO: 20, a
peptide consisting of an amino acid sequence of SEQ ID NO: 21 and a
peptide consisting of an amino acid sequence of SEQ ID NO: 22.
19. The antibody according to claim 17, which specifically
recognizes a cell adhesion activity-related region of a periostin
isoform having cell adhesion activity and neutralizes the cell
adhesion activity of the periostin isoform.
20. The antibody according to claim 17, which is a polyclonal
antibody, a monoclonal antibody, a chimeric antibody, a humanized
antibody or a human antibody.
21. The antibody according to claim 20, which is a monoclonal
antibody.
22. The antibody according to claim 21, which is produced by a
hybridoma cell line designated as NITE BP-01546.
23. An antibody fragment consisting of a partial fragment of the
monoclonal antibody according to claim 21.
24. An antibody derivative comprising a protein or low molecular
weight drug linked to the antibody according to claim 17.
25. An antibody derivative comprising a protein or low molecular
weight drug linked to the antibody fragment according to claim
23.
26. A hybridoma cell line designated as NITE BP-01546.
27. A method for producing the antibody according to claim 20, the
method comprising immunizing a non-human mammal with a peptide
consisting of an amino acid sequence selected from the group
consisting of an amino acid sequence of SEQ ID NO: 6, an amino acid
sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO:
18, or the peptide having a Cys residue added to the N-terminus,
fusing an antibody-producing cell of the animal with a myeloma cell
to form a hybridoma, and culturing the hybridoma.
28. A method for detecting or quantifying a periostin isoform
having cell adhesion activity in a biological sample by using the
antibody according to claim 17.
29. A method for detecting or quantifying a periostin isoform
having cell adhesion activity in a biological sample by using the
antibody fragment according to claim 23.
30. A method for detecting or quantifying a periostin isoform
having cell adhesion activity in a biological sample by using the
antibody derivative according to claim 24.
31. The antibody according to claim 17, which is produced using as
an antigen a periostin Exon-21 peptide alone.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antibody against a
peptide encoded by Exon-21 of periostin and a pharmaceutical
composition for preventing or treating inflammation-associated
diseases containing the same.
BACKGROUND ART
[0002] Periostin is an extracellular matrix protein consisting of a
polypeptide of a molecular weight of about 90,000. The polypeptide
chain contains a signal sequence, a cysteine-rich domain, a
fourfold repeated domain, and a C-terminal domain.
[0003] Periostin was initially designated osteoblast-specific
factor-2 (OSF-2) and was isolated and identified as a gene
specifically expressed in the mouse osteoblast cell line MC3T3-E1
(Patent Literature 1, Non Patent Literature 1). The protein was
later renamed periostin and was reported to have adhesion-promoting
activity in osteoblast cells (Non Patent Literature 2).
[0004] In early studies, periostin was considered to be an
extracellular matrix specifically expressed in bone tissue.
However, it has been revealed that periostin is highly expressed
not only in bone tissue but also at the onset of heart failure (Non
Patent Literature 3, Non Patent Literature 4), aneurysm (Non Patent
Literature 5), cancers (Non Patent Literature 6 to 8), preeclampsia
(Non Patent Literature 9), vascular restenosis (Non Patent
Literature 10 to 15), inflammatory diseases ((i) esophagitis (Non
Patent Literature 16), (ii) sinusitis and asthma (Non Patent
Literature 17), (iii) asthma (Non Patent Literature 18), (iv)
angiogenesis (Non Patent Literature 6, Non Patent Literature 19 to
22)), etc. and that the protein is very slightly expressed in
normal tissue. It has also been revealed that some periostin splice
variants are expressed in osteoblasts (Non Patent Literature 1 and
2, Non Patent Literature 23, Patent Literature 2).
[0005] As to the functions of periostin, a periostin splice variant
of 811 amino acids (corresponding to PN-2 in FIG. 1) (Non Patent
Literature 2) and a periostin splice variant of 783 amino acids
(corresponding to PN-4 in FIG. 1) (Non Patent Literature 24) have
been reported to have cell adhesion properties. In contrast, some
periostin splice variants lack cell adhesion properties and they
include a periostin splice variant of 838 amino acids
(corresponding to PN-1 in FIG. 1) (Patent Literature 2) and a
periostin splice variant of 810 amino acids (corresponding to PN-3
in FIG. 1) (Patent Literature 4).
[0006] As regards cancers, cancer metastasis is mediated by
processes such as invasion of cancer cells from the primary tumor
into blood vessels or lymph vessels, selective migration of cancer
cells to metastatic organs, invasion of cancer cells from blood
vessels into metastatic organs, growth of cancer cells supported by
the microenvironment where metastasis occurred, and
angiogenesis-associated growth of a tumor whose diameter exceeds
several millimeters (Non Patent Literature 25 and 26). Among these
complex processes for metastasis establishment, invasion and
metastasis induced by the enhanced motility of cancer cells are
very important stages (Non Patent Literature 27). Until now, it has
been reported that highly metastatic cancer cells produce an
autocrine motility factor by themselves to enhance their own motion
(Non Patent Literature 28). Inhibitory substances against this
malignant factor are expected as metastasis inhibitors, but no
specific inhibitor has been found at present.
[0007] Various reports have been issued on high level expression of
periostin in highly metastatic cancers (pancreatic cancer (Non
Patent Literature 29), oral cancer (Non Patent Literature 21),
pancreatic cancer (Non Patent Literature 30), breast cancer (Non
Patent Literature 31), head and neck cancer (Non Patent Literature
32), colon cancer (Non Patent Literature 19), breast cancer (Non
Patent Literature 6, 8 and 33), thymic cancer (Non Patent
Literature 34 and 35), non-small cell lung cancer (Non Patent
Literature 36 and 37), ovarian cancer (Non Patent Literature 38),
prostate cancer (Non Patent Literature 39), liver cancer and bile
duct cancer (Non Patent Literature 40), esophagus squamous cancer
(Non Patent Literature 41), prostate cancer (Non Patent Literature
42), thyroid cancer (Non Patent Literature 43)). The high level
expression of the transcription factor Twist in highly metastatic
cancers has been reported (Non Patent Literature 44 and 45) and
received attention. There has been a report showing that Twist is
also located in the promoter region of periostin (Non Patent
Literature 46). In addition, it has been reported that the invasion
ability of the human embryonic kidney epithelial cell line 293T is
enhanced when the periostin gene is introduced into the cell line
(Non Patent Literature 47). It has also been reported that a
periostin splice variant of 811 amino acids (corresponding to PN-2
in FIG. 1) was less expressed in various cancer cells, and
introduction of the periostin gene into melanoma cells inhibited
their metastasis to the lung (Non Patent Literature 48).
[0008] As regards vascular restenosis, a bare metal stent (BMS) has
been widely used. Three to eight months after BMS implantation,
in-stent restenosis (ISR) occurs in 10 to 40% of cases. The
mechanism of ISR is considered to be principally neointimal
hyperplasia associated with migration of smooth muscle cells from
the tunica media of the coronary artery into the stent and
subsequent proliferation of the cells (Non Patent Literature 49).
To overcome the drawback, a drug-eluting stent (DES), which is a
stent with the surface coated with a drug, was developed as a
sirolimus-eluting stent (SES) by Sousa et al. in 1999. However,
late stent thrombosis caused by DES has been reported, and
accordingly a drug for safely inhibiting restenosis has been
desired. Various papers have reported high level expression of
periostin in vessel smooth muscle of an animal model with balloon
injury-induced restenosis (Non Patent Literature 10 to 15).
[0009] As regards inflammations, antiinflammatory drugs have been
clinically used and they include steroidal and non-steroidal
antiinflammatory drugs for acute and chronic inflammatory diseases,
and immunosuppressants and gold preparations for chronic
progressive inflammatory diseases (for example, rheumatism,
osteoarthritis, etc.). The main mechanism of action of
non-steroidal antiinflammatory drugs is the inhibition of
inflammatory mediators. The drugs provide symptomatic treatment and
are effective for acute diseases, but less effective for chronic
inflammatory diseases. Steroidal antiinflammatory drugs are highly
effective for acute and chronic inflammatory diseases, but have
been reported to concomitantly cause serious side effects.
Accordingly, care should be taken when the drugs are used. Gold
preparations are not applied to acute inflammation diseases, but
are used for chronic rheumatism. Gold preparations have
immunoregulatory activity and thus exert delayed effects. Gold
preparations, however, also have been reported to cause side
effects, including mucosal and cutaneous symptoms,
myelosuppression, renal dysfunction and respiratory dysfunction.
Accordingly, as with the case of steroidal antiinflammatory drugs,
sufficient care should be taken when gold preparations are used.
Some of immunosuppressants have also received attention in terms of
clinical application to chronic rheumatism, but the side effects
characteristic of immunosuppressants are of a concern.
[0010] Enhanced expression of periostin in inflammatory diseases
has been reported (Non Patent Literature 16 to 18, 50 and 51).
[0011] Angiogenesis is closely associated with, in addition to
cancers, aggravation of some diseases including diabetic
retinopathy, atherosclerosis, periodontosis, scleroderma, glaucoma,
age-related macular degeneration, and diabetes mellitus type II.
Angiogenesis also plays pivotal roles in the onset and aggravation
of rheumatoid arthritis, Kaposi sarcoma, psoriasis and Basedow
disease (Non Patent Literature 52). It has been also shown that
expansion of adipose tissue depends on angiogenesis, and the
inhibition of angiogenesis has been reported to be effective for
the prevention of obesity etc. (Non Patent Literature 53). In
Alzheimer's disease, cerebral endothelial cells activated by
angiogenesis secrete a precursor substrate for .beta.-amyloid and a
neurotoxic peptide that selectively kills cortical neurons, and
hence the inhibition of angiogenesis has been reported to be
effective for the prevention and treatment of Alzheimer's disease
(Non Patent Literature 54). Based on these studies, angiogenesis
inhibitors have been used to treat and prevent the above diseases
in recent years, and there has been a demand for substances
effective for inhibiting angiogenesis.
[0012] Angiogenesis inhibitors that have been found are angiostatin
(Non Patent Literature 55 and 56); endostatin (Non Patent
Literature 57); fumagillin derived from Aspergillus fumigatus and
its synthetic derivative TNP-470 (Non Patent Literature 58);
cytogenin (Non Patent Literature 59); synthetic chemical
substances, such as metalloproteinase inhibitors, batimastat
(BB-94) and marimastat (BB-2516) (Non Patent Literature 60 and 61);
and monoclonal antibodies that inhibit the binding of angiogenesis
factors (EGF, TGF-.alpha., VEGF, etc.) to the corresponding
receptors (Non Patent Literature 62). These substances, however,
require careful consideration of the side effects, and the safety
of the substances to a human body is not fully guaranteed.
[0013] Expression of periostin has been reported to be closely
related to angiogenesis in the onset of cancers (Non Patent
Literature 6, Non Patent Literature 19, Non Patent Literature 21
and 22). Induction of angiogenesis by periostin has been reported
to be achieved through the expression of VEGF receptor-2
(Flk-1/KDR) in vascular endothelial cells (Non Patent Literature
20).
[0014] As described above, it has been indicated that periostin
gene expression is related to vascular restenosis conditions,
cancers, inflammations and angiogenesis conditions. Reports have
also been made on an antibody relating to the inhibition of cell
migration mediated by periostin (Non Patent Literature 12) and an
antibody having inhibitory activity against periostin-induced cell
growth (Non Patent Literature 13). However, the relation of the
structure of a periostin splice variant to vascular restenosis,
cancers, inflammations and angiogenesis still remains unclear.
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SUMMARY OF INVENTION
Technical Problem
[0081] An object of the present invention is to provide an
inhibitor of a periostin isoform having cell adhesion activity.
Another object of the present invention is to provide a novel agent
for preventing or treating vascular restenosis, cancers,
inflammations, angiogenesis and arteriosclerosis, the agent having
a mechanism different from that of existing agents and being
capable of improving the quality of life and long-term prognosis.
Further, another object of the present invention is to provide a
therapeutic method, a diagnostic method and a diagnostic reagent
for vascular restenosis, cancers, inflammations and
angiogenesis.
Solution to Problem
[0082] The inventors forced periostin splice variants PN-2 and PN-4
to be expressed, purified the proteins, and coated a plate with
each of the proteins to investigate the adhesion of fibroblasts to
the plate. The adhesion of the fibroblasts varied with the types of
the splice variants. Periostin splice variant PN-2 showed
significantly stronger cell adhesion activity than the negative
control albumin (BSA) and the uncoated group, whereas periostin
splice variant PN-4 showed weaker cell adhesion activity than the
uncoated group and merely had very weak cell adhesion activity
compared with the negative control albumin (BSA) (FIG. 2). The
difference in adhesion ability indicated that the Exon-21 region is
involved in cell adhesion. Based on this, the inventors presumed
that an anti-periostin Exon-21 polyclonal antibody produced by
using the Exon-21 region as an antigen will inhibit the cell
adhesion by PN-2.
[0083] Analysis of periostin splice variants highly expressed in
vascular restenosis, cancers, inflammatory colitis or angiogenesis
conditions revealed that the C-terminal domain from which the
splice variants are derived consists of Exons 15 to 23, and that
rats have the following variants (1) to (4) (see FIG. 1).
(1) a variant retaining all the exons (called PN-1; consisting of
838 amino acids of SEQ ID NO: 1; the cDNA sequence is shown in SEQ
ID NO: 2) (2) a variant lacking Exon-17 (called PN-2; consisting of
811 amino acids of SEQ ID NO: 3; 27 amino acids (Exon-17) of SEQ ID
NO: 4 are deleted from PN-1; the cDNA sequence is shown in SEQ ID
NO: 5) (3) a variant lacking Exon-21 (called PN-3; consisting of
810 amino acids; the amino acids at positions 785 to 812 from the
N-terminus (28 amino acids of SEQ ID NO: 6 (Exon-21)) are deleted
from PN-1) (4) a variant lacking Exon-17 and Exon-21 (called PN-4;
consisting of 783 amino acids of SEQ ID NO: 7; 28 amino acids
(Exon-21) of SEQ ID NO: 6 are deleted from PN-2; the cDNA sequence
is shown in SEQ ID NO: 8)
[0084] In addition to the rat splice variants, mouse and human PN-2
and PN-4 were also found (mouse PN-2 (SEQ ID NO: 9 (amino acid
sequence), SEQ ID NO: 10 (cDNA sequence)); mouse PN-4 (SEQ ID NO:
11 (amino acid sequence), SEQ ID NO: 12 (cDNA sequence)); human
PN-2 (SEQ ID NO: 13 (amino acid sequence), SEQ ID NO: 14 (cDNA
sequence)); human PN-4 (SEQ ID NO: 15 (amino acid sequence), SEQ ID
NO: 16 (cDNA sequence))).
[0085] The inventors attempted to produce an antibody specifically
recognizing the amino acid residues encoded by Exon-21 as an
inhibitor of the exon, which exon is the structural difference
between PN-2 and PN-4 and exclusively found in PN-2.
[0086] In order to produce an antibody, the material used as an
immunogen must be hydrophilic, and when part of a large
polypeptide, such as a protein, is used to produce an antibody, the
part to be used as an immunogen must be exposed on the surface of
the protein and form an epitope. Thus, in order to examine the
possibility of using the Exon-21 peptide chain as an antigen, an
epitope search was initially performed using Accelrys software Mac
Vector 7.2, which is widely used in the bioinformatics field. The
exon region showed some "hydrophilicity", but it was suggested from
the "surface probability" and "antigenicity" that the amino acid
sequence of the Exon-21 region of SEQ ID NO: 6
(EVSKVTKFIEGGDGHLFEDEAIKRLLQG) is very unlikely to be exposed on
the surface of the protein molecule and has no immunogenicity. It
was therefore presumed that the exon region cannot be used as an
immunogen to produce an antibody, and that it would be difficult to
practically produce an antibody against the exon region.
[0087] However, based on the belief that the use of an antibody
against the polypeptide region encoded by Exon-21 found in PN-2
would be optimal for the specific inhibition of the PN-2 protein
functions, the inventors attempted to produce an antibody against
the amino acid sequence encoded by Exon-21. The inventors
synthesized a peptide consisting of 28 amino acids constituting the
peptide encoded by the Exon-21 region, immunized rabbits with the
peptide, and purified an IgG fraction from the serum, thereby
succeeding in the production of an anti-rat Exon-21 peptide
polyclonal antibody.
[0088] The inventors conducted an investigation to determine
whether the anti-rat Exon-21 polyclonal antibody has inhibitory
activity against the adhesion activity of periostin. Mouse PN-2
protein was coated on rat vascular smooth muscle cells that had
been cultured to subconfluence, and monocyte-derived THP-1 cells
were seeded thereon, resulting in the adhesion of the THP-1 cells.
Separately, PN-2 protein was mixed with the antibody, and the
mixture and THP-1 cells were seeded, resulting in the inhibition of
the adhesion of the THP-1 cells. The overall results confirmed that
the anti-rat Exon-21 polyclonal antibody has inhibitory activity
against the adhesion activity of periostin. The anti-rat Exon-21
polyclonal antibody inhibits the adhesion of THP-1 cells, thereby
inhibiting the differentiation of the cells into macrophages. Hence
the antibody has an inhibitory function against macrophage invasion
into organs. Macrophages are often associated with various
inflammatory diseases, and the anti-rat Exon-21 polyclonal
antibody, therefore, was shown to have anti-inflammatory activity
(FIG. 3).
[0089] The inventors investigated the inhibitory effect on vascular
intimal hyperplasia using an animal model. Injury was induced in
the left carotid artery of SD rats with the use of a balloon
catheter, and simultaneously the anti-rat Exon-21 polyclonal
antibody was intravascularly administered. After a certain period
of time, the blood vessels were harvested, fixed, and stained with
HE. The area ratio of the intima to the media was calculated and
the severity of intimal hyperplasia was examined. No significant
difference was found in the area of the media, but a significant
difference was observed in the intima/media ratio between the
anti-rat Exon-21 polyclonal antibody administration group and the
scratching-alone group. The results showed the inhibition of
intimal hyperplasia (FIG. 4A).
[0090] Based on the report of the association of PN-2 protein with
inflammations, the inventors investigated the effect on
arteriosclerosis by the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) (the polyclonal antibody against the peptide encoded by
Exon-21 of rat periostin). The investigation was performed using a
model of ApoE knockout (KO) mice, which are generally considered to
be an arteriosclerosis-prone model, loaded with a high-fat diet for
three months. The anti-rat Exon-21 polyclonal antibody
(ex21PoAb)-administered mice showed significant inhibition of
arteriosclerosis in the thoracic aorta (the upper part of the
aorta), as evaluated by Oil red O staining, compared with the
rabbit control IgG antibody (rIgG)-administered mice. The results
revealed the arteriosclerosis inhibitory effect of the anti-rat
Exon-21 polyclonal antibody (FIG. 5).
[0091] The inventors investigated the anti-inflammatory effect of
an anti-human Exon-21 monoclonal antibody (ex21MoAb) in a model
with Crohn's disease, which is inflammatory colitis typifying
inflammatory diseases. The anti-human Exon-21 monoclonal antibody
(ex21MoAb) or a mouse control IgG antibody (mIgG) was administered
to male C57B6 mice at 8 weeks of age, and 1.75% dextran sulfate
sodium (DDS) was administered via drinking water. Two weeks later,
the length of the large intestine was significantly well maintained
in the anti-human Exon-21 monoclonal antibody (ex21MoAb)
administration group as compared with the mouse control IgG
antibody (mIgG) administration group (FIG. 6). The anti-human
Exon-21 monoclonal antibody (ex21MoAb) showed anti-inflammatory
activity in the mouse colitis model.
[0092] Based on the report of the association of PN-2 protein with
angiogenesis, the inventors investigated the angiogenesis
inhibitory effect of the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) and the anti-human Exon-21 monoclonal antibody
(ex21MoAb) in an animal model. After confirmation of the expression
of the PN-2 gene in a lower extremity ischemia mouse model, the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) was administered,
as a result of which a significant inhibitory effect on
angiogenesis was observed (FIG. 7B). Immunostaining of the blood
vessels in the tissue with a CD31 antibody showed significant
inhibition of angiogenesis in the anti-rat Exon-21 polyclonal
antibody (ex21PoAb) administration group (FIG. 8). The anti-human
Exon-21 monoclonal antibody (ex21MoAb) also showed an inhibitory
effect on the lower extremity blood flow (FIG. 9). The angiogenesis
inhibitory effect of the anti-Exon-21 antibodies suggested
potential inhibitory effect of the anti-Exon-21 antibodies on
pathological angiogenesis.
[0093] The inventors performed Matrigel assay using cultured cells.
The supernatant of MDA-MB231 human breast cancer cells
significantly increased angiogenesis in human endothelial cells,
whereas addition of the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) or the anti-human Exon-21 monoclonal antibody (ex21MoAb)
significantly inhibited angiogenesis (FIGS. 10A and 10B). It was
also shown that PN-2 induced angiogenesis in a dose-dependent
manner (FIGS. 11A and 11B). The results showed that the
anti-Exon-21 antibodies inhibit angiogenesis induced by a
cancer.
[0094] The inventors investigated the effect of the anti-rat
Exon-21 polyclonal antibody (ex21PoAb) on the proliferative
capacity of cultured mouse 4T1 breast cancer cells, and as a
result, significant cytostatic activity was observed by MTS assay,
as compared with the administration of a rabbit control IgG
antibody (rIgG) (FIG. 12A). The inventors also investigated the
effect of the anti-human Exon-21 monoclonal antibody (ex21MoAb) on
the necrosis of mouse 4T1 breast cancer cells, and as a result,
significant necrosis induction activity was observed by the
measurement of LDH in the supernatant, as compared with the
administration of a mouse control IgG antibody (mIgG). The results
confirmed the direct proliferation inhibitory effect and direct
necrosis induction effect of the anti-Exon-21 antibodies on mouse
411 breast cancer cells (FIG. 12B).
[0095] Mouse 4T1 breast cancer cells were injected into the foot
pad of mice to establish lung metastasis model mice, and then the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) was administered to
the model mice once a week. Three to five weeks after the
establishment of the model, significant inhibition of primary tumor
growth as well as of the number of lung metastatic colonies from
the primary tumors was observed, as compared with a rabbit control
IgG antibody (rIgG) administration group (FIGS. 13A and 13B). The
anti-human Exon-21 monoclonal antibody (ex21MoAb) was administered
in the same manner as above, and as a result, significant
inhibition of lung metastasis was observed as compared with a mouse
control IgG antibody (mIgG) administration group (FIG. 14).
[0096] B16F10 mouse melanoma cells were injected into the foot pad
of mice to establish lung metastasis model mice, and then the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) was administered to
the model mice once a week. Three weeks after the establishment of
the model, significant inhibition of primary tumor growth as well
as of the number of lung metastatic colonies from the primary
tumors was observed, as compared with a rabbit control IgG antibody
(rIgG) administration group (FIGS. 15A and 15B). The neutralizing
antibodies against PN-2 (the anti-Exon-21 antibodies) were assumed
to inhibit the PN-2 functions, such as the promoting effect for the
adhesion of macrophages and the angiogenic effect, and thereby to
inhibit the growth and lung metastasis of breast cancer cells or
melanoma cells. This assumption suggested the potential of the
neutralizing antibodies as novel therapeutic agents. The
experimental results revealed that the anti-Exon-21 antibodies have
inhibitory activity on primary tumor growth, which proceeds along
with the progression of the cancer conditions, and also have
inhibitory activity on lung metastasis from the primary tumors.
[0097] The inventors investigated the effect of the anti-human
Exon-21 monoclonal antibody (ex21MoAb) on arteriosclerosis. The
anti-human Exon-21 monoclonal antibody (ex21MoAb) was administered
to an aneurysm mouse model once a week, and as a result,
significant inhibition of the expansion of the diameter of the
aorta was observed as measured with an ultrasound scanner, as
compared with a mouse control IgG antibody (mIgG) administration
group. The results revealed the aneurysm inhibitory effect of the
anti-human Exon-21 monoclonal antibody (ex21MoAb) (FIG. 16). Thus
the inventors completed the present invention.
[0098] The present invention solves the above problems. The present
invention provides an antibody against a periostin isoform having
cell adhesion activity that is specifically expressed in various
inflammation-associated conditions including cancers. In
particular, the present invention provides a composition for
treating various inflammation-associated diseases including
cancers, the composition comprising an antibody that recognizes the
splice site of the periostin isoform as an antigen.
[0099] That is, the present invention includes the following.
(1) An antibody binding to one or more peptides selected from the
group consisting of a peptide consisting of an amino acid sequence
of SEQ ID NO: 6, a peptide consisting of an amino acid sequence of
SEQ ID NO: 17 and a peptide consisting of an amino acid sequence of
SEQ ID NO: 18. (2) An antibody specifically binding to one or more
peptides selected from the group consisting of a peptide consisting
of an amino acid sequence of SEQ ID NO: 19, a peptide consisting of
an amino acid sequence of SEQ ID NO: 20, a peptide consisting of an
amino acid sequence of SEQ ID NO: 21 and a peptide consisting of an
amino acid sequence of SEQ ID NO: 22. (3) The antibody according to
the above (1) or (2), which specifically recognizes a cell adhesion
activity-related region of a periostin isoform having cell adhesion
activity and neutralizes the cell adhesion activity of the
periostin isoform. (4) The antibody according to any one of the
above (1) to (3), which is a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a humanized antibody or a human
antibody. (5) The antibody according to the above (4), which is a
monoclonal antibody. (6) The antibody according to the above (5),
which is produced by a hybridoma cell line designated as NITE
BP-01546. (7) An antibody fragment consisting of a partial fragment
of the monoclonal antibody according to the above (5) or (6). (8)
An antibody derivative comprising a protein or low molecular weight
drug linked to the antibody according to any one of the above (1)
to (6) or the antibody fragment according to the above (7). (9) A
hybridoma producing the antibody according to any one of the above
(1) to (5). (10) A hybridoma cell line designated as NITE BP-01546.
(11) A method for producing the antibody according to the above
(4), the method comprising
[0100] immunizing a non-human mammal with a peptide consisting of
an amino acid sequence selected from the group consisting of an
amino acid sequence of SEQ ID NO: 6, an amino acid sequence of SEQ
ID NO: 17 and an amino acid sequence of SEQ ID NO: 18, or the
peptide having a Cys residue added to the N-terminus,
[0101] fusing an antibody-producing cell of the animal with a
myeloma cell to form a hybridoma, and
[0102] culturing the hybridoma.
(12) The production method according to the above (11), wherein the
hybridoma is a hybridoma cell line designated as NITE BP-01546.
(13) A pharmaceutical composition for inhibiting a periostin
isoform having cell adhesion activity, the composition comprising
the antibody according to any one of the above (1) to (6), the
antibody fragment according to the above (7) or the antibody
derivative according to the above (8). (14) A pharmaceutical
composition for preventing or treating an inflammation-associated
disease in which a periostin isoform having cell adhesion activity
is involved, the composition comprising the antibody according to
any one of the above (1) to (6), the antibody fragment according to
the above (7) or the antibody derivative according to the above
(8). (15) A pharmaceutical composition for inhibiting vascular
intimal hyperplasia in which a periostin isoform having cell
adhesion activity is involved, treating a cancer in which a
periostin isoform having cell adhesion activity is involved,
inhibiting angiogenesis in which a periostin isoform having cell
adhesion activity is involved, or preventing or treating aneurysm
in which a periostin isoform having cell adhesion activity is
involved,
[0103] the composition comprising the antibody according to any one
of the above (1) to (6), the antibody fragment according to the
above (7) or the antibody derivative according to the above
(8).
(16) A method for detecting or quantifying a periostin isoform
having cell adhesion activity in a biological sample by using the
antibody according to any one of the above (1) to (6), the antibody
fragment according to the above (7) or the antibody derivative
according to the above (8).
[0104] The term "an amino acid sequence of SEQ ID NO: XX" herein
includes an amino acid sequence of SEQ ID NO: XX having deletion,
substitution or addition of one to several amino acids. The term
"several" means usually 2 to 8, preferably 2 to 5, more preferably
2 to 3.
Advantageous Effects of Invention
[0105] An inhibitor of a periostin isoform having cell adhesion
activity, the inhibitor comprising the antibody of the present
invention, is used to inhibit a particular periostin variant highly
expressed in vascular intimal hyperplasia, cancers, inflammations
including inflammatory colitis, diseases accompanied by
angiogenesis, or the like, thereby inhibiting the exacerbation of
the conditions of the diseases and treating the diseases. The
antibody can also be used for the measurement of the amount of such
a periostin variant in a patient sample to determine the presence
or absence of a disease and the progression of the disease
conditions.
BRIEF DESCRIPTION OF DRAWINGS
[0106] FIG. 1 is a schematic view showing mouse periostin splice
variants.
[0107] FIG. 2 is a chart showing the assay results of the cell
adhesion properties of rat PN-2 and PN-4 proteins in Example 1.
[0108] FIGS. 3A and 3B are images and a chart showing the results
of the study in Example 4. The images and chart indicates that
mouse PN-2 protein promotes the adhesion of THP-1 cells and induces
differentiation into macrophages, whereas an anti-rat Exon-21
polyclonal antibody inhibits the adhesion of THP-1 cells and
inhibits differentiation into macrophages.
[0109] FIGS. 4A and 4B are a chart and images showing the results
of the study on the neointimal hyperplasia inhibitory effect of the
anti-rat Exon-21 polyclonal antibody in a rat carotid artery
balloon injury model in Example 7.
[0110] FIG. 5 is a chart showing the results of the study on the
arteriosclerosis inhibitory effect of the anti-rat Exon-21
polyclonal antibody in an ApoE KO arteriosclerosis mouse model in
Example 8.
[0111] FIG. 6 is a chart showing the results of the study on the
inflammation inhibitory effect of an anti-human Exon-21 monoclonal
antibody in a mouse colitis model in Example 9.
[0112] FIG. 7A is a chart showing the timing of the administration
of an antibody in Example 10. FIG. 7B is a chart showing the
results of the study on the angiogenesis inhibitory effect of the
anti-rat Exon-21 polyclonal antibody in an arteriosclerosis
obliterans model in Example 10.
[0113] FIG. 8 is a chart showing the results of the study on the
angiogenesis inhibitory effect of the anti-rat Exon-21 polyclonal
antibody in an arteriosclerosis obliterans model in Example 10.
[0114] FIG. 9 is a chart showing the results of the study on the
angiogenesis inhibitory effect of the anti-human Exon-21 monoclonal
antibody in an arteriosclerosis obliterans model in Example 10.
[0115] FIGS. 10A and 10B are images and a chart showing the results
of the study on the angiogenesis inhibitory effect of the anti-rat
Exon-21 polyclonal antibody and the anti-human Exon-21 monoclonal
antibody in a Matrigel angiogenesis model using human endothelial
cells in Example 11. In the chart, the term "sham" indicates a sham
treatment group.
[0116] FIGS. 11A and 11B are images and a chart showing the results
of the study on the angiogenesis inhibitory effect of the anti-rat
Exon-21 polyclonal antibody and the anti-human Exon-21 monoclonal
antibody in a Matrigel angiogenesis model using human endothelial
cells in Example 11. In the chart, the term "sham" indicates a sham
treatment group.
[0117] FIG. 12A is a chart showing the results of the study on the
cancer cell proliferation inhibitory effect of the anti-rat Exon-21
polyclonal antibody on mouse 4T1 breast cancer cells in Example 12.
FIG. 12B is a chart showing the results of the study on the
necrosis induction effect of the anti-human Exon-21 monoclonal
antibody on mouse 4T1 breast cancer cells in Example 11.
[0118] FIG. 13A is a chart showing the results of the study on the
effect of the anti-rat Exon-21 polyclonal antibody using lung
metastasis model mice of mouse 4T1 breast cancer cells in Example
13 (injection of mouse 4T1 breast cancer cells, followed by
measurement of the volume of the primary tumors in the lower
extremities three weeks after the injection). FIG. 13B is a chart
showing the results of the study on the effect of the anti-rat
Exon-21 polyclonal antibody using lung metastasis model mice of
mouse 4T1 breast cancer cells in Example 13 (injection of mouse 4T1
breast cancer cells, followed by the counting of metastatic
colonies five weeks after the injection).
[0119] FIG. 14 is a chart showing the results of the study on the
effect of the anti-human Exon-21 monoclonal antibody using lung
metastasis model mice of mouse 4T1 breast cancer cells in Example
13 (injection of mouse 4T1 breast cancer cells, followed by the
counting of metastatic colonies five weeks after the
injection).
[0120] FIG. 15A is a chart showing the results of the study on the
effect of the anti-rat Exon-21 polyclonal antibody using lung
metastasis model mice of mouse melanoma B16-F10 cells in Example 14
(injection of mouse melanoma cells, followed by measurement of the
volume of the primary tumors three weeks after the injection). FIG.
15B is a chart showing the results of the study on the effect of
the anti-rat Exon-21 polyclonal antibody using lung metastasis
model mice of mouse melanoma B16-F10 cells in Example 14 (injection
of mouse melanoma cells, followed by the counting of metastatic
colonies five weeks after the injection).
[0121] FIG. 16 is a chart showing the results of the study on the
inhibitory effect of the anti-human Exon-21 monoclonal antibody on
the expansion of the diameter of the aorta in an aneurysm model in
Example 15.
DESCRIPTION OF EMBODIMENTS
[0122] An embodiment of the present invention provides an antibody
against a periostin isoform having cell adhesion activity.
Periostin is one of extracellular matrix proteins and several
splice variants of periostin are known. Some of periostin splice
variants are specifically expressed in cancer conditions etc. In
general, antibodies are highly specific, are safe for humans and
have other advantages, and therefore, in the present invention, an
antibody can be used as a substance for inhibition (i.e., an
inhibitory drug) against the functions of periostin splicing
variants specifically expressed in cancer conditions etc. In the
present invention, an antibody can be produced using, as an
antigen, a chemically synthesized peptide consisting of the amino
acid sequence encoded by Exon-21 in the C-terminal domain from
which splice variants specific to cancer conditions and the like
are derived. However, such a peptide can also be produced by
enzymatic digestion of periostin proteins or by genetic engineering
techniques, and the origin is not particularly limited.
[0123] The term "having cell adhesion activity" herein means the
possession of cell adhesion-promoting activity. An investigation to
determine whether a protein has cell adhesion activity can be
performed as follows. A 10 .mu.g/mL sample is placed in a petri
dish to allow a protein to adhere to the surface overnight.
Cultured cells such as cardiac fibroblasts are then added to the
dish. Three to six hours later, the dish is washed and detached
cells are removed. The remaining cells are dyed. The state of the
remaining adherent cells is examined.
[0124] In the present invention, the periostin isoform having cell
adhesion activity is not particularly limited, but preferred are a
periostin isoform consisting of an amino acid sequence of SEQ ID
NO: 3 (rat periostin PN-2, 811 amino acids), a periostin isoform
consisting of an amino acid sequence of SEQ ID NO: 9 (mouse
periostin PN-2, 811 amino acids), a periostin isoform consisting of
an amino acid sequence of SEQ ID NO: 13 (human periostin PN-2, 809
amino acids), which is easily predicted to have adhesion activity
since the amino acid sequence of human periostin is almost
identical to those of mouse and rat periostins, a periostin splice
variant having the amino acids constituting the peptide encoding
Exon-21 but lacking the amino acids constituting the peptide
encoding Exon-17, etc.
[0125] Periostin isoforms having cell adhesion activity include
periostin isoforms having an amino acid sequence of SEQ ID NO: 6
(28 amino acids encoded by Exon-21 of rat periostin), SEQ ID NO: 17
(28 amino acids encoded by Exon-21 of mouse periostin) or SEQ ID
NO: 18 (28 amino acids encoded by Exon-21 of human periostin).
[0126] Periostin isoforms that can serve as an epitope for an
antibody include a periostin isoform having an amino acid sequence
of SEQ ID NO: 19 (6 amino acids at positions 2 to 7 from the
N-terminus of the amino acid sequence encoded by Exon-21 of human
periostin (SEQ ID NO: 18)), a periostin isoform having an amino
acid sequence of SEQ ID NO: 20 (7 amino acids at positions 17 to 23
from the N-terminus of the amino acid sequence encoded by Exon-21
of human periostin (SEQ ID NO: 18)), a periostin isoform having an
amino acid sequence of SEQ ID NO: 21 (5 amino acids at positions 3
to 7 from the N-terminus of the amino acid sequence encoded by
Exon-21 of human periostin (SEQ ID NO: 18)), a periostin isoform
having an 8-amino acid sequence of SEQ ID NO: 22 consisting of 2
amino acids at positions 27 and 28 from the N-terminus of the amino
acid sequence encoded by Exon-21 of human periostin (SEQ ID NO: 18)
and the subsequent 6 amino acids, etc.
[0127] The regions responsible for the cell adhesion activity of
periostin include, for example, Exon-21. Specific examples of the
regions include the amino acid residues of SEQ ID NO: 6
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 3 (the amino acids at positions 758 to 785
of SEQ ID NO: 3), the amino acid residues of SEQ ID NO: 17
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 9 (the amino acids at positions 758 to 785
of SEQ ID NO: 9), the amino acid residues of SEQ ID NO: 18
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 13 (the amino acids at positions 756 to 783
of SEQ ID NO: 13), etc.
[0128] In a preferred embodiment of the present invention, the
phrase "specifically recognizes a site involved in cell adhesion"
means to specifically recognize preferably a cell adhesion-related
region of a periostin isoform containing Exon-21. Preferred
antibodies that specifically recognize a site involved in the cell
adhesion activity of a periostin isoform include, for example,
antibodies against the amino acid residues of SEQ ID NO: 6
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 3 (the amino acids at positions 758 to 785
of SEQ ID NO: 3), the amino acid residues of SEQ ID NO: 17
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 9 (the amino acids at positions 758 to 785
of SEQ ID NO: 9), the amino acid residues of SEQ ID NO: 18
representing a portion of a periostin isoform having an amino acid
sequence of SEQ ID NO: 13 (the amino acids at positions 756 to 783
of SEQ ID NO: 13), or part of any of the amino acid residues.
Further examples of the antibodies include antibodies against a
polypeptide having an amino acid sequence of SEQ ID NO: 19 or 20,
an amino acid sequence of SEQ ID NO: 21 or 22, or part of the amino
acid sequences.
[0129] The phrase "inhibits a region involved in the cell adhesion
activity of periostin" means inhibition of the effect or activity
of the above-described "region involved in the cell adhesion
activity of periostin". In particular, for example, the phrase
means inhibition of the effect or activity of periostin using the
above-described antibody that specifically recognizes a site
involved in the cell adhesion activity.
[0130] In an embodiment, the antibody of the present invention
includes a monoclonal antibody and a polyclonal antibody produced
by using any of the antigens as described above. The term
"monoclonal antibody" herein refers to any monoclonal antibody
reactive against any of the antigens as described above. The
"monoclonal antibody" include natural antibodies produced by
immunizing mammals such as mice, rats, hamsters, guinea pigs and
rabbits with any of the antigens; antibodies that can be produced
by using genetic recombination techniques, such as chimeric
monoclonal antibodies (chimeric antibodies) and humanized
monoclonal antibodies (humanized antibodies, i.e., CDR-grafted
antibodies); and human monoclonal antibodies (human antibodies)
that can be produced by using human antibody-producing transgenic
animals or the like. The antibody of the present invention include
monoclonal antibodies of any isotype, such as IgG (IgG1, IgG2,
IgG3, IgG4), IgM, IgA, IgD and IgE. The antibody of the present
invention is preferably IgG (IgG1, IgG2, IgG3, IgG4) or IgM.
[0131] When any of the peptides described above is to be used as an
antigen, the peptide can be used alone as an antigen.
Alternatively, to increase its antigenicity, the peptide can be
adsorbed to a macromolecular material such as polyvinyl
pyrrolidone, latex and polymethyl methacrylate and used for
immunization, or can be conjugated to a carrier protein such as KLH
(keyhole limpet hemocyanin) and BSA (bovine serum albumin), and any
method can be used to increase the antigenicity. Generally, the
peptide is preferably conjugated to a carrier protein by known
methods (e.g., see "Zoku Iyakuhin no Kaihatsu, vol. 14,
Hirokawa-Shoten Ltd., 1991").
[0132] For directional conjugation of the peptide to a carrier
protein, a cysteine residue is added to the C- or N-terminus of the
peptide, and via the cysteine residue, the peptide is conjugated to
the carrier protein. As long as conjugation suitable for this
purpose is achieved, any crosslinker commonly used in the art can
be used. Suitable crosslinkers include
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(hereinafter abbreviated to "SMCC"), 3-maleimidobenzoic
acid-N-hydroxysuccinimide ester (MBS), etc. The monoclonal antibody
is produced by generating a hybridoma by the cell fusion method of
Kohler and Milstein (G. Kohler et al Nature (1975) 256, 495-7),
culturing the hybridoma to allow the hybridoma to secrete an
antibody, and isolating the antibody from the culture. In
particular, a mammal is immunized with a peptide having an amino
acid sequence encoded by Exon-21 or the like and then the
antibody-producing cells of the animal are fused to myeloma cells
to generate a hybridoma. Screening for a hybridoma producing an
antibody binding to Exon-21 is performed by, e.g., enzyme-linked
immunosorbent assay (hereinafter abbreviated to "ELISA") on the
hybridoma supernatant using a microplate on which the antigen has
been immobilized.
[0133] The animal to be immunized is not particularly limited, and
include various mammals such as mice, rats, guinea pigs, rabbits,
sheep, goats, cats, dogs, etc. Of the listed animals for
immunization, Balb/c mice are generally used for production of
monoclonal antibodies because of ease of handling or other
advantages, but other strains of mice can also be used. The
concentration of the antigen used for immunization is determined so
that a sufficient amount of antigenically stimulated lymphocytes
are produced. Preferably, 1 to 100 .mu.g of the antigen is diluted
to an appropriate concentration in physiological saline or the
like, suspended in Freund's complete adjuvant or Freund's
incomplete adjuvant or the like, and administered to an animal by
intraperitoneal or subcutaneous injection or other means. The
administration is performed once to several times at intervals of 2
to 4 weeks. The final immunization is normally performed by
administering a solution of 1 to 100 .mu.g of the antigen in
physiological saline by intravenous or subcutaneous injection or
other means. Several days after the final immunization,
antibody-producing cells such as lymphocytes, preferably spleen
cells or lymph node cells, are harvested from the immunized animal
for cell fusion.
[0134] Cell fusion using spleen cells as antibody-producing cells
will be explained below, but antibody-producing cells other than
spleen cells can also be used for cell fusion. Spleen cells
prepared from the spleen aseptically removed 3 to 4 days after the
final immunization are fused to appropriate myeloma cells in the
presence of a fusion promoter. The myeloma cells used for fusion
may be any myeloma cells as long as they are derived from mammals,
but generally preferred are those derived from the same species as
the animal used for immunization. Various cell lines are already
known. For example, preferred cell lines used for mice are
SP2/0-Ag14 (SP2) [Nature, 276, 269 (1978)], NS-1-Ag4/1 (NS-1),
P3-X63Ag8U.1 (P3U1) [Curr. Top. Microbiol. Immunol. 81, 1-7 (1978),
available from ATCC under ATCC No. CRL-1597], P3-NS1-1-Ag4-1,
P3-X63Ag8 (P3), FO, X63Ag8.653 (X63.653), 210.RCY3.Ag1.2.3,
S194/5XXO.BUl, SKO-007, GM15006TG-A12, etc. Preferred cell lines
used for rats are Y3.Ag1.2.3 etc. Preferred fusion promoters
include polyethylene glycol (PEG) having a molecular weight of
1,000 to 6,000 and Sendai virus. Generally, the ratio of spleen
cells and myeloma cells for cell fusion is preferably 10:1 to
2:1.
[0135] Hybridomas can be separated from fused cells by culturing a
mixture of unfused spleen cells, unfused myeloma cells and fused
cells in a selective medium that inhibits the survival of unfused
myeloma cells for an appropriate period of time until unfused cells
die (about 1 week). The selective medium may be, for example, HAT
medium (a medium containing hypoxanthine, aminopterin and
thymidine). In this selective medium, unfused myeloma cells die,
and non-tumorous cells, i.e., unfused spleen cells die after a
certain period of time (after about 1 week), as a result of which
hybridomas are selected as viable cells. The hybridomas can be
subjected to conventional limiting dilution for screening to select
a strain producing the desired antibody and for cloning of the
strain. Thus obtained hybridoma producing a monoclonal antibody of
the present invention can be grown in medium suitable for the
growth and can be easily stored in a deep freezer or liquid
nitrogen for a long period of time.
[0136] The thus obtained hybridoma can be grown in nutrient medium
or in the abdominal cavity of a mammal for antibody production. The
produced antibodies can be purified from the culture supernatant or
the ascites or serum of the mammal.
[0137] As a hybridoma of the present invention, a hybridoma that
was internationally deposited with Incorporated Administrative
Agency, National Institute of Technology and Evaluation, Patent
Microorganisms Depositary (NPMD) (Deposit date: Feb. 26, 2013,
Accession No.: NITE BP-01546, Identification Reference: KS-0259#8,
080611 Kohjin Bio) can be used.
[0138] Purification of the antibodies can be performed by
conventional isolation/purification methods such as centrifugation,
dialysis, salting out with ammonium sulfate or the like, ion
exchange chromatography using a DEAE column or the like, gel
filtration, affinity chromatography, etc.
[0139] The isotype and subclass determination of the thus obtained
monoclonal antibody can be performed by an identification method
such as the Ouchterlony method, ELISA and RIA. The Ouchterlony
method is convenient but requires the condensation of the
monoclonal antibody when the concentration is low. When ELISA or
RIA is used, the isotype and subclass of the monoclonal antibody
can be identified by direct reaction of the culture supernatant
with an antigen-adsorbed solid phase, followed by reaction with
antibodies against different immunoglobulin isotypes and subclasses
as secondary antibodies. More conveniently, commercially available
identification kits (e.g., Mouse Typer Kit (Bio-Rad)) or the like
can be used. Protein quantification can be performed by the
Folin-Lowry method or by calculation from the absorbance at 280 nm
[1.4 (OD 280)=1 mg/mL immunoglobulin].
[0140] The thus obtained monoclonal antibody of the present
invention specifically recognizes a periostin isoform having an
amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 13
(PN-2); a periostin isoform having an amino acid sequence of SEQ ID
NO: 6, SEQ ID NO: 17 or SEQ ID NO: 18; a periostin isoform having
an amino acid sequence of SEQ ID NO: 19 or 20; a peptide consisting
of an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID
NO: 18; or a peptide having an amino acid sequence of SEQ ID NO: 19
or 20. Preferably, the monoclonal antibody of the present invention
specifically recognizes and binds to a peptide (SEQ ID NO: 19)
consisting of the amino acid residues from valine (V) at position 2
to lysine (K) at position 7 from the N-terminus of the amino acid
sequence of the human periostin Exon-21 peptide chain (SEQ ID NO:
18), and a peptide (SEQ ID NO: 20) consisting of the amino acid
residues from phenylalanine (F) at position 17 to lysine (K) at
position 23 from the N-terminus of the amino acid sequence of the
human periostin Exon-21 peptide chain (SEQ ID NO: 18). That is, the
monoclonal antibody of the present invention specifically
recognizes the amino acid sequence VTKVTK (SEQ ID NO: 19)
consisting of the amino acid residues from valine at position 2 to
lysine at position 7 from the N-terminus of the amino acid sequence
of the human periostin Exon-21 peptide chain (SEQ ID NO: 18), the
amino acid sequence FEDEEIK (SEQ ID NO: 20) consisting of the amino
acid residues from phenylalanine (F) at position 17 to lysine (K)
at position 23 from the N-terminus of the amino acid sequence of
the human periostin Exon-21 peptide chain (SEQ ID NO: 18), or part
of the amino acid sequences.
[0141] The monoclonal antibody of the present invention inhibits or
prevents the cell adhesion properties of human periostin-1 protein,
i.e., the monoclonal antibody has the activity to neutralize the
cell adhesion properties of human periostin-1 protein. The
monoclonal antibody of the present invention also inhibits
invasion, inflammations and angiogenesis in cancer conditions or
the like, and prevents or treats inflammation-associated
diseases.
[0142] When a polyclonal antibody is used as the antibody of the
present invention, the polyclonal antibody can be produced by
conventional methods such as the method described in "Shin
Seikagaku Jikken Koza, vol. 12, edited by the Japanese Biochemical
Society, Tokyo Kagaku Dozin Co. Ltd., 1992".
[0143] The animal to be immunized is not particularly limited, and
include horses, goats, sheep, rabbits, guinea pigs, mice, chickens,
etc. When a rabbit is to be immunized, an antigen is diluted to an
appropriate concentration in physiological saline or the like and
suspended in Freund's complete adjuvant, Freund's incomplete
adjuvant, aluminum hydroxide adjuvant, or the like, and injected at
a dose of 10 to 1,000 .mu.g/animal per injection, followed by 1 to
3 booster injections 2 to 4 weeks after the immunization to give
antisera. Multi-site subcutaneous injection is preferred.
Preparation of polyclonal antibodies from antisera can be performed
in the same manner as described for the purification of the
monoclonal antibody.
[0144] The thus obtained polyclonal antibody of the present
invention specifically recognizes a periostin isoform having an
amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 9 or SEQ ID NO: 13
(PN-2); a periostin isoform having an amino acid sequence of SEQ ID
NO: 6, SEQ ID NO: 17 or SEQ ID NO: 18; a periostin isoform having
an amino acid sequence of SEQ ID NO: 19 or 20; a peptide consisting
of an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 17 or SEQ ID
NO: 18; or a peptide having an amino acid sequence of SEQ ID NO: 19
or 20. Preferably, the polyclonal antibody of the present invention
specifically recognizes and binds to a peptide (SEQ ID NO: 19)
consisting of the amino acid residues from lysine (K) at position 7
to glycine (G) at position 11 from the N-terminus of the amino acid
sequence of the human periostin Exon-21 peptide chain (SEQ ID NO:
18), and a peptide (SEQ ID NO: 20) consisting of the amino acid
residues from lysine (K) at position 23 to leucine (L) at position
26 from the N-terminus of the amino acid sequence of the human
periostin Exon-21 peptide chain (SEQ ID NO: 18). The polyclonal
antibody of the present invention specifically recognizes the amino
acid sequence TKVTK (SEQ ID NO: 21) consisting of the amino acid
residues from position 3 to position 7 from the N-terminus of the
amino acid sequence of the human periostin Exon-21 peptide chain
(SEQ ID NO: 18), the amino acid sequence QGDTPVRK (SEQ ID NO: 22)
consisting of the amino acid residues at positions 27 and 28 from
the N-terminus of the amino acid sequence of the human periostin
Exon-21 peptide chain (SEQ ID NO: 18) and the subsequent 6 amino
acids, or part of the amino acid sequences.
[0145] The polyclonal antibody of the present invention inhibits or
prevents the cell adhesion properties of human periostin-1 protein,
i.e., the polyclonal antibody has the activity to neutralize the
cell adhesion properties of human periostin-1 protein. The
polyclonal antibody of the present invention also inhibits
invasion, inflammations and angiogenesis in cancer conditions or
the like, and prevents or treats inflammation-associated
diseases.
[0146] The present invention also provides a composition for
inhibiting vascular intimal hyperplasia, treating a cancer or
inhibiting angiogenesis, the composition comprising an
anti-periostin antibody that recognizes a periostin splice variant
having cell adhesion properties.
Production of Humanized Antibodies
[0147] Immunoglobulin G (hereinafter simply referred to as "IgG")
consists of two light polypeptide chains having a molecular weight
of about 23,000 (hereinafter referred to as "light chains") and two
heavy polypeptide chains having a molecular weight of about 50,000
(hereinafter referred to as "heavy chains"). The heavy and light
chains both have repeating units of a conserved amino acid sequence
of about 110 residues, and these units are the basic elements of
the three-dimensional structure of IgG (hereinafter referred to as
"domains"). The heavy and light chains consist of 4 and 2
successive domains, respectively. The amino terminal domains of the
heavy and light chains are more variable in amino acid sequence
between antibody molecules than other domains, and thus the amino
terminal domains are called the variable domains (hereinafter
referred to as "V domains"). At each amino terminal domain of IgG,
the heavy chain V domain and the light chain V domain
complementarily associate to form a variable region. The remaining
domains collectively form a constant region. The sequence of the
constant region is divergent between animal species. For example,
the constant region of mouse IgG differs from the constant region
of human IgG, and hence mouse IgG is recognized as a foreign body
by the human immune system, resulting in a Human Anti-Mouse
Antibody (hereinafter referred to as "HAMA") response (Schroff R W.
et al. Cancer Res. (1985) 45, 879-85). Mouse antibodies thus cannot
be repeatedly administered to humans. In order to administer such
antibodies to humans, the antibody molecules must be modified to
prevent a HAMA response while maintaining the specificity of the
antibodies.
[0148] According to the results of X-ray crystal structural
analysis, domains are generally in the form of a long cylindrical
structure made up of a stack of two antiparallel beta sheets
consisting of 3 to 5 beta chains. In terms of the variable region,
three loops are assembled to form an antigen-binding site in each
of the V domains of the heavy and light chains. These loops are
called complementarity determining regions (hereinafter referred to
as "CDRs"), which are most variable in amino acid sequence. The
remaining parts of the variable region other than the CDRs serve to
maintain the structures of the CDRs and are called "framework".
Kabatt et al. collected a large number of the primary sequences of
heavy and light chain variable regions, and provided a table in
which the primary sequences are classified into CDRs and frameworks
on the basis of sequence conservation (Kabatt et al. SEQUENCES OF
IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242,
E.A.).
[0149] The frameworks were further classified into a plurality of
subgroups based on shared amino acid sequence patterns. The
existence of consensus framework between human and mouse was also
found. Such studies on the structural features of IgG led to the
development of the methods for producing humanized antibodies
described below. At an early stage of the studies, chimeric
antibodies having a variable region from a mouse antibody fused to
a constant region from a human antibody were proposed (Morrison S
L. et al Proc Natl Acad Sci USA. (1984) 81, 6851-5). However, since
such chimeric antibodies still contain many non-human amino acid
residues, the antibodies may induce a HAMA response, especially
when they are administered for a long term (Begent et al., Br. J.
Cancer, (1990) 62, 487).
[0150] A method for further reducing amino acid residues derived
from a non-human mammal that may induce a HAMA response to humans
was proposed, and the method involved grafting only the CDRs into a
human antibody (Peter T et al. Nature, (1986) 321, 522-5). However,
grafting of only the CDRs was normally insufficient to exhibit
immunoglobulin activity against an antigen. In 1987, Chothia et al.
used X-ray crystal structural analysis data to find the following:
(a) the amino acid sequences of the CDRs contain the sites that
directly bind to an antigen and the sites that maintain the
structures of the CDRs, and possible three-dimensional structures
of the CDRs are classified into several typical patterns (canonical
structures); and (b) the canonical structure classes are determined
by not only the CDRs but also the types of the amino acids located
at specific positions in the framework (Chothia C. et al. J. Mol.
Biol. (1987) 196, 901-17). These findings suggested that, when CDR
grafting is performed, part of the amino acid residues in the
framework should also be grafted into a human antibody in addition
to the CDR sequences (JP 4-502408 T).
[0151] An non-human mammal antibody having a CDR (s) to be grafted
to a human antibody is in general defined as "donor", and the human
antibody into which the CDR(s) is (are) to be grafted is defined as
"acceptor". In CDR grafting, the structure (s) of the CDR(s) should
be conserved as much as possible to ensure the retention of the
activity of the immunoglobulin molecule. To achieve this, key
points to note are: (a) from which subgroup the acceptor should be
selected, and (b) which amino acid residues should be selected from
the donor framework.
[0152] Queen et al. proposed a method for designing a humanized
antibody, involving grafting the amino acid residues from a donor
framework together with the CDR sequences into an acceptor with the
proviso that the amino acid residues from the donor framework
satisfy at least one of the following criteria (JP 4-502408 T):
(a) the amino acids to be substituted are rare for their positions
in the acceptor framework region, and the corresponding amino acids
from the donor are common for their positions in the acceptor
framework region; (b) the amino acids are immediately adjacent to
one of the CDRs; and (c) the amino acids are predicted to have a
side chain atom within about 3 angstroms of the CDRs in a
three-dimensional immunoglobulin model and to be capable of
interacting with an antigen or with the CDRs of the humanized
antibody.
[0153] The DNA encoding the heavy or light chain of an anti-Exon-21
monoclonal antibody of the present invention can be produced by
preparing mRNA from hybridoma cells that produce the anti-Exon-21
monoclonal antibody, converting the mRNA to cDNA with a reverse
transcriptase, and isolating the DNA encoding the heavy or light
chain of the antibody.
Production of Human Antibodies
[0154] The term "human antibody" or "human immunoglobulin" herein
means an immunoglobulin in which its constituent regions, including
the heavy chain variable regions (VH) and the heavy chain constant
regions (CH) as well as the light chain variable regions (VL) and
the light chain constant regions (CL), are all derived from a gene
encoding a human immunoglobulin. In other words, the term means an
antibody in which the heavy chains are derived from a human
immunoglobulin heavy chain gene and the light chains are derived
from a human immunoglobulin light chain gene.
[0155] A human antibody can be produced by conventional methods.
For example, at least a human immunoglobulin gene is integrated
into a locus of the gene of a non-human mammal such as a mouse to
generate a transgenic animal, and the transgenic animal is
immunized with an antigen, followed by the same procedures as
described above for the production of monoclonal antibodies.
Transgenic mice producing human antibodies can be generated, for
example, by the methods described in prior documents (Mendez M J et
al. Nature Genetics (1997) 15, 146-56; Green L L et al. Nature
Genetics (1994) 7, 13-21; JP 4-504365 T; WO 94/25585; Nikkei
Science, June, pp. 40-50, 1995; Nils Lonberg et al. Nature (1994)
368, 856-9; and JP 6-500233 T).
[0156] The antibody used in the present invention is not limited to
the whole antibody molecule and may be an antibody fragment or
derivative as long as the fragment or derivative neutralizes the
activity of a periostin isoform having cell adhesion activity.
[0157] The antibody fragment may be, for example, a Fab, a
F(ab').sub.2, Fv, a single chain antibody (scFv), a
disulfide-stabilized antibody (dsFv), a CDR-containing peptide, or
the like.
[0158] The antibody fragment Fab, F(ab').sub.2, or the like of the
present invention can be produced by treating an antibody
inhibiting the cell adhesion activity of periostin with a protease
such as papain or pepsin, or alternatively, can be produced by
constructing a gene encoding the antibody fragment and introducing
the construct into an expression vector, followed by expression in
an appropriate host cell.
[0159] The antibody fragment scFv of the present invention can be
produced by linking an H chain V region with an L chain V region
from an antibody inhibiting the cell adhesion activity of periostin
via an appropriate peptide linker etc. Alternatively, the scFv can
be produced by constructing DNA segments encoding the entire
sequences or desired amino acid sequences of genes encoding the H
chain or H chain V region and encoding the L chain or L chain V
region of the antibody, and introducing the constructs into an
expression vector, followed by expression in an appropriate host
cell.
[0160] The antibody fragment dsFv of the present invention is an
antibody fragment produced by preparing the H chain V region and
the L chain V region from an antibody inhibiting the cell adhesion
activity of periostin, subjecting the regions to substitution of
one amino acid residue for a cysteine residue to give two modified
polypeptides, and linking the polypeptides between the cysteine
residues via a disulfide linkage. The amino acid residue to be
substituted for a cysteine residue can be selected using protein
structure prediction of the antibody. Alternatively, the dsFv can
be produced by constructing a DNA segment encoding the entire
sequence or desired amino acid sequence of a gene encoding the
antibody fragment, and introducing the construct into an expression
vector, followed by expression in an appropriate host cell.
[0161] The antibody fragment CDR-containing peptide of the present
invention is produced so as to comprise at least one or more CDR
regions selected from the CDR regions in the H or L chains of an
antibody inhibiting the cell adhesion activity of periostin.
Alternatively, several CDR regions may be linked by techniques
using an appropriate peptide linker or the like. The CDR-containing
peptide can also be produced by constructing a DNA segment encoding
the entire sequence or desired amino acid sequence of a gene
encoding the peptide, and introducing the construct into an
expression vector, followed by expression in an appropriate host
cell. Alternatively, the CDR-containing peptide can also be
produced by chemical synthesis such as the Fmoc or tBoc method.
[0162] In the present invention, a derivative comprising the above
antibody or antibody fragment linked to a protein or low-molecular
compound can also be used. Such modification may be accomplished by
known techniques.
[0163] A DNA encoding the antibody, the antibody fragment or their
protein-linked derivative of the present invention can be
determined by a conventional method. The DNA can be used to produce
a recombinant vector containing the DNA by a conventional method,
and the recombinant vector can be introduced into a host cell by a
conventional method to give a transformant. The transformant can be
cultured by a conventional method to produce the antibody, the
antibody fragment or their protein-linked derivative in the
culture. From the culture, the antibody, the antibody fragment or
their protein-linked derivative can be harvested. In this manner,
the antibody, the antibody fragment, and their protein-linked
derivative can be produced.
[0164] In an embodiment of the present invention, the antibody, the
antibody fragment and/or the antibody derivative of the present
invention can be used to prevent or treat inflammation-associated
diseases in which a periostin isoform having cell adhesion activity
is involved.
[0165] The term "inflammation-associated diseases in which a
periostin isoform having cell adhesion activity is involved" refers
to diseases during which the gene of a periostin isoform having
cell adhesion activity is highly expressed and during which the
production of the protein isoform encoded by the gene is increased.
The term also refers to diseases whose pathology is exacerbated by
an increase in the gene or protein expression.
[0166] Such inflammation-associated diseases in which a periostin
isoform having cell adhesion activity is involved are not
particularly limited, and include diseases of which the primary
cause is vascular intimal hyperplasia, cancers and other
inflammation-associated diseases. Examples of the diseases of which
the primary cause is vascular intimal hyperplasia include
arteriosclerosis, restenosis primarily caused by vascular intimal
hyperplasia observed after coronary angioplasty, etc. Cancers to
which the antibody, the antibody fragment and/or the antibody
derivative of the present invention can be applied include, but are
not limited to, for example, brain tumor, leukemia, osteosarcoma,
breast cancer, colorectal cancer, melanoma, bone cancer, stomach
cancer, lung cancer, liver cancer, renal cancer, pancreatic cancer,
gall bladder cancer, skin cancer, uterine cancer, ovarian cancer,
rectal cancer, colon cancer, tubal cancer, esophagus cancer, small
intestine cancer, thyroid cancer, parathyroid cancer, adrenal
cancer, prostate cancer, bladder cancer, malignant lymphoma, etc.
Particularly suitable cancers are breast cancer, colorectal cancer,
lung cancer and melanoma. Examples of said other
inflammation-associated diseases include autoimmune arthritis,
atopic dermatitis, asthma, pulmonary emphysema, Behcet disease,
multiple sclerosis, spinocerebellar degeneration, uveitis,
Guillain-Barre syndrome, Fisher syndrome, chronic inflammatory
demyelinating polyneuropathy, polymyositis, scleroderma, autoimmune
hepatitis, sarcoidosis, chronic pancreatitis, inflammatory
enterocolitis, Crohn's disease, solid cancers, multiple myeloma,
angiofibroma, atherosclerosis, arteriovenous malformations,
granuloma, hemangioma, hypertrophic scars, keloids, progeria,
psoriasis, pyrogenic granuloma, verrucae, hemarthrosis, ununited
fractures, rheumatoid arthritis (e.g., malignant rheumatoid
arthritis etc.), osteoarthritis, follicular cysts, ovarian
hypertrophy syndrome, polycystic ovary syndrome, age-related
macular degeneration, diabetic retinopathy, neovascular glaucoma,
trachoma, pulmonary emphysema, chronic bronchitis, obesity,
periodontosis, angiogenesis associated with corneal graft,
aneurysm, etc. Angiogenesis is involved in many of the above
inflammation-associated diseases.
[0167] Another embodiment of the present invention includes a
diagnostic reagent for inflammation-associated diseases in which a
periostin isoform having cell adhesion activity is involved, the
diagnostic reagent being produced by labeling any of the above
antibodies with a marker. The marker is not particularly limited
and examples thereof include enzymes, radioisotopes, fluorescent
dyes, etc. The enzymes used herein are not particularly limited as
long as they satisfy certain conditions, such as having a high
turnover number, remaining stable even after conjugation, and
specifically reacting with their substrates to develop color, etc.
Enzymes used in conventional enzyme immunoassay (EIA) can be used.
Examples of preferred enzymes include peroxidases,
.beta.-galactosidases, esterase, glucose-6-phosphate dehydrogenase,
malate dehydrogenase, etc. Enzyme inhibitors and coenzymes etc. can
also be used.
[0168] Conjugation of the enzyme and antibody can be performed by a
known method using a known crosslinker such as maleimide compounds.
Substrates that can be used are known substances selected depending
on the enzyme used. For example, when the enzyme used is a
peroxidase, 3,3',5,5'-tetramethylbenzidine can be used. When the
enzyme used is an alkaline phosphatase, paranitrophenol or the like
can be used. Radioisotopes that can be used as a marker include
those used in conventional radioimmunoassay (RIA) such as .sup.123I
and .sup.3H. Fluorescent dyes that can be used are those used in
conventional fluoroimmunoassay such as fluorescence isothiocyanate
(FITC) and tetramethyl rhodamine isothiocyanate (TRITC). The
present diagnostic reagent can also be used for immunohistological
staining that specifically stains cancer cells and the surrounding
fibroblasts. When the diagnostic reagent is labeled with a
radioisotope, the diagnostic reagent can also be internally
administered for imaging of cancer lesions etc.
[0169] Another embodiment of the present invention provides a
method for detecting or quantifying a periostin isoform having cell
adhesion activity in a biological sample, i.e., serum, prepared
from human or animal blood, the method using the antibody, the
antibody fragment and/or the antibody derivative of the present
invention. The present invention also provides a method for
diagnosing an inflammation-associated disease (e.g., heart failure
etc.) in which a periostin isoform having cell adhesion activity is
involved, the method comprising detecting or quantifying the
periostin isoform. In the present methods, a periostin isoform
having cell adhesion activity can be detected by so-called sandwich
ELISA (enzyme-linked immunosorbent assay). A diagnostic kit of the
present invention is used as follows. An anti-periostin primary
antibody is immobilized on a plate, a sample is contacted with the
plate to form a complex with the primary antibody, then an
anti-periostin secondary antibody labeled with a marker is allowed
to bind to the complex, and the signal intensity of the marker in
the ternary complex is measured to detect and quantify a periostin
isoform having cell adhesion activity. Since a periostin isoform
having cell adhesion activity is a splice variant that is
specifically expressed in conditions such as cancers, monitoring of
the production of the periostin isoform can be used to diagnose the
conditions such as cancers.
[0170] As described above, the antibody of the present invention
can be labeled and used as a secondary antibody.
[0171] Another embodiment of the present invention provides a
pharmaceutical composition for inhibiting a periostin isoform
having cell adhesion activity, the composition comprising the
antibody, the antibody fragment and/or the antibody derivative of
the present invention as an active ingredient. Since the antibody,
the antibody fragment and/or the derivative has an inhibitory
activity on a periostin isoform having cell adhesion activity, the
pharmaceutical composition inhibits angiogenesis, inhibits vascular
intimal hyperplasia, treats a cancer, and prevents or treats
aneurysm. That is, the present invention provides a composition for
inhibiting vascular intimal hyperplasia, treating a cancer,
inhibiting angiogenesis, or preventing or treating aneurysm, the
composition comprising an anti-periostin antibody that recognizes a
periostin splice variant having cell adhesion properties. The
composition of the present invention can be used to treat and
prevent restenosis that is primarily caused by vascular intimal
hyperplasia observed after coronary angioplasty, to prevent or
treat a cancer, to treat or prevent diseases accompanied by
angiogenesis, and to prevent or treat aneurysm.
[0172] Another embodiment of the present invention provides a
pharmaceutical composition for preventing or treating an
inflammation-associated disease in which a periostin isoform having
cell adhesion activity is involved, the composition comprising the
antibody, the antibody fragment and/or the derivative of the
present invention as an active ingredient. Examples of the
embodiment of the present invention include a pharmaceutical
composition for inhibiting vascular intimal hyperplasia, the
composition comprising the antibody, the antibody fragment and/or
the antibody derivative as an active ingredient; a pharmaceutical
composition for treating a cancer, the composition comprising the
antibody, the antibody fragment and/or the antibody derivative as
an active ingredient; a pharmaceutical composition for inhibiting
angiogenesis, the composition comprising the antibody, the antibody
fragment and/or the antibody derivative as an active ingredient; a
pharmaceutical composition for preventing or treating aneurysm, the
composition comprising the antibody, the antibody fragment and/or
the antibody derivative as an active ingredient; etc.
[0173] The composition of the present invention for treating a
cancer exhibits the effects of inhibiting the growth of cancer foci
and of inhibiting the metastasis of a cancer. The composition,
hence, can be used with the intended purpose of inhibiting the
growth of cancer foci or of inhibiting the metastasis of a cancer,
or both purposes.
[0174] The pharmaceutical composition comprising the antibody, the
antibody fragment and/or the antibody derivative of the present
invention as an active ingredient is prepared with the use of known
pharmacologically acceptable additives that are commonly used in a
conventional preparation method, including a carrier, an excipient,
and other additives.
[0175] The active ingredient of the pharmaceutical composition
according to the present invention is preferably administered in
admixture with a known pharmacologically acceptable carrier,
excipient, diluent, or the like by any mode of administration
commonly used in the pharmaceutical field (for example, by oral
administration or parenteral administration such as intravenous,
intramuscular and subcutaneous administrations). The pharmaceutical
composition of the present invention can be prepared by, for
example, mixing the active ingredient with a pharmacologically
acceptable carrier, flavor, excipient, stabilizer, diluent,
emulsifier, solution, suspension, syrup, or the like, as needed.
The dosage form of the pharmaceutical composition of the present
invention is not particularly limited and examples thereof include
tablets, powders, granules, solutions, etc. Additives that can be
incorporated into tablets or the like include, for example, binders
such as gelatin and lubricants such as corn starch. The
pharmaceutical composition may be coated with a sugar or a gastric
or enteric film. When the dosage form is a capsule, the composition
can further comprises a liquid carrier. The composition can be
formulated into an injectable sterile composition with a
conventional pharmaceutical formula. Injectable aqueous vehicles
include isotonic solutions containing glucose etc., and such
isotonic solutions may be used in combination with an appropriate
solubilizer such as polyethylene glycol. The pharmaceutical
composition may be incorporated with a buffer, a stabilizer, a
preservative, an antioxidant, a soothing agent, or the like. For
oral administration, when the active ingredient is likely to be
decomposed in the digestive tract, the composition may be made into
a formulation that are resistant to decomposition in the digestive
tract (for example, liposome microcapsules encapsulating the active
ingredient) and then orally administered. Other modes of
administration for absorption through mucous membrane other than
the digestive tract are also possible, including rectal,
intranasal, sublingual and transpulmonary routes. In these cases,
the composition can be administered in the form of a suppository, a
nose drop, a sublingual tablet, a transpulmonary agent, or the
like.
[0176] When the pharmaceutical composition of the present invention
is used for therapeutic purposes, the dosage is determined so as to
be therapeutically effective. The therapeutically effective dosage
varies with, e.g., the age, the body weight and the severity of the
symptoms of a subject to which the composition is to be
administered, and the route of administration. For these reasons,
the dosage is determined on an individual basis. In general, the
daily dosage for an adult by oral administration is about 0.1 to
1,000 mg, and the dosage is given as a single dose or one to
several divided doses (twice, three times, etc.). For continuous
intravenous administration, the composition can be administered at
a dosage of 0.01 .mu.g/kgmin to 1.0 .mu.g/kgmin, desirably 0.025
.mu.g/kgmin to 0.1 .mu.g/kgmin.
[0177] Other embodiments of the present invention provide "a method
for inhibiting a periostin isoform having cell adhesion activity by
using an antibody against the periostin isoform", "a method for
preventing or treating an inflammation-associated disease in which
a periostin isoform having cell adhesion activity is involved, the
method comprising administering, to a cancer patient, a
therapeutically effective amount of an anti-periostin antibody that
recognizes a periostin splice variant having cell adhesion
properties", etc. Other embodiments of the present invention
provide "use of an antibody against a periostin isoform having cell
adhesion activity for production of a pharmaceutical composition
for inhibiting the periostin isoform", "use of an antibody against
a periostin isoform having cell adhesion activity for production of
a pharmaceutical composition for preventing or treating an
inflammation-associated disease in which the periostin isoform is
involved", etc. The anti-periostin antibody includes the
above-described antibodies, antibody fragments and/or antibody
derivatives. The inflammation-associated disease includes the
above-described inflammation-associated diseases.
EXAMPLES
[0178] The present invention will be described in more detail with
reference to Examples, but is not limited to thereto. Various
modifications may be made by a person having ordinary skill in the
art, without departing from the technical idea of the present
invention.
Preparation Example 1: Search for Periostin by Subtraction
1-1 Establishment of Pathological Model Rats of Heart Failure and
Harvest of Left Ventricular Samples
[0179] Male Dahl salt-sensitive rats (Dahl-S) (Shimizu Laboratory
Supplies Co., Ltd.) were raised on an 8% high salt diet from 6
weeks of age, and the left ventricles were harvested from three
animals at the cardiac hypertrophy stage (11 weeks of age) and from
three animals at the heart failure stage (14 weeks of age).
1-2 Preparation of mRNAs
[0180] Total RNAs were prepared from about 500 mg of each left
ventricle using ISOGEN (Nippon Gene) as instructed by the
manufacturer. The total RNAs from the three animals at the cardiac
hypertrophy stage and the total RNAs from the three animals at the
heart failure stage were separately combined. mRNAs were purified
from about 400 .mu.g of each of the combined total RNAs using Fast
Track 2.0 Kit (Invitrogen) as instructed by the manufacturer to
recover about 3 .mu.g of mRNA from each stage.
1-3 cDNA Subtraction
[0181] cDNA subtraction was performed using PCR-Select cDNA
Subtraction Kit (Clontech) as instructed by the manufacturer.
Briefly, cDNAs were synthesized from 2 .mu.g of each mRNA obtained
in the above section 1-2 and digested with restriction enzyme RsaI.
The cDNA synthesized from the animals at 14 weeks of age was used
as tester cDNA and the cDNA synthesized from the animals at 11
weeks of age was used as driver cDNA. To the tester cDNA, two types
of adapters included in the kit were separately ligated.
Subtraction hybridization was performed. PCR was then performed
using primers complementary to the adapters to specifically amplify
differentially expressed cDNA fragments to give amplification
product 1.
[0182] Another subtraction operation was performed in the same
manner as above except that the cDNA synthesized from the animals
at 11 weeks of age was used as tester cDNA and that the cDNA
synthesized from the animals at 14 weeks of age was used as driver
cDNA to give amplification product 2.
1-4 Dot Blot Screening
A. Preparation of Dot Blots
[0183] Amplification product 1 was TA cloned into PCR II vector
(Invitrogen) and clones with the insert fragment were selected. The
insert fragment of each clone was amplified by PCR reaction, and 1
.mu.L of each amplified product was heat-treated, then dot-blotted
on two nylon membrane filters (Boehringer) and fixed with a UV
crosslinker (Stratagene).
B. Preparation of cDNA Probes
[0184] Amplification product 1 was digested with restriction
enzymes RsaI, EaeI and SmaI to remove the adapters. The product was
then subjected to random prime labeling with DIG-dUTP using DIG
High Prime DNA Labeling and Detection Kit II (Boehringer) as
instructed by the manufacturer to prepare cDNA probe 1. In the same
manner, cDNA probe 2 was prepared from amplification product 2.
C. Screening
[0185] One of the dot blot membranes prepared in the above section
A was hybridized with cDNA probe 1 and the other was hybridized
with cDNA probe 2. Specifically, hybridization was performed in a
hybridization solution (DIG Easy Hyb solution) at 42.degree. C.
overnight using DIG High Prime DNA Labeling and Detection Kit II
(Boehringer) as instructed by the manufacturer. The membranes were
washed twice with 2.times.SSC and 0.1% SDS at room temperature for
5 minutes and then twice with 0.1.times.SSC and 0.1% SDS at
68.degree. C. for 15 minutes. The membranes were then reacted with
the alkaline phosphatase-conjugated DIG antibody in blocking buffer
included in the kit. A chemiluminescence substrate (CSPD
ready-to-use) was added to allow chemiluminescent reaction to
proceed. The membranes were exposed to X-ray films. Clones showing
a stronger signal with cDNA probe 1 than with cDNA probe 2 were
selected as positive clones and sequenced.
1-5 Sequencing
[0186] The nucleotide sequences were determined by analysis on an
automated DNA sequencer, Model 373A (PE Applied Biosystems), using
a dye terminator sequencing kit (trade name: Thermo Sequenase.TM.
II Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia)). The
obtained gene sequences were compared with sequences available from
the GenBank databank. One of the clones (SF014) was found to have a
gene having 86% homology to mouse periostin (GenBank Accession No.
D13664).
Preparation Example 2: Cloning of Rat Periostin-1 cDNA
[0187] Rat periostin cDNA was isolated as follows. A rat aorta cDNA
library (Clontech) was inserted into .lamda.gt 11 vector to
generate 10 phage subpools of about 4,000 clones (a total of about
40,000 clones). The phage subpools were screened by PCR using
primers (1) 5'-GTTCATTGAAGGTGGCGATGGTC-3' (SEQ ID NO: 23) and (2)
5'-GAGATAAAATCCCTGCATGGTCCT-3' (SEQ ID NO: 24) that were designed
based on the nucleotide sequence of SF014. As a result of the
screening, three positive subpools were obtained. One of the
subpools was screened by hybridization using the above fragment
amplified by PCR as a probe, labeled with alkaline phosphatase
using AlkPhos Direct.TM. (Amersham Pharmacia), to give one positive
clone rat periostin #1. Its insert fragment was subcloned into the
EcoRI site of pBluescript II (Stratagene) and the complete
nucleotide sequence was determined by the method described in 1-5
of Preparation example 1.
[0188] The resulting clone had a length of about 3 kb,
corresponding to the nucleotides from position 292 to the 3' end of
mouse periostin (GenBank Accession No. D13664). The results suggest
that the clone was a 5'-truncated clone.
[0189] A SMART RACE cDNA amplification kit (trade name: SMART.TM.
RACE cDNA Amplification Kit (Clontech)) was used as instructed by
the manufacturer to perform 5'-RACE reaction using rat aorta cDNA
as a template and primers (2) 5'-GAGATAAAATCCCTGCATGGTCCT-3' (SEQ
ID NO: 24) as described above and (3)
5'-CACGGTCGATGACATGGACAACACC-3' (SEQ ID NO: 25) designed based on
the nucleotide sequence of rat periostin #1. The resulting PCR
product was TA cloned into PCR II vector (Invitrogen) to give a
clone. The clone was designated as rat periostin 5' RACE #1. The
nucleotide sequence was determined by the method described in 1-5
of Preparation example 1.
[0190] The results showed that rat periostin 5' RACE #1 was a clone
of which the nucleotide sequence is longer than that of the
initially obtained rat periostin #1 by about 300 bp in the 5'
direction, and that the 5' end of rat periostin 5' RACE #1 is
longer by 15 bp than the 5' end of mouse periostin (GenBank
Accession No. D13664). The above ten phage subpools of about 40,000
clones (a total of about 400,000 clones) prepared from the rat
aorta cDNA library were screened by PCR using primers (4)
5'-ACGGAGCTCAGGGCTGAAGATG-3' (SEQ ID NO: 26) designed based on the
nucleotide sequence of rat periostin 5' RACE #1 and (3)
5'-CACGGTCGATGACATGGACAACACC-3' (SEQ ID NO: 25) as described above,
to give two positive subpools. One of the subpools was screened by
hybridization using the above fragment amplified by PCR as a probe
to give one positive clone. The clone was designated as rat
periostin #2. Its insert fragment was subcloned into the EcoRI site
of pBluescript II (Stratagene) and the nucleotide sequence was
determined by the method described in 1-5 of Preparation example
1.
[0191] The resulting clone had a length of about 2.6 kb. The 5' end
was identical to that of the clone obtained from the 5'-RACE
reaction and the 3' end corresponded to the nucleotides up to
position 2410 of mouse periostin (GenBank Accession No. D13664).
The nucleotide sequence of rat periostin 5' RACE #1 previously
obtained was identical to the nucleotide sequence of the relevant
region of rat periostin #2. The full length of rat periostin cDNA
was completed by rat periostin #1 and rat periostin #2. The
nucleotide sequence of the full-length cDNA is shown in SEQ ID NO:
2, and the amino acid sequence translated from the nucleotide
sequence is shown in SEQ ID NO: 1.
Preparation Example 3: Cloning of cDNAs of Rat Periostins-2 and
-4
[0192] PCR was performed from the cDNA of the Dahl rats at the
cardiac hypertrophy stage used in Preparation example 1 and the
gene sequence cloned in Preparation example 2 using primers
5'-AAGCTAGCGAAGATGGTTCCTCTCCTGCCCT-3' (SEQ ID NO: 27) and
5'-CTTTGGGTTTTTCCAGCCTC-3' (SEQ ID NO: 28). The PCR product was TA
cloned into pCR4 Blunt TOPO vector (Invitrogen). From the resulting
colonies, 96 colonies were selected and transferred to a 96-well
plate so as to be 1 colony/well. Screening for candidate rat
periostin-2 and -4 genes was performed using primers
5'-CCCCATGACTGTCTATAGACCT-3' (SEQ ID NO: 29) and
5'-ATTTCCCTTAAAAATCAGATTG-3' (SEQ ID NO: 30). The selected clones
were further screened by sequencing for clones with no PCR
errors.
Preparation Example 4: Construction of Baculovirus Expression
Vectors
[0193] The plasmids pCR4 Blunt TOPO/rat periostin-2 and pCR4 Blunt
TOPO/rat periostin-4 prepared in Preparation example 3 were
digested with restriction enzymes Spe I and Not I to excise rat
periostin-2 and -4 fragments. The fragments were separately ligated
using a ligation kit (Takara Bio Inc.) to a pFastBacHTc
(Invitrogen) vector fragment digested with restriction enzymes pe I
and Not I to give expression vectors. The vectors were designated
as pFastBac/rat periostin-2 and pFastBac/rat periostin-4,
respectively. The nucleotide sequences of the inserts were
confirmed by the method described in 1-5 of Preparation example
1.
Preparation Example 5: Preparation and Culture of Recombinant
Baculoviruses
[0194] Escherichia coli DH10BAC cells were transformed separately
with each of pFastBac/rat periostin-2 and pFastBac/rat periostin-4
prepared in Preparation example 4 to produce recombinant
baculoviruses. The insertion of the desired inserts in the
resulting baculoviruses was confirmed by electrophoresis and
PCR.
[0195] Insect Sf9 cells (2.times.10.sup.6 cells/mL) were infected
separately with each of the recombinant baculoviruses at MOI=0.1,
and then cultured in serum-free medium (2,000 mL of Sf-900 II SFM
(Invitrogen) containing 50 .mu.g/mL gentamicin) at 28.degree. C.
for 4 to 5 days. The culture supernatants were harvested.
Preparation Example 6: Purification of Rat Periostin Proteins
[0196] To SP Sepharose Fast Flow columns (10 mL bed volume)
equilibrated with equilibration buffer (50 mM sodium acetate
buffer, pH 6.0, 0.1 M sodium chloride) were separately applied
2,000 mL of each of the culture supernatants obtained in
Preparation example 5, and the resulting flow-through fractions
were separately pooled to give SP Sepharose flow-through
fractions.
[0197] The columns were washed with the equilibration buffer (about
100 mL) until the absorbance at 280 nm approached zero to give SP
Sepharose wash fractions.
[0198] The columns were eluted with 100 mL of elution buffer (50 mM
sodium dihydrogen phosphate (pH 8.0), 0.5 M sodium chloride, 5 mM
imidazole) to give SP Sepharose eluate fractions.
[0199] The SP Sepharose eluate fractions each in an amount of 100
mL were separately applied to Ni-NTA agarose columns (5 mL bed
volume) equilibrated with 50 mM sodium phosphate buffer, pH 8.0,
0.5 M sodium chloride and 5 mM imidazole. The resulting
flow-through fractions were separately pooled to give Ni-NTA
agarose flow-through fractions.
[0200] The columns were washed with about 50 mL of washing buffer
(50 mL sodium dihydrogen phosphate, pH 8.0, 0.5 M sodium chloride,
5 mM imidazole) to give Ni-NTA agarose wash fractions.
[0201] The columns were eluted with about 25 mL of each of the
following elution buffers: (1) 50 mM sodium dihydrogen phosphate,
0.5 M sodium chloride, 20 mM imidazole, followed by elution buffers
with the same formulations except that the imidazole concentrations
were (2) 30 mM, (3) 40 mM, (4) 50 mM and (5) 60 mM, to give Ni-NTA
agarose eluate fractions (1) to (5).
[0202] The fractions shown to contain the desired proteins by
Western blotting were concentrated to 1 mL or less.
[0203] The concentrated samples were applied to gel filtration
columns (Sephacryl S-200HR, 11 mm diameter.times.95 cm; 90 bed
volume) equilibrated with degassed PBS (-) (137 mM NaCl, 8.1 mM
Na.sub.2HPO.sub.4, 2.68 mM KCl, 1.47 mM KH.sub.2PO.sub.4), and were
eluted with PBS (-). The eluates were lyophilized to give purified
rat periostin proteins.
Example 1: In Vitro Study of the Presence or Absence of Cell
Adhesion Activity of Rat PN-2 and PN-4 Proteins
[0204] To a 96-well cell culture multi-well plate, 10 .mu.g/mL
fibronectin, 100 .mu.g/mL BSA, 10 .mu.g/mL PN-2 protein, and 10
.mu.g/mL PN-4 protein were separately added and incubated at
4.degree. C. overnight so that the wells were coated with each
protein. After removal of the protein solution from the wells, rat
neonatal cardiac fibroblasts suspended in DMEM (10% BSA, PC/SM)
were added at 10.sup.4 cells/well and cultured in a 37.degree. C.
incubator for 3 hours. The level of cell adhesion was measured as
follows. After removal of the culture supernatant, the cells were
fixed in 2.5% glutaraldehyde for 30 minutes, stained with 0.02%
crystal violet and then measured for their absorbance at OD 550 nm
with a plate reader (BIO-RAD, Model 680 MICRO PLATE READER).
Uncoated wells were stained as background samples and used to
correct the absorbance values for comparison purposes. Data
analysis was made by the Fisher's PLSD test (FIG. 2). The positive
control fibronectin (indicated by FN in the figure) showed cell
adhesion, whereas the negative control BSA showed no cell adhesion.
The wells without protein coating also showed cell adhesion, but
the level was smaller than that of the positive control
fibronectin. The group to which rat PN-2 protein was added showed a
cell adhesion level equal to or higher than that of the fibronectin
group, whereas the group to which rat PN-4 protein was added showed
a cell adhesion level merely equal to that of the uncoated
wells.
Example 2: Synthesis of Rat Exon-21 Peptide Chain and Production of
Polyclonal Antibodies
[0205] In Example 1, rat PN-4 protein was shown to have lower cell
adhesion properties than rat PN-2 protein. From this fact and the
results of sequence comparison of the proteins, the structure
specific to rat PN-2 protein was revealed to be the Exon-21
sequence. Based on this, a peptide having a Cys residue added to
the N-terminus of the amino acid sequence constituting Exon-21 was
chemically synthesized in a 10 mg yield at a purity of 80% or more.
Rabbits (Kbl:JW) were immunized with the polypeptide conjugated to
6 mg of a carrier protein KLH. FCA (Freund's complete adjuvant) was
used in the primary immunization (administration), and FIA
(Freund's incomplete adjuvant) was used in the secondary and
subsequent immunizations. Administration was performed at 20 dorsal
subcutaneous sites at weeks 2, 4 and 6 after the primary
administration, using a peptide dose of 800 .mu.g/animal in the
primary immunization and 400 .mu.g/animal in the secondary and
subsequent immunizations. The antibody titer was determined by
ELISA and the total serum was collected at week 7 after the
initiation of the administration. An affinity column was prepared
by use of the synthetic peptide, and the antibodies specifically
reacting to the Exon-21 peptide were isolated. The polyclonal
antibodies against the peptide encoded by Exon-21 of rat periostin
were referred to as anti-rat Exon-21 polyclonal antibodies.
Example 3: Confirmation of the Binding Capacity to Rat Periostin
Protein (PN-2)
[0206] The two types of polyclonal antibodies (No. 1 and No. 3)
produced in Example 2 were assayed by dot blotting to confirm their
binding capacity to rat periostin protein (PN-2). The purified
proteins (30 .mu.g/mL) produced in Preparation example were each
spotted in a 5 .mu.L volume on a Hybond-ECL nitrocellulose membrane
(GE Healthcare Bio-Sciences KK) and the membrane was washed once
with TBS solution (10 mM Tris-HCl (pH 8.0), 150 mM NaCl). Blocking
buffer (Block Ace, Snow Brand Milk Products Co., Ltd.) was added
and the membrane was shaken at room temperature for 1 hour. After a
1 .mu.g/mL solution of each monoclonal antibody (primary antibody)
was added to the membrane, the membrane was shaken for 3 hours and
washing with TBS solution under 10-minute shaking was repeated four
times. After a 0.4 .mu.g/mL solution of an HRP-conjugated
anti-rabbit IgG antibody (Promega) (secondary antibody) was added
to the membrane, the membrane was shaken at room temperature for 1
hour and washing with TBS solution under 10-minute shaking was
repeated four times. A detection reagent (ECL Plus Western Blotting
Detection System, GE Healthcare Bio-Sciences KK) was added and
reacted for 1 minute to detect chemiluminescence. The assay
confirmed that the two types of polyclonal antibodies bind to rat
periostin PN-2.
Example 4: In Vitro Study of the Neutralizing Activity of Anti-Rat
Exon-21 Polyclonal Antibody
[0207] DB1X rat vascular smooth muscle cells A7r5 (Cat. No.
09-1444, Sumitomo Dainippon Pharma Co., Ltd.) were seeded in a
96-well cell culture multi-well plate at a density of 0.5 to
1.times.10.sup.4 cells/well and cultured to subconfluence, and then
the medium was replaced with a serum (-) medium. Separately, in
order to fluorescently label THP-1 cells (human acute monocytic
leukemia, Cat. No. 06-202, Sumitomo Dainippon Pharma Co., Ltd.), 2
nM BCECF-AM (BCECF-AM special packaging, Cat. No. B221, Dojindo
Laboratories Co., Ltd.) was added to the THP-1 cells, incubated for
30 minutes, centrifuged, and washed twice with PBS (-). For the
measurement of the adhesion of the fluorescently labeled THP-1
cells, the anti-rat Exon-21 polyclonal antibody (ex21PoAb) or a
rabbit control IgG antibody (rIgG) was mixed with PN-2 protein and
reacted at room temperature for 1 hour, and then the reacted
mixture and 1.times.10.sup.4 cells/well of the fluorescently
labeled THP-1 cells were added to the A7r5-seeded wells. As
controls, (1) fibronectin, (2) PN-2, (3) PN-2+rabbit control IgG
antibody (rIgG), and (5) THP-1 alone were used. The above mixture
and the controls were incubated for 6 hours. RPMI 1640 medium was
then slowly added to each well to fill it up, and the plate was
covered with parafilm to exclude air. The plate was turned upside
down and incubated for 30 minutes. The parafilm was then carefully
removed, the medium was aspirated, and the plate was washed three
times with PBS (-). As a measure of cell adhesion, the fluorescence
intensity was determined with a plate reader (Wallac 1420
ARVOmx/light (Perkin-Elmer)) at an excitation wavelength of 485 nm
and a fluorescence wavelength of 535 nm. As shown in FIGS. 3A and
3B, cell adhesion was observed in (1) fibronectin group, (2) PN-2
group and (3) PN-2+rabbit control IgG antibody (rIgG) group,
whereas no significant adhesion was observed in (4) anti-rat
Exon-21 polyclonal antibody (ex21PoAb) treatment group as with the
case of (5) THP-1 alone. The results revealed that the anti-rat
Exon-21 polyclonal antibody has inhibitory activity, i.e.,
neutralizing activity, against the cell adhesion properties of
PN-2.
Example 5: Production of Monoclonal Antibodies Against Human
Periostin Exon-21 Peptide Chain
(1) Antigen Production
[0208] A peptide (antigen peptide; CEVTKVTKFIE GGDGHLFEDE EIKRLLQG
(SEQ ID NO: 31)) having a Cys residue added to the N-terminus of
the amino acid sequence constituting human periostin Exon-21 (SEQ
ID NO: 18) was chemically synthesized by the Fmoc method in a 10 mg
yield at a purity of 90% or more. Then, 5 mg of a carrier protein
KLH (CALBIOCHEM) was conjugated to 5 mg of the antigen peptide to
give an antigen solution. Briefly, KLH was dissolved in PBS (0.01
M) to a concentration of 3.3 mg/mL. To this, a 0.2524 mg/mL MBS
solution (GE Healthcare Bio-Sciences KK) was added dropwise, and
the mixture was reacted with stirring at room temperature for 60
minutes. Dichloromethane was used to remove free MBS to give
KLH-MB. Then, 5 mg of KLH-MB was mixed with a solution of 5 mg of
the antigen peptide in 0.01 M sodium phosphate buffer (pH 7.2), and
the mixture was reacted with stirring at 4.degree. C. for 12 hours
to give an antigen solution.
(2) Immunization
[0209] The antigen solution (50 .mu.L) containing 100 .mu.g of the
KLH-conjugated antigen peptide obtained in the above (1) was mixed
with FCA (Freund's complete adjuvant, 50 .mu.L) to prepare an
emulsion. The whole volume of the emulsion was subcutaneously
injected into the hind paws of three female BALB/c mice at 6 weeks
of age. The mice were then received additional injections twice at
an interval of 2 weeks into the hind paws with a mixed emulsion of
the above antigen solution and FIA (Freund's incomplete adjuvant)
prepared at the time of use. The mice were then sacrificed by
cervical dislocation and the lymph nodes in the paws were
aseptically harvested.
[0210] The above lymph nodes were crushed in RPMI medium (Kohjin
Bio Co., Ltd.) and passed through a mesh of about 10 .mu.m pore
size to give a suspension of the lymph node cells in RPMI medium.
The suspension was centrifuged at 1,000 rpm for 10 minutes to give
a pellet fraction of the lymph node cells. The red blood cells
contained in the pellet fraction was hemolyzed with 1 mL of a
solution prepared by adding 20 mM HEPES buffer (pH 7.4) to a 0.84%
ammonium chloride solution. After the removal of the red blood
cells, the fraction was centrifuged at 1,000 rpm for 5 minutes. The
resulting pellet fraction (cell fraction) was washed several times
with RPMI medium and then used for cell fusion.
(3) Preparation of Myeloma Cells
[0211] The mouse myeloma cell line P3X63Ag8U.1 (P3U1), which is
resistant to 8-azaguanine and does not secrete immunoglobulin, was
cultured in RPMI medium containing 20% fetal calf serum (FCS) in a
10% CO.sub.2 incubator at 37.degree. C. The cells in the
logarithmic growth phase were collected and centrifuged at 1,000
rpm for 5 minutes to separate the cells as a pellet fraction. The
pellet fraction was suspended in RPMI medium.
(4) Cell Fusion
[0212] The RPMI medium containing 1.times.10.sup.8 to
3.times.10.sup.8 immunized lymph node cells prepared in the above
(2) was mixed with the RPMI medium containing 10.sup.8 myeloma
cells prepared in the above (3). The mixture was centrifuged at
1,000 rpm for 10 minutes. The supernatant was gently removed to
leave the cells as a pellet fraction, followed by addition of 1 mL
of 25% (w/v) polyethylene glycol 1500 (PEG 1500, Boehringer). To
this, RPMI medium was slowly added to a total volume of 10 mL. To
this, 20% FCS-containing RPMI medium (10 mL) was added and allowed
to stand for a while, followed by centrifugation at 1,000 rpm for 5
minutes. The resulting pellet fraction (cell fraction) was adjusted
to a cell density of 10.sup.6 cells/mL by addition of 20%
FCS-containing RPMI medium. The cell suspension was dispensed at
200 .mu.L/well in 96-well culture plates (Corning). After culture
of the cells in a 5% CO.sub.2 incubator at 37.degree. C. for 24
hours, HAT solution (Invitrogen) was added and culture was
continued for additional 2 weeks.
(5) Screening by ELISA
[0213] Screening for positive wells containing the culture
supernatant that showed reactivity with the antigen peptide was
performed. An antigen solution used for the assay was a conjugate
prepared by coupling the antigen peptide (2 mg) produced in the
above (1) to ovalbumin (OVA) as a carrier protein.
[0214] Each well of a 96-well microtiter plate (Falcon 353912) was
coated with the conjugate (1 .mu.g/mL) at 4.degree. C. overnight.
After the plate was washed, 50 .mu.L of the culture supernatant
from the above (4) (containing monoclonal antibodies) was added
dropwise to each well and allowed to stand in a 37.degree. C.
incubator for 2 hours, followed by washing with PBS (-) (phosphate
buffered saline). After addition of alkaline phosphatase-conjugated
sheep anti-mouse IgG antibody (Zymed), the plate was allowed to
stand in a 37.degree. C. incubator for 1 hour, and washed with PBS
(-). A color development substrate (ALP) was added and color
developed for 20 minutes. The absorbance (antibody titer) at OD 490
nm was measured for each well with a plate reader (BIO-RAD, Model
680 MICRO PLATE READER) to evaluate the reactivity with the antigen
peptide and thereby to determine positive wells containing the
culture supernatant that showed reactivity with the antigen
peptide.
(6) Cloning of Antibody-Producing Cells
[0215] Cloning of antibody-producing cell lines from the cells in
the positive wells for which reactivity with the antigen peptide
was confirmed by ELISA in the above (5) was performed by limiting
dilution. Briefly, the cells in the positive wells were seeded into
each well of a 96-well culture plate and cultured in a 5% CO.sub.2
incubator at 37.degree. C. for 2 weeks. For the culture supernatant
in each well, reactivity with the antigen peptide was examined by
ELISA in the same manner as in the above (5). For each positive
well, cloning by limiting dilution was performed again to select 30
cells having a high reactivity with the antigen peptide and showing
good colony growth. The cells were transferred to 24-well culture
plates and cultured in a 5% CO.sub.2 incubator at 37.degree. C. for
2 weeks. For each culture supernatant, reactivity with the antigen
peptide (antibody titer) was examined again by ELISA in the same
manner as in the above (5). Cells in two wells showing a high
absorbance at OD 490 nm, i.e., two hybridoma cell lines (No. 8 and
No. 10) were determined to be useful as antibody-producing cells
and were selected.
[0216] The thus obtained antibody-producing cells constantly
produce anti-human Exon-21 monoclonal antibodies, i.e., the
antibodies of the present invention, and hence the supernatant of
the medium in which these antibody-producing cells have been
cultured can be directly used as an antibody solution of the
present invention.
[0217] The above antibody-producing cell line (hybridoma) No. 8,
which produces an anti-human Exon-21 monoclonal antibody, was
internationally deposited with Incorporated Administrative Agency,
National Institute of Technology and Evaluation, Patent
Microorganisms Depositary (NPMD) (Deposit Date: Feb. 26, 2013,
Accession No.: NITE BP-01546, Identification Reference: KS-0259#8,
080611 Kohjin Bio).
(7) Confirmation of the Binding Capacity to Periostin Protein
(PN-2)
[0218] Antibodies produced by the two antibody-producing cells
obtained in the above (6) were assayed by dot blotting to confirm
their binding capacity to mouse periostin protein (PN-2), based on
the fact that mouse periostin has an almost identical amino acid
sequence to that of human periostin. Briefly, mouse PN-2 protein
(Recombinant Mouse Periostin/OSF-2, R&D SYSTEMS) (100 .mu.g/mL)
was spotted in 5 .mu.L volumes on a Hybond-ECL nitrocellulose
membrane (GE Healthcare Bio-Sciences KK) and the membrane was
washed once with TBS solution (10 mM Tris-HCl (pH 8.0), 150 mM
NaCl). Blocking buffer (Block Ace, Snow Brand Milk Products Co.,
Ltd.) was added and the membrane was shaken at room temperature for
1 hour. After a 1 .mu.g/mL solution of each monoclonal antibody
(primary antibody) obtained in the above (6) was added to the
membrane, the membrane was shaken for 3 hours and washing with TBS
solution under 10-minute shaking was repeated four times. After a
0.4 .mu.g/mL solution of an HRP-conjugated anti-mouse IgG antibody
(Promega) (secondary antibody) was added to the membrane, the
membrane was shaken at room temperature for 1 hour and washing with
TBS solution under 10-minute shaking was repeated four times. A
detection reagent (ECL Plus Western Blotting Detection System, GE
Healthcare Bio-Sciences KK) was added and reacted for 1 minute to
detect chemiluminescence. The assay confirmed that the antibodies
produced by the two antibody-producing cells cloned in the above
(6) bind to human periostin PN-2.
(8) Mass Production and Purification of Monoclonal Antibodies
[0219] To BALB/c mice, 0.5 mL of pristane
[2,6,10,14-tetramethylpentadecane (Wako Pure Chemical Industries,
Ltd.)] was intraperitoneally administered and the mice were kept
for 2 to 3 weeks. The monoclonal antibody-producing hybridomas No.
8 and No. 10 that had been maintained at the logarithmic growth
phase were collected and centrifuged for removal of the culture
supernatant. To each of the cell pellet fractions, FCS-free RPMI
medium was added to prepare a cell suspension at a cell density of
1.times.10.sup.7 cells/mL. The cell suspension was
intraperitoneally injected into the BALB/c mice pretreated with
pristane. About three weeks later, the exuded ascites was collected
from the abdominal with a syringe. The collected ascites was passed
through a filter with a pore diameter of 0.22 .mu.m, the filtrate
was purified in a conventional manner by affinity chromatography on
a Protein G-Sepharose column (Millipore, 11511324) to prepare two
types of anti-human Exon-21 monoclonal antibodies.
Example 6: Epitope Analysis of Anti-Human Exon-21 Monoclonal
Antibodies
[0220] Replitope (an array of 37 types of peptides fixed on a glass
slide) was produced and used to identify the epitopes for the
anti-human periostin Exon-21 peptide monoclonal antibodies (two
types) and for the anti-rat periostin Exon-21 peptide polyclonal
antibodies (two types).
[0221] As primary antibodies, the rabbit IgG No. 1 (0.51 mg/mL) and
No. 3 (0.62 mg/mL) polyclonal antibodies, and the No. 8 Mouse IgG2b
(2.21 mg/mL) and No. 10 Mouse IgG1 (1.35 mg/mL) monoclonal
antibodies were used. As secondary antibodies, Cy5-conjugated
anti-rabbit IgG (H+L) (JIR 111-175-144) and Dylight 649-conjugated
anti-mouse IgG (Pierce, #35515) were used. The peptides were
synthesized on cellulose membranes. The peptides were then
transferred to microliter plates and spotted on a glass surface
with a nano-pipetting system for peptide arrays (JPT Peptide
Technologies GmbH, Berlin, Germany). The peptide microarray was
incubated with blocking buffer (Pierce International, Superblock
TBS, order #37536) for 2 hours. The microarray was incubated with
the primary antibodies (10 .mu.g/mL, in blocking buffer, total
assay volume 200 .mu.L) or blocking buffer alone in a microarray
hybridization station (TECAN HS400 microarray hybridization
station). The microarray was washed with TBS-buffer (50 mM
TBS-buffer+0.1% Tween 20 (JPT), pH 7.2), and incubated with the
fluorochrome-conjugated secondary antibodies (anti-rabbit IgG or
anti-mouse IgG) at a final concentration of 1 .mu.g/mL in blocking
buffer. The microarray was washed three times with TBS-buffer and
then washed with SSC-buffer (3 mM SSC-buffer (JPT), pH 7.0) and
dried under nitrogen stream. The microarray was finally analyzed
with a high-resolution fluorescence scanner (trade name: GenePix
4200AL, Axon, Inc.). Spot analysis was performed using a software
GenePix 6.0.
[0222] As a result of the assay, the epitopes for the anti-rat
Exon-21 polyclonal antibodies were identified to be the sequences
TKVTK (SEQ ID NO: 21) and QGDTPVRK (SEQ ID NO: 22), and the
epitopes for the anti-human Exon-21 monoclonal antibodies were
identified to be the sequences VTKVTK (SEQ ID NO: 19) and FEDEEIK
(SEQ ID NO: 20).
Example 7: Study of Inhibitory Effect of Anti-Rat Exon-21
Polyclonal Antibody on Vascular Intimal Hyperplasia Using Rat
Carotid Artery Balloon Injury Model
[0223] Injury was induced in SD rats by scratching the left common
carotid artery with a 2F (French) Fogarty balloon catheter, and
simultaneously the anti-rat Exon-21 polyclonal antibody (ex21PoAb)
or a rabbit control IgG antibody (rIgG) was intravascularly
administered. One day, three days, one week, two weeks and three
weeks after the production of the intimal injury models, five rats
were fixed by perfusion for each time period. The blood vessels
were harvested, and the balloon injury-induced carotid arteries as
samples and the carotid arteries with no injury as controls were
dissected in a length of about 1 cm from the blood vessels. About 2
to 3 mm segment of each of the dissected blood vessels was fixed in
4% paraformaldehyde, and the rest of each vessel was used for
extraction of total RNA for cDNA synthesis. From the fixed tissue,
sections were prepared and stained with HE (hematoxylin-eosine)
(FIG. 4B). The areas of the tunica intima and the tunica media in
all the HE stained sections were determined using a graphical
analysis software (trade name: Graphic converter ver. 4.02). From
the determined areas, the intima/media ratio was calculated, and
the severity of intimal hyperplasia was examined. As shown in FIG.
4A, a significant difference of p=0.0362 (N=8) was observed in the
intima/media ratio between the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) administration group and the rabbit control IgG antibody
(rIgG) group. The results showed the inhibition of intimal
hyperplasia (FIGS. 4A and 4B).
Example 8: Study of Inhibitory Effect of Anti-Rat Exon-21
Polyclonal Antibody on Vascular Intimal Hyperplasia Using ApoE
Knockout Mice
[0224] ApoE knockout mice, an arteriosclerosis-prone model, were
stimulated with a high-fat diet, and simultaneously with the start
of high-fat diet feeding, the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) or a rabbit control IgG antibody (rIgG) was
intraperitoneally administered at a dose of 100 .mu.g/mL once a
week. Three months later, the aorta was dissected. The developed
arteriosclerotic lesions were compared in terms of the following
two parameters: the severity of arteriosclerosis defined as the
ratio of Oil red O stained positive area to the total blood vessel
area; and the % change in the severity of arteriosclerosis defined
as the severity of arteriosclerosis divided by the IgG
administration group's average value of the severity of
arteriosclerosis. The results showed significant inhibition of
aortic arteriosclerosis in the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) administration group as compared with the rabbit control
IgG antibody (rIgG) administration group (N=3) (p<0.05) (FIG.
5).
Example 9: Study of Anti-Inflammatory Effect of Anti-Human Exon-21
Monoclonal Antibody Using Mouse Colitis Model
[0225] A study was performed to investigate the effect of the
anti-human Exon-21 monoclonal antibody (ex21MoAb) or a mouse
control IgG antibody (mIgG) on a mouse colitis model with 1.75%
dextran sulfate sodium (DDS)-induced Crohn's disease.
Simultaneously with DDS administration, each antibody was
intraperitoneally administered at a dose of 100 .mu.g/animal once a
week. Two weeks later, the large intestines were harvested, and the
lengths of the large intestines were measured and compared. Due to
DDS administration, the lengths of the large intestines were
significantly shortened as compared with the sham group (N=6)
(p<0.01). However, the anti-human Exon-21 monoclonal antibody
(ex21MoAb) administration group showed significant inhibition of
the shortening of the length of the large intestine, as compared
with the mouse control IgG antibody (mIgG) administration group and
the DDS-alone administration group (N=6) (p<0.05) (FIG. 6).
Example 10: Study of Inhibitory Effect of Anti-Rat Exon-21
Polyclonal Antibody on Angiogenesis Using Lower Extremity Ischemia
Model
[0226] The lower extremity femoral artery of C57BL6N mice was
ligated to prepare a lower extremity ischemia model. Three days
before the ligation, the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) or a rabbit control IgG antibody (rIgG) was
intraperitoneally administered at a dose of 40 .mu.g/mL (twice a
week). Zero day (immediately before the administration), and 1, 7,
14, 21 and 28 days after the administration, the lower extremity
blood flow in the unaffected side and the affected side was
measured with Laser Doppler Imager (LDI; Moor Instruments). The
relative perfusion rate defined as the affected side
flow/unaffected side flow was calculated for the evaluation of the
lower extremity blood flow, and the groups were compared. The
relative perfusion rate 28 days later showed a significant decrease
in the lower extremity blood flow in the anti-rat Exon-21
polyclonal antibody (ex21PoAb) administration group as compared
with the rabbit control IgG antibody (rIgG) administration group
(p<0.05) (FIG. 7). The adducent muscles of the mice were
harvested, and frozen sections were prepared, immunostained with a
CD31 antibody, and subjected to the evaluation of the blood
vessels. The number of the blood vessels per unit area was
significantly smaller in the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) administration group than in the rabbit control IgG
antibody (rIgG) administration group, which revealed significant
inhibition of angiogenesis (p<0.01) (N=6) (FIG. 8). The same set
of experiments was performed using the anti-human Exon-21
monoclonal antibody (ex21MoAb). As with the case of the polyclonal
antibody, the relative perfusion rate computed with LDI indicated a
significant decrease in the blood flow as compared with the mouse
control IgG antibody (mIgG) administration group, which revealed
inhibition of angiogenesis (FIG. 9).
Example 11: In Vitro Inhibitory Effect of Anti-Rat Exon-21
Polyclonal Antibody on Tube Formation
[0227] The supernatant of 4T1 breast cancer cells was added to
Matrigel (BD Bioscience, No. 356231). To this, a mouse control IgG
antibody (mIgG), the anti-human Exon-21 monoclonal antibody
(ex21MoAb), a rabbit control IgG antibody (rIgG), and the anti-rat
Exon-21 polyclonal antibody (ex21PoAb), each at a concentration of
1/100, were separately added. Each mixture was added to a 96-well
plate in an amount of 70 .mu.L/well. The mixtures were incubated
for 30 minutes to allow gel formation, and HUVECs (normal human
umbilical vein endothelial cells) were seeded at a cell density of
1.5.times.10.sup.4 cells/well. The cells were incubated for 5
hours, stained with a crystal violet solution, and photographed
under a microscope. The number of tubes was counted. The angiogenic
properties of the supernatant of 4T1 breast cancer cells were
significantly inhibited by the anti-rat Exon-21 polyclonal antibody
and the anti-human Exon-21 monoclonal antibody (p<0.05) (FIGS.
10A and 10B). Matrigel to which PN-2 protein in various
concentrations was added was processed and evaluated in the same
manner as above. The evaluation showed that, at concentrations of 5
.mu.g/mL or more, PN-2 protein significantly induced tube formation
in a dose-dependent manner, which indicated the angiogenic
properties of PN-2 protein (p<0.05) (FIGS. 11A and 11B). The
results of the study showed that the anti-Exon-21 antibodies have
an angiogenesis inhibitory effect. The results also indicated the
angiogenic properties of PN-2 protein.
Example 12: In Vitro Study of Influence of Anti-Rat Exon-21
Polyclonal Antibody on Cell Growth
[0228] Mouse 4T1 breast cancer cells were seeded in DMEM (serum
free, PC/SM) in a 96-well cell culture multi-well plate at a
density of 10.sup.3 cells/well and cultured in a 37.degree. C.
incubator overnight. After removal of the culture supernatant, to
the wells was added DMEM (serum free, PC/SM) medium to which the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) or a rabbit control
IgG antibody (rIgG) (each at 200 .mu.g/mL) was added at a final
concentration of 1 .mu.g/mL. The cells were then cultured
overnight. On the following day, the medium in all the wells was
replaced with DMEM (10% BSA, PC/SM), and 20 .mu.L of CellTiter
Reagent of a cell proliferation assay kit (trade name: CellTiter 96
AQueous One Solution Cell Proliferation Assay kit, Promega) was
added to 100 .mu.L of the medium in the wells. The cells were
cultured at 37.degree. C. for 1 hour. The staining intensity of the
cells was measured with a plate reader (BIO-RAD, Model 680 MICRO
PLATE READER) at 490 nm (FIG. 12A). The results revealed that the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) exhibited cell
growth inhibitory activity at a high concentration as compared with
the rabbit control IgG antibody (rIgG) (p<0.05).
[0229] Another investigation was performed to determine whether the
anti-human Exon-21 monoclonal antibody has cytotoxicity against
mouse 4T1 breast cancer cells. Mouse F4T1 breast cancer cells were
seeded in DMEM (serum free, PC/SM) in a 96-well cell culture
multi-well plate at a density of 1.times.10.sup.4 cells/well and
cultured in a 37.degree. C. incubator overnight. After removal of
the culture supernatant, to the wells was added DMEM (serum free,
PC/SM) medium to which the anti-human Exon-21 monoclonal antibody
(ex21MoAb) or a mouse control IgG antibody (mIgG) (each at 100
.mu.g/mL) was added at a final concentration of 1 .mu.g/mL. The
cells were then cultured for 6 hours. The culture supernatant was
collected and lactate dehydrogenase (LDH) contained in the
supernatant was measured by ELISA. For the measurement, a
cytotoxicity assay kit (trade name: LDH Cytotoxicity Detection Kit,
Takara Bio, Inc.) was used. LDH is an enzyme present in the
cytoplasm and usually does not permeate through the cell membrane.
When the cell membrane is damaged, LDH is released outside the
cell, i.e., into the medium. LDH, therefore, can serve as an
indicator of cytotoxicity. The supernatant was subjected to
measurement with a plate reader (BIO-RAD, Model 680 MICRO PLATE
READER) at 490 nm. The results revealed that the anti-human Exon-21
monoclonal antibody (ex21MoAb) exhibited significant cell growth
inhibitory activity at a high concentration as compared with the
mouse control IgG antibody (mIgG) (P<0.01) (FIG. 12B).
Example 13: Study of Effect of Anti-Rat Exon-21 Polyclonal Antibody
Using Lung Metastasis Model Mice of Mouse 4T1 Breast Cancer
Cells
[0230] Mouse 4T1 cells (ATCC) were seeded on 10 cm Tissue Culture
Dishes (Greiner) containing RPMI 1640 (Gibco) containing 10% bovine
serum albumin (FBS) (Biowest) and Penicillin-streptomycin Mixed
solution (Nacalai Tesque), and the cells were cultured in a
37.degree. C. incubator for 24 hours. The culture supernatant was
removed, and the cells were washed with PBS and then treated with
trypsin/EDTA. The floating cells were collected and centrifuged at
1,500 rpm for 3 minutes. Subsequently, 1.5.times.10.sup.5 cells
were passaged and cultured in a 37.degree. C. incubator for 72
hours. The cells in the logarithmic growth phase were suspended in
100 .mu.L of PBS so as to be 1.times.10.sup.6 cells/animal. The
prepared cells were injected into the foot pad of female BALB/c
mice at 8 weeks of age using an insulin syringe equipped with an
injection needle, 29 G Myjector (TERMO), to establish lung
metastasis model mice. To the model mice, the anti-rat Exon-21
polyclonal antibody (ex21PoAb) or a rabbit control IgG antibody
(rIgG) was intraperitoneally administered at 100 .mu.g/animal per
week using an insulin syringe equipped with an injection needle, 29
G Myjector. For the experiment using the anti-rat Exon-21
polyclonal antibody (ex21PoAb) (1 mg/mL), the cell injection was
performed simultaneously with the antibody administration, and one
week after and two weeks after the cell injection, further antibody
administrations were performed. The rabbit control IgG antibody
(rIgG) used was Normal Rabbit IgG (R&D Systems). After the cell
injection, the diameter of the swelling lesions in the lower
extremities were measured with a caliper to evaluate the volume of
the primary tumors. The evaluation was performed in accordance with
Dethlefsen L A. et al. J. Natl. Cancer Inst., 40, 389 (1968), using
the formula: (length of foot sole).times.(width of foot sole).sub.
2/2. The mice were dissected three weeks after the cell injection,
and body weight measurement, an examination of the presence or
absence of lung metastasis from the primary tumors, and the
counting of the lung metastatic colonies were performed. The
obtained data were analyzed by Student's t-test. The volume of the
primary tumors in the lower extremities three weeks after the cell
injection was significantly reduced by the administration of the
anti-rat Exon-21 polyclonal antibody (ex21PoAb) (p<0.05) (FIG.
13A). A comparison of the number of the lung metastatic colonies
five weeks after the cell injection revealed significant reduction
in the number of the colonies by the administration of the anti-rat
Exon-21 polyclonal antibody (ex21PoAb) (P<0.05) (FIG. 13B). The
same set of experiments was performed using the anti-human Exon-21
monoclonal antibody (ex21MoAb). As with the case of the polyclonal
antibody, significant reduction in the number of the lung
metastatic colonies was observed, as compared with a mouse control
IgG antibody (mIgG) administration group and with a PBS (-)
administration group (FIG. 14). The results revealed the inhibitory
effect of the anti-Exon-21 antibodies on the primary tumors and
metastatic foci of 4T1 breast cancer cells.
Example 14: Study of Effect of Anti-Rat Exon-21 Polyclonal Antibody
Using Lung Metastasis Model Mice of Mouse B16F10 Melanoma Cells
[0231] Mouse B16F10 melanoma cells (mouse melanoma cell line
B16F10) were cultured in a 37.degree. C. incubator, washed with
PBS, and treated with trypsin/EDTA. The floating cells were
collected and centrifuged at 1, 500 rpm for 3 minutes. The
collected cells were suspended in 100 .mu.L of PBS so as to be
5.times.10.sup.3 cells/animal. The prepared cells were injected
into the foot pad of male C57BL/6N mice at 8 weeks of age using an
insulin syringe equipped with an injection needle, 29 G Myjector
(TERMO), to establish lung metastasis model mice. To the model
mice, the anti-rat Exon-21 polyclonal antibody (ex21PoAb) or a
rabbit control IgG antibody (rIgG) was administered at 100
.mu.g/animal per week using an insulin syringe equipped with an
injection needle, 29 G Myjector. The control used was Normal Rabbit
IgG (R&D Systems).
[0232] To reproduce the conditions close to the clinical situation,
the administration of the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) (1 mg/mL) or a rabbit control IgG antibody (rIgG) (1
mg/mL) as a control at 100 .mu.g/animal per week was performed one
week after the injection of the mouse B16F10 melanoma cells. The
primary tumors were measured with a caliper to evaluate the volume
of the primary tumors. The evaluation was performed in accordance
with Dethlefsen L A. et al. J. Natl. Cancer Inst., 40, 389 (1968),
using the formula: (length of foot sole).times.(width of foot
sole).sup. 2/2. Three weeks after the cell injection, the
administration of the anti-rat Exon-21 polyclonal antibody
significantly inhibited the growth of the primary tumors as
compared the administration of the rabbit control IgG antibody
(rIgG) (p<0.05) (FIG. 15A). The number of lung metastatic
colonies was counted by visual inspection and compared. The results
showed that, five weeks after the cell injection, the
administration of the anti-rat Exon-21 polyclonal antibody
(ex21PoAb) significantly inhibited lung metastasis as compared with
the administration of the rabbit control IgG antibody (rIgG)
(p<0.05) (FIG. 15B). The data analysis of the number of the lung
metastatic colonies was performed by Mann-Whitney test.
Example 15: Study of Effect of Anti-Human Exon-21 Monoclonal
Antibody Using Aneurysm Model Mice
[0233] The study was conducted using aged (6 months or more) ApoE
knockout mice. Angiotensin II was dissolved in physiological
saline. The angiotensin II (Sigma Aldrich, A9525) solution was
loaded into an osmotic pump for continuous administration (Alzet,
Muromachi Kikai Co., Ltd., Model: 2004). The dosage was set at
1,000 ng/kgday. The adjusted osmotic pump was primed with
physiological saline for a night and day. Vaporized isoflurane was
administered to the mice by inhalation at a concentration of 2% and
a flow rate of 1 L/min. After sufficient anesthesia was achieved,
the mice were maintained in a prone position. The neck skin was
sterilized with a 70% ethanol solution and a small incision was
made. From the incision site, scissors were inserted under the
mouse skin, and the skin was peeled off. A space for placing the
pump was created, and the osmotic pump was implanted under the
mouse skin. After implantation, the incision was closed with
staples for small animal surgery (Fine Science Tools, Muromachi
Kikai Co., Ltd., Model: 12040-01).
[0234] A mouse control IgG antibody (mIgG, 100 .mu.g/animal) or the
anti-human Exon-21 monoclonal antibody (ex21MoAb; 100 .mu.g/animal)
was intraperitoneally administered once a week, starting from zero
week. Four weeks later, the diameter of the aorta was measured with
an ultrasound scanner (Toshiba Medical Systems Corporation, Aplio
XV). The results revealed that the anti-human Exon-21 monoclonal
antibody (ex21MoAb) significantly inhibited the expansion of the
diameter of the aorta (FIG. 16).
INDUSTRIAL APPLICABILITY
[0235] An antibody against a periostin isoform having cell adhesion
activity can be used to prevent and treat inflammation-associated
diseases including cancers. The antibody can also be used for the
measurement of the amount of such a periostin isoform in a patient
sample to determine the presence or absence of a cancer and the
progression of the disease conditions.
Accession No.
Identification of the Microorganism
[0236] Identification Reference: KS-0259#8, 080611 Kohjin Bio
Accession No.: NITE BP-01546
Deposit Date
[0237] Feb. 26, 2013
International Depositary Authority
[0238] Name: Incorporated Administrative Agency, National Institute
of Technology and Evaluation, Patent Microorganisms Depositary
[0239] Address: Room 122, 2-5-8, Kazusakamatari, Kisarazu-shi,
Chiba 292-0818 JAPAN
Sequence CWU 1
1
311838PRTRattus sp. 1Met Val Pro Leu Leu Pro Leu Ser Ala Leu Leu
Leu Leu Phe Leu Cys1 5 10 15Asp Val Asp Pro Ala Asn Ala Asn Ser Tyr
Tyr Asp Lys Val Leu Ala 20 25 30His Ser Arg Ile Arg Gly Arg Asp Gln
Gly Pro Asn Val Cys Ala Leu 35 40 45Gln Gln Ile Leu Gly Thr Lys Lys
Lys Tyr Phe Ser Ser Cys Lys Asn 50 55 60Trp Tyr Gln Gly Ala Ile Cys
Gly Lys Lys Thr Thr Val Leu Tyr Glu65 70 75 80Cys Cys Pro Gly Tyr
Met Arg Met Glu Gly Met Lys Gly Cys Pro Ala 85 90 95Val Met Pro Ile
Asp His Val Tyr Gly Thr Leu Gly Ile Val Gly Ala 100 105 110Thr Thr
Thr Gln His Tyr Ser Asp Val Ser Lys Leu Arg Glu Glu Ile 115 120
125Glu Gly Lys Gly Ser Tyr Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp
130 135 140Asp Asn Leu Asp Ser Asp Ile Arg Arg Gly Leu Glu Asn Asn
Val Asn145 150 155 160Val Glu Leu Leu Asn Ala Leu His Ser His Met
Val Asn Lys Arg Met 165 170 175Leu Thr Lys Asp Leu Lys His Gly Met
Val Ile Pro Ser Met Tyr Asn 180 185 190Asn Leu Gly Leu Phe Ile Asn
His Tyr Pro Asn Gly Val Val Thr Val 195 200 205Asn Cys Ala Arg Val
Ile His Gly Asn Gln Ile Ala Thr Asn Gly Val 210 215 220Val His Val
Ile Asp Arg Val Leu Thr Gln Ile Gly Thr Ser Ile Gln225 230 235
240Asp Phe Ile Glu Ala Glu Asp Glu Leu Ser Ser Phe Arg Ala Ala Ala
245 250 255Ile Thr Ser Asp Leu Leu Glu Ser Leu Gly Arg Asp Gly His
Phe Thr 260 265 270Leu Phe Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu
Pro Arg Gly Val 275 280 285Leu Glu Arg Ile Met Gly Asp Lys Val Ala
Ser Glu Ala Leu Met Lys 290 295 300Tyr His Ile Leu Asn Thr Leu Gln
Cys Ser Glu Ala Ile Thr Gly Gly305 310 315 320Ala Val Phe Glu Thr
Met Glu Gly Asn Thr Ile Glu Ile Gly Cys Glu 325 330 335Gly Asp Ser
Ile Ser Ile Asn Gly Ile Lys Met Val Asn Lys Lys Asp 340 345 350Ile
Val Thr Lys Asn Gly Val Ile His Leu Ile Asp Glu Val Leu Ile 355 360
365Pro Asp Ser Ala Lys Gln Val Ile Glu Leu Ala Gly Lys Gln Gln Thr
370 375 380Thr Phe Thr Asp Leu Val Ala Gln Leu Gly Leu Ala Ser Ser
Leu Lys385 390 395 400Pro Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val
Asn Asn Ala Phe Ser 405 410 415Asp Asp Thr Leu Ser Met Asp Gln Arg
Leu Leu Lys Leu Ile Leu Gln 420 425 430Asn His Ile Leu Lys Val Lys
Val Gly Leu Ser Asp Leu Tyr Asn Gly 435 440 445Gln Ile Leu Glu Thr
Ile Gly Gly Lys Gln Leu Arg Val Phe Val Tyr 450 455 460Arg Thr Ala
Ile Cys Ile Glu Asn Ser Cys Met Val Arg Gly Ser Lys465 470 475
480Gln Gly Arg Asn Gly Ala Ile His Ile Phe Arg Glu Ile Ile Gln Pro
485 490 495Ala Glu Lys Ser Leu His Glu Lys Leu Arg Gln Asp Lys Arg
Phe Ser 500 505 510Ile Phe Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys
Asp Leu Leu Thr 515 520 525Gln Pro Gly Asp Trp Thr Leu Phe Ala Pro
Thr Asn Asp Ala Phe Lys 530 535 540Gly Met Thr Asn Glu Glu Arg Glu
Ile Leu Ile Gly Asp Lys Asn Ala545 550 555 560Leu Gln Asn Ile Ile
Leu Tyr His Leu Thr Pro Gly Val Tyr Ile Gly 565 570 575Lys Gly Phe
Glu Pro Gly Val Thr Asn Ile Leu Lys Thr Thr Gln Gly 580 585 590Ser
Lys Ile Tyr Val Lys Gly Val Asn Glu Thr Leu Leu Val Asn Glu 595 600
605Leu Lys Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly Val Ile His
610 615 620Val Val Asp Lys Leu Leu Tyr Pro Ala Asp Ile Pro Val Gly
Asn Asp625 630 635 640Gln Leu Leu Glu Leu Leu Asn Lys Leu Ile Lys
Tyr Ile Gln Ile Lys 645 650 655Phe Val Arg Gly Ser Thr Phe Lys Glu
Ile Pro Met Thr Val Tyr Thr 660 665 670Thr Lys Ile Ile Thr Lys Leu
Val Glu Pro Lys Ile Lys Val Ile Gln 675 680 685Gly Ser Leu Gln Pro
Ile Ile Lys Thr Glu Gly Pro Ala Met Thr Lys 690 695 700Ile His Ile
Glu Gly Glu Pro Asp Phe Arg Leu Ile Lys Glu Gly Glu705 710 715
720Thr Val Thr Glu Val Ile His Gly Glu Pro Val Ile Lys Lys Tyr Thr
725 730 735Lys Ile Ile Asp Gly Val Pro Val Glu Ile Thr Glu Lys Glu
Thr Arg 740 745 750Glu Glu Arg Ile Ile Thr Gly Pro Glu Ile Lys Tyr
Thr Arg Ile Ser 755 760 765Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu
Gln Lys Phe Leu Gln Lys 770 775 780Glu Val Ser Lys Val Thr Lys Phe
Ile Glu Gly Gly Asp Gly His Leu785 790 795 800Phe Glu Asp Glu Ala
Ile Lys Arg Leu Leu Gln Gly Asp Thr Pro Ala 805 810 815Lys Lys Ile
Gln Ala Asn Lys Arg Val Gln Gly Ser Arg Arg Arg Ser 820 825 830Arg
Glu Gly Arg Ser Gln 83522517DNARattus sp. 2atggttcctc tcctgccctt
atctgctctg ctgctgctgt tcctgtgtga cgttgacccc 60gcaaatgcca acagttacta
tgacaaggtc ctagctcaca gccgcatcag gggtcgggat 120cagggcccaa
atgtctgtgc cctccagcag attctgggca ccaaaaagaa atacttcagc
180tcctgtaaga actggtatca aggtgctatc tgcgggaaga aaaccactgt
gctatatgaa 240tgctgccccg gctatatgag aatggaaggg atgaaaggct
gcccagcagt gatgcccatt 300gaccatgttt atggcacgct gggcatcgtg
ggagccacga ccactcaaca ctattctgat 360gtctcgaagc tcagggaaga
gattgaagga aaagggtcct acacatactt cgcgccgagt 420aacgaagctt
gggacaacct ggattccgac atccgcagag gactagagaa caatgtcaat
480gttgagttac tgaacgcttt acacagccac atggttaata agagaatgct
aaccaaggac 540ctgaaacacg gcatggttat tccttcaatg tacaacaatc
tggggctttt tatcaatcat 600tatcccaatg gggttgtcac tgtgaactgt
gctcgagtaa tccacgggaa ccagattgcc 660acaaatggtg ttgtccatgt
catcgaccgt gtcctgacac aaattggcac ctccatccaa 720gacttcattg
aagcagaaga tgagctttca tcattcagag cggctgccat cacttctgac
780cttttggagt cccttggaag agacggtcac ttcacactct ttgctcccac
caatgaggct 840ttcgagaaac tcccacgagg agtcctagaa aggatcatgg
gagacaaagt ggcttctgaa 900gctctcatga agtaccacat cctgaatacc
ctccagtgct ctgaggctat cacaggagga 960gcggtgtttg agaccatgga
aggaaacact attgaaatag ggtgtgaggg agacagcatc 1020tccattaacg
gaatcaagat ggtgaacaag aaagacattg tgacgaagaa tggtgtcatc
1080cacctgattg atgaagtcct cattcctgat tctgctaaac aagttattga
gctggctgga 1140aaacagcaaa ccactttcac ggacctggta gcccagttag
ggttggcgtc ttctctgaag 1200ccggatggag agtacacgct gttagcgcct
gtgaacaatg cgttctctga tgacactctg 1260agcatggacc agcgccttct
taagctaatt ctgcaaaatc acatattgaa agtaaaagtc 1320ggccttagtg
atctctacaa tggacagatt ctggagacca ttggaggcaa acaactccgt
1380gtcttcgtgt atcggacggc tatctgcata gaaaactcat gcatggtgag
aggaagcaag 1440caggggagga acggtgccat tcacatattc cgagagatca
tccaaccggc ggagaagtcc 1500ctgcacgaaa aactgcgcca agataagcgc
ttcagcatct tcctcagcct cctcgaagct 1560gcagatctga aagatcttct
gacacagccc ggagattgga ccttgtttgc accaaccaat 1620gatgccttca
agggaatgac taatgaagaa agggagattc tgattgggga taaaaatgct
1680ctccaaaaca tcattcttta ccacctgacc ccaggggttt atattggaaa
gggatttgaa 1740cccggagtca ccaacatcct gaagaccaca cagggaagca
aaatctatgt gaaaggagtc 1800aatgagacgc ttttggtgaa tgagttgaag
tccaaagaat ctgacatcat gacaacaaac 1860ggcgtcattc acgttgtgga
caaactcctc tatccagcag acattccggt tggaaatgat 1920cagctcttgg
aattactgaa caaactgata aaatacatcc aaattaagtt cgttcgtggc
1980agcaccttca aagaaatccc catgactgtc tatacaacta aaattataac
caaactcgtg 2040gaaccaaaaa ttaaagtcat tcaaggcagt cttcagccta
ttatcaaaac agaaggacct 2100gcaatgacga agatccacat tgaaggcgag
cctgacttca ggctgattaa agaaggtgaa 2160acagtgacag aagtgatcca
cggagaacca gtcattaaaa agtacaccaa aatcatagac 2220ggggttcctg
ttgaaataac tgaaaaagag acccgggaag aacgcatcat cacaggtcct
2280gagataaaat acactaggat ttccacagga ggtggggaaa cagaagagac
cctgcagaaa 2340ttcttgcaaa aagaggtctc caaggtcaca aagttcattg
aaggtggcga tggtcactta 2400tttgaagatg aggcgattaa aagactgctt
cagggagaca cacctgcaaa gaagatacaa 2460gccaacaaaa gggttcaagg
gtctagaagg cgatcaagag aaggccgttc tcagtga 25173811PRTRattus sp. 3Met
Val Pro Leu Leu Pro Leu Ser Ala Leu Leu Leu Leu Phe Leu Cys1 5 10
15Asp Val Asp Pro Ala Asn Ala Asn Ser Tyr Tyr Asp Lys Val Leu Ala
20 25 30His Ser Arg Ile Arg Gly Arg Asp Gln Gly Pro Asn Val Cys Ala
Leu 35 40 45Gln Gln Ile Leu Gly Thr Lys Lys Lys Tyr Phe Ser Ser Cys
Lys Asn 50 55 60Trp Tyr Gln Gly Ala Ile Cys Gly Lys Lys Thr Thr Val
Leu Tyr Glu65 70 75 80Cys Cys Pro Gly Tyr Met Arg Met Glu Gly Met
Lys Gly Cys Pro Ala 85 90 95Val Met Pro Ile Asp His Val Tyr Gly Thr
Leu Gly Ile Val Gly Ala 100 105 110Thr Thr Thr Gln His Tyr Ser Asp
Val Ser Lys Leu Arg Glu Glu Ile 115 120 125Glu Gly Lys Gly Ser Tyr
Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp 130 135 140Asp Asn Leu Asp
Ser Asp Ile Arg Arg Gly Leu Glu Asn Asn Val Asn145 150 155 160Val
Glu Leu Leu Asn Ala Leu His Ser His Met Val Asn Lys Arg Met 165 170
175Leu Thr Lys Asp Leu Lys His Gly Met Val Ile Pro Ser Met Tyr Asn
180 185 190Asn Leu Gly Leu Phe Ile Asn His Tyr Pro Asn Gly Val Val
Thr Val 195 200 205Asn Cys Ala Arg Val Ile His Gly Asn Gln Ile Ala
Thr Asn Gly Val 210 215 220Val His Val Ile Asp Arg Val Leu Thr Gln
Ile Gly Thr Ser Ile Gln225 230 235 240Asp Phe Ile Glu Ala Glu Asp
Glu Leu Ser Ser Phe Arg Ala Ala Ala 245 250 255Ile Thr Ser Asp Leu
Leu Glu Ser Leu Gly Arg Asp Gly His Phe Thr 260 265 270Leu Phe Ala
Pro Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val 275 280 285Leu
Glu Arg Ile Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys 290 295
300Tyr His Ile Leu Asn Thr Leu Gln Cys Ser Glu Ala Ile Thr Gly
Gly305 310 315 320Ala Val Phe Glu Thr Met Glu Gly Asn Thr Ile Glu
Ile Gly Cys Glu 325 330 335Gly Asp Ser Ile Ser Ile Asn Gly Ile Lys
Met Val Asn Lys Lys Asp 340 345 350Ile Val Thr Lys Asn Gly Val Ile
His Leu Ile Asp Glu Val Leu Ile 355 360 365Pro Asp Ser Ala Lys Gln
Val Ile Glu Leu Ala Gly Lys Gln Gln Thr 370 375 380Thr Phe Thr Asp
Leu Val Ala Gln Leu Gly Leu Ala Ser Ser Leu Lys385 390 395 400Pro
Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser 405 410
415Asp Asp Thr Leu Ser Met Asp Gln Arg Leu Leu Lys Leu Ile Leu Gln
420 425 430Asn His Ile Leu Lys Val Lys Val Gly Leu Ser Asp Leu Tyr
Asn Gly 435 440 445Gln Ile Leu Glu Thr Ile Gly Gly Lys Gln Leu Arg
Val Phe Val Tyr 450 455 460Arg Thr Ala Ile Cys Ile Glu Asn Ser Cys
Met Val Arg Gly Ser Lys465 470 475 480Gln Gly Arg Asn Gly Ala Ile
His Ile Phe Arg Glu Ile Ile Gln Pro 485 490 495Ala Glu Lys Ser Leu
His Glu Lys Leu Arg Gln Asp Lys Arg Phe Ser 500 505 510Ile Phe Leu
Ser Leu Leu Glu Ala Ala Asp Leu Lys Asp Leu Leu Thr 515 520 525Gln
Pro Gly Asp Trp Thr Leu Phe Ala Pro Thr Asn Asp Ala Phe Lys 530 535
540Gly Met Thr Asn Glu Glu Arg Glu Ile Leu Ile Gly Asp Lys Asn
Ala545 550 555 560Leu Gln Asn Ile Ile Leu Tyr His Leu Thr Pro Gly
Val Tyr Ile Gly 565 570 575Lys Gly Phe Glu Pro Gly Val Thr Asn Ile
Leu Lys Thr Thr Gln Gly 580 585 590Ser Lys Ile Tyr Val Lys Gly Val
Asn Glu Thr Leu Leu Val Asn Glu 595 600 605Leu Lys Ser Lys Glu Ser
Asp Ile Met Thr Thr Asn Gly Val Ile His 610 615 620Val Val Asp Lys
Leu Leu Tyr Pro Ala Asp Ile Pro Val Gly Asn Asp625 630 635 640Gln
Leu Leu Glu Leu Leu Asn Lys Leu Ile Lys Tyr Ile Gln Ile Lys 645 650
655Phe Val Arg Gly Ser Thr Phe Lys Glu Ile Pro Met Thr Val Tyr Arg
660 665 670Pro Ala Met Thr Lys Ile His Ile Glu Gly Glu Pro Asp Phe
Arg Leu 675 680 685Ile Lys Glu Gly Glu Thr Val Thr Glu Val Ile His
Gly Glu Pro Val 690 695 700Ile Lys Lys Tyr Thr Lys Ile Ile Asp Gly
Val Pro Val Glu Ile Thr705 710 715 720Glu Lys Glu Thr Arg Glu Glu
Arg Ile Ile Thr Gly Pro Glu Ile Lys 725 730 735Tyr Thr Arg Ile Ser
Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu Gln 740 745 750Lys Phe Leu
Gln Lys Glu Val Ser Lys Val Thr Lys Phe Ile Glu Gly 755 760 765Gly
Asp Gly His Leu Phe Glu Asp Glu Ala Ile Lys Arg Leu Leu Gln 770 775
780Gly Asp Thr Pro Ala Lys Lys Ile Gln Ala Asn Lys Arg Val Gln
Gly785 790 795 800Ser Arg Arg Arg Ser Arg Glu Gly Arg Ser Gln 805
810427PRTRattus sp. 4Thr Thr Lys Ile Ile Thr Lys Val Val Glu Pro
Lys Ile Lys Val Ile1 5 10 15Glu Gly Ser Leu Gln Pro Ile Ile Lys Thr
Glu 20 2552436DNARattus sp. 5atggttcctc tcctgccctt atctgctctg
ctgctgctgt tcctgtgtga cgttgacccc 60gcaaatgcca acagttacta tgacaaggtc
ctagctcaca gccgcatcag gggtcgggat 120cagggcccaa atgtctgtgc
cctccagcag attctgggca ccaaaaagaa atacttcagc 180tcctgtaaga
actggtatca aggtgctatc tgcgggaaga aaaccactgt gctatatgaa
240tgctgccccg gctatatgag aatggaaggg atgaaaggct gcccagcagt
gatgcccatt 300gaccatgttt atggcacgct gggcatcgtg ggagccacga
ccactcaaca ctattctgat 360gtctcgaagc tcagggaaga gattgaagga
aaagggtcct acacatactt cgcgccgagt 420aacgaagctt gggacaacct
ggattccgac atccgcagag gactagagaa caatgtcaat 480gttgagttac
tgaacgcttt acacagccac atggttaata agagaatgct aaccaaggac
540ctgaaacacg gcatggttat tccttcaatg tacaacaatc tggggctttt
tatcaatcat 600tatcccaatg gggttgtcac tgtgaactgt gctcgagtaa
tccacgggaa ccagattgcc 660acaaatggtg ttgtccatgt catcgaccgt
gtcctgacac aaattggcac ctccatccaa 720gacttcattg aagcagaaga
tgagctttca tcattcagag cggctgccat cacttctgac 780cttttggagt
cccttggaag agacggtcac ttcacactct ttgctcccac caatgaggct
840ttcgagaaac tcccacgagg agtcctagaa aggatcatgg gagacaaagt
ggcttctgaa 900gctctcatga agtaccacat cctgaatacc ctccagtgct
ctgaggctat cacaggagga 960gcggtgtttg agaccatgga aggaaacact
attgaaatag ggtgtgaggg agacagcatc 1020tccattaacg gaatcaagat
ggtgaacaag aaagacattg tgacgaagaa tggtgtcatc 1080cacctgattg
atgaagtcct cattcctgat tctgctaaac aagttattga gctggctgga
1140aaacagcaaa ccactttcac ggacctggta gcccagttag ggttggcgtc
ttctctgaag 1200ccggatggag agtacacgct gttagcgcct gtgaacaatg
cgttctctga tgacactctg 1260agcatggacc agcgccttct taagctaatt
ctgcaaaatc acatattgaa agtaaaagtc 1320ggccttagtg atctctacaa
tggacagatt ctggagacca ttggaggcaa acaactccgt 1380gtcttcgtgt
atcggacggc tatctgcata gaaaactcat gcatggtgag aggaagcaag
1440caggggagga acggtgccat tcacatattc cgagagatca tccaaccggc
ggagaagtcc 1500ctgcacgaaa aactgcgcca agataagcgc ttcagcatct
tcctcagcct cctcgaagct 1560gcagatctga aagatcttct gacacagccc
ggagattgga ccttgtttgc accaaccaat 1620gatgccttca agggaatgac
taatgaagaa agggagattc tgattgggga taaaaatgct 1680ctccaaaaca
tcattcttta ccacctgacc ccaggggttt atattggaaa gggatttgaa
1740cccggagtca ccaacatcct gaagaccaca cagggaagca aaatctatgt
gaaaggagtc 1800aatgagacgc ttttggtgaa tgagttgaag tccaaagaat
ctgacatcat gacaacaaac 1860ggcgtcattc acgttgtgga caaactcctc
tatccagcag acattccggt tggaaatgat 1920cagctcttgg aattactgaa
caaactgata aaatacatcc aaattaagtt cgttcgtggc 1980agcaccttca
aagaaatccc catgactgtc tatagacctg caatgacgaa gatccacatt
2040gaaggcgagc ctgacttcag gctgattaaa gaaggtgaaa cagtgacaga
agtgatccac 2100ggagaaccag tcattaaaaa gtacaccaaa atcatagacg
gggttcctgt tgaaataact 2160gaaaaagaga cccgggaaga acgcatcatc
acaggtcctg agataaaata cactaggatt 2220tccacaggag gtggggaaac
agaagagacc ctgcagaaat
tcttgcaaaa agaggtctcc 2280aaggtcacaa agttcattga aggtggcgat
ggtcacttat ttgaagatga ggcgattaaa 2340agactgcttc agggagacac
acctgcaaag aagatacaag ccaacaaaag ggttcaaggg 2400tctagaaggc
gatcaagaga aggccgttct cagtga 2436628PRTRattus sp. 6Glu Val Ser Lys
Val Thr Lys Phe Ile Glu Gly Gly Asp Gly His Leu1 5 10 15Phe Glu Asp
Glu Ala Ile Lys Arg Leu Leu Gln Gly 20 257783PRTRattus sp. 7Met Val
Pro Leu Leu Pro Leu Ser Ala Leu Leu Leu Leu Phe Leu Cys1 5 10 15Asp
Val Asp Pro Ala Asn Ala Asn Ser Tyr Tyr Asp Lys Val Leu Ala 20 25
30His Ser Arg Ile Arg Gly Arg Asp Gln Gly Pro Asn Val Cys Ala Leu
35 40 45Gln Gln Ile Leu Gly Thr Lys Lys Lys Tyr Phe Ser Ser Cys Lys
Asn 50 55 60Trp Tyr Gln Gly Ala Ile Cys Gly Lys Lys Thr Thr Val Leu
Tyr Glu65 70 75 80Cys Cys Pro Gly Tyr Met Arg Met Glu Gly Met Lys
Gly Cys Pro Ala 85 90 95Val Met Pro Ile Asp His Val Tyr Gly Thr Leu
Gly Ile Val Gly Ala 100 105 110Thr Thr Thr Gln His Tyr Ser Asp Val
Ser Lys Leu Arg Glu Glu Ile 115 120 125Glu Gly Lys Gly Ser Tyr Thr
Tyr Phe Ala Pro Ser Asn Glu Ala Trp 130 135 140Asp Asn Leu Asp Ser
Asp Ile Arg Arg Gly Leu Glu Asn Asn Val Asn145 150 155 160Val Glu
Leu Leu Asn Ala Leu His Ser His Met Val Asn Lys Arg Met 165 170
175Leu Thr Lys Asp Leu Lys His Gly Met Val Ile Pro Ser Met Tyr Asn
180 185 190Asn Leu Gly Leu Phe Ile Asn His Tyr Pro Asn Gly Val Val
Thr Val 195 200 205Asn Cys Ala Arg Val Ile His Gly Asn Gln Ile Ala
Thr Asn Gly Val 210 215 220Val His Val Ile Asp Arg Val Leu Thr Gln
Ile Gly Thr Ser Ile Gln225 230 235 240Asp Phe Ile Glu Ala Glu Asp
Glu Leu Ser Ser Phe Arg Ala Ala Ala 245 250 255Ile Thr Ser Asp Leu
Leu Glu Ser Leu Gly Arg Asp Gly His Phe Thr 260 265 270Leu Phe Ala
Pro Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val 275 280 285Leu
Glu Arg Ile Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys 290 295
300Tyr His Ile Leu Asn Thr Leu Gln Cys Ser Glu Ala Ile Thr Gly
Gly305 310 315 320Ala Val Phe Glu Thr Met Glu Gly Asn Thr Ile Glu
Ile Gly Cys Glu 325 330 335Gly Asp Ser Ile Ser Ile Asn Gly Ile Lys
Met Val Asn Lys Lys Asp 340 345 350Ile Val Thr Lys Asn Gly Val Ile
His Leu Ile Asp Glu Val Leu Ile 355 360 365Pro Asp Ser Ala Lys Gln
Val Ile Glu Leu Ala Gly Lys Gln Gln Thr 370 375 380Thr Phe Thr Asp
Leu Val Ala Gln Leu Gly Leu Ala Ser Ser Leu Lys385 390 395 400Pro
Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser 405 410
415Asp Asp Thr Leu Ser Met Asp Gln Arg Leu Leu Lys Leu Ile Leu Gln
420 425 430Asn His Ile Leu Lys Val Lys Val Gly Leu Ser Asp Leu Tyr
Asn Gly 435 440 445Gln Ile Leu Glu Thr Ile Gly Gly Lys Gln Leu Arg
Val Phe Val Tyr 450 455 460Arg Thr Ala Ile Cys Ile Glu Asn Ser Cys
Met Val Arg Gly Ser Lys465 470 475 480Gln Gly Arg Asn Gly Ala Ile
His Ile Phe Arg Glu Ile Ile Gln Pro 485 490 495Ala Glu Lys Ser Leu
His Glu Lys Leu Arg Gln Asp Lys Arg Phe Ser 500 505 510Ile Phe Leu
Ser Leu Leu Glu Ala Ala Asp Leu Lys Asp Leu Leu Thr 515 520 525Gln
Pro Gly Asp Trp Thr Leu Phe Ala Pro Thr Asn Asp Ala Phe Lys 530 535
540Gly Met Thr Asn Glu Glu Arg Glu Ile Leu Ile Gly Asp Lys Asn
Ala545 550 555 560Leu Gln Asn Ile Ile Leu Tyr His Leu Thr Pro Gly
Val Tyr Ile Gly 565 570 575Lys Gly Phe Glu Pro Gly Val Thr Asn Ile
Leu Lys Thr Thr Gln Gly 580 585 590Ser Lys Ile Tyr Val Lys Gly Val
Asn Glu Thr Leu Leu Val Asn Glu 595 600 605Leu Lys Ser Lys Glu Ser
Asp Ile Met Thr Thr Asn Gly Val Ile His 610 615 620Val Val Asp Lys
Leu Leu Tyr Pro Ala Asp Ile Pro Val Gly Asn Asp625 630 635 640Gln
Leu Leu Glu Leu Leu Asn Lys Leu Ile Lys Tyr Ile Gln Ile Lys 645 650
655Phe Val Arg Gly Ser Thr Phe Lys Glu Ile Pro Met Thr Val Tyr Arg
660 665 670Pro Ala Met Thr Lys Ile His Ile Glu Gly Glu Pro Asp Phe
Arg Leu 675 680 685Ile Lys Glu Gly Glu Thr Val Thr Glu Val Ile His
Gly Glu Pro Val 690 695 700Ile Lys Lys Tyr Thr Lys Ile Ile Asp Gly
Val Pro Val Glu Ile Thr705 710 715 720Glu Lys Glu Thr Arg Glu Glu
Arg Ile Ile Thr Gly Pro Glu Ile Lys 725 730 735Tyr Thr Arg Ile Ser
Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu Gln 740 745 750Lys Phe Leu
Gln Lys Asp Thr Pro Ala Lys Lys Ile Gln Ala Asn Lys 755 760 765Arg
Val Gln Gly Ser Arg Arg Arg Ser Arg Glu Gly Arg Ser Gln 770 775
78082352DNARattus sp. 8atggttcctc tcctgccctt atctgctctg ctgctgctgt
tcctgtgtga cgttgacccc 60gcaaatgcca acagttacta tgacaaggtc ctagctcaca
gccgcatcag gggtcgggat 120cagggcccaa atgtctgtgc cctccagcag
attctgggca ccaaaaagaa atacttcagc 180tcctgtaaga actggtatca
aggtgctatc tgcgggaaga aaaccactgt gctatatgaa 240tgctgccccg
gctatatgag aatggaaggg atgaaaggct gcccagcagt gatgcccatt
300gaccatgttt atggcacgct gggcatcgtg ggagccacga ccactcaaca
ctattctgat 360gtctcgaagc tcagggaaga gattgaagga aaagggtcct
acacatactt cgcgccgagt 420aacgaagctt gggacaacct ggattccgac
atccgcagag gactagagaa caatgtcaat 480gttgagttac tgaacgcttt
acacagccac atggttaata agagaatgct aaccaaggac 540ctgaaacacg
gcatggttat tccttcaatg tacaacaatc tggggctttt tatcaatcat
600tatcccaatg gggttgtcac tgtgaactgt gctcgagtaa tccacgggaa
ccagattgcc 660acaaatggtg ttgtccatgt catcgaccgt gtcctgacac
aaattggcac ctccatccaa 720gacttcattg aagcagaaga tgagctttca
tcattcagag cggctgccat cacttctgac 780cttttggagt cccttggaag
agacggtcac ttcacactct ttgctcccac caatgaggct 840ttcgagaaac
tcccacgagg agtcctagaa aggatcatgg gagacaaagt ggcttctgaa
900gctctcatga agtaccacat cctgaatacc ctccagtgct ctgaggctat
cacaggagga 960gcggtgtttg agaccatgga aggaaacact attgaaatag
ggtgtgaggg agacagcatc 1020tccattaacg gaatcaagat ggtgaacaag
aaagacattg tgacgaagaa tggtgtcatc 1080cacctgattg atgaagtcct
cattcctgat tctgctaaac aagttattga gctggctgga 1140aaacagcaaa
ccactttcac ggacctggta gcccagttag ggttggcgtc ttctctgaag
1200ccggatggag agtacacgct gttagcgcct gtgaacaatg cgttctctga
tgacactctg 1260agcatggacc agcgccttct taagctaatt ctgcaaaatc
acatattgaa agtaaaagtc 1320ggccttagtg atctctacaa tggacagatt
ctggagacca ttggaggcaa acaactccgt 1380gtcttcgtgt atcggacggc
tatctgcata gaaaactcat gcatggtgag aggaagcaag 1440caggggagga
acggtgccat tcacatattc cgagagatca tccaaccggc ggagaagtcc
1500ctgcacgaaa aactgcgcca agataagcgc ttcagcatct tcctcagcct
cctcgaagct 1560gcagatctga aagatcttct gacacagccc ggagattgga
ccttgtttgc accaaccaat 1620gatgccttca agggaatgac taatgaagaa
agggagattc tgattgggga taaaaatgct 1680ctccaaaaca tcattcttta
ccacctgacc ccaggggttt atattggaaa gggatttgaa 1740cccggagtca
ccaacatcct gaagaccaca cagggaagca aaatctatgt gaaaggagtc
1800aatgagacgc ttttggtgaa tgagttgaag tccaaagaat ctgacatcat
gacaacaaac 1860ggcgtcattc acgttgtgga caaactcctc tatccagcag
acattccggt tggaaatgat 1920cagctcttgg aattactgaa caaactgata
aaatacatcc aaattaagtt cgttcgtggc 1980agcaccttca aagaaatccc
catgactgtc tatagacctg caatgacgaa gatccacatt 2040gaaggcgagc
ctgacttcag gctgattaaa gaaggtgaaa cagtgacaga agtgatccac
2100ggagaaccag tcattaaaaa gtacaccaaa atcatagacg gggttcctgt
tgaaataact 2160gaaaaagaga cccgggaaga acgcatcatc acaggtcctg
agataaaata cactaggatt 2220tccacaggag gtggggaaac agaagagacc
ctgcagaaat tcttgcaaaa agacacacct 2280gcaaagaaga tacaagccaa
caaaagggtt caagggtcta gaaggcgatc aagagaaggc 2340cgttctcagt ga
23529811PRTMus musculus 9Met Val Pro Leu Leu Pro Leu Tyr Ala Leu
Leu Leu Leu Phe Leu Cys1 5 10 15Asp Ile Asn Pro Ala Asn Ala Asn Ser
Tyr Tyr Asp Lys Val Leu Ala 20 25 30His Ser Arg Ile Arg Gly Arg Asp
Gln Gly Pro Asn Val Cys Ala Leu 35 40 45Gln Gln Ile Leu Gly Thr Lys
Lys Lys Tyr Phe Ser Ser Cys Lys Asn 50 55 60Trp Tyr Gln Gly Ala Ile
Cys Gly Lys Lys Thr Thr Val Leu Tyr Glu65 70 75 80Cys Cys Pro Gly
Tyr Met Arg Met Glu Gly Met Lys Gly Cys Pro Ala 85 90 95Val Met Pro
Ile Asp His Val Tyr Gly Thr Leu Gly Ile Val Gly Ala 100 105 110Thr
Thr Thr Gln His Tyr Ser Asp Val Ser Lys Leu Arg Glu Glu Ile 115 120
125Glu Gly Lys Gly Ser Tyr Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp
130 135 140Glu Asn Leu Asp Ser Asp Ile Arg Arg Gly Leu Glu Asn Asn
Val Asn145 150 155 160Val Glu Leu Leu Asn Ala Leu His Ser His Met
Val Asn Lys Arg Met 165 170 175Leu Thr Lys Asp Leu Lys His Gly Met
Val Ile Pro Ser Met Tyr Asn 180 185 190Asn Leu Gly Leu Phe Ile Asn
His Tyr Pro Asn Gly Val Val Thr Val 195 200 205Asn Cys Ala Arg Val
Ile His Gly Asn Gln Ile Ala Thr Asn Gly Val 210 215 220Val His Val
Ile Asp Arg Val Leu Thr Gln Ile Gly Thr Ser Ile Gln225 230 235
240Asp Phe Leu Glu Ala Glu Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala
245 250 255Ile Thr Ser Asp Leu Leu Glu Ser Leu Gly Arg Asp Gly His
Phe Thr 260 265 270Leu Phe Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu
Pro Arg Gly Val 275 280 285Leu Glu Arg Ile Met Gly Asp Lys Val Ala
Ser Glu Ala Leu Met Lys 290 295 300Tyr His Ile Leu Asn Thr Leu Gln
Cys Ser Glu Ala Ile Thr Gly Gly305 310 315 320Ala Val Phe Glu Thr
Met Glu Gly Asn Thr Ile Glu Ile Gly Cys Glu 325 330 335Gly Asp Ser
Ile Ser Ile Asn Gly Ile Lys Met Val Asn Lys Lys Asp 340 345 350Ile
Val Thr Lys Asn Gly Val Ile His Leu Ile Asp Glu Val Leu Ile 355 360
365Pro Asp Ser Ala Lys Gln Val Ile Glu Leu Ala Gly Lys Gln Gln Thr
370 375 380Thr Phe Thr Asp Leu Val Ala Gln Leu Gly Leu Ala Ser Ser
Leu Lys385 390 395 400Pro Asp Gly Glu Tyr Thr Leu Leu Ala Pro Val
Asn Asn Ala Phe Ser 405 410 415Asp Asp Thr Leu Ser Met Asp Gln Arg
Leu Leu Lys Leu Ile Leu Gln 420 425 430Asn His Ile Leu Lys Val Lys
Val Gly Leu Ser Asp Leu Tyr Asn Gly 435 440 445Gln Ile Leu Glu Thr
Ile Gly Gly Lys Gln Leu Arg Val Phe Val Tyr 450 455 460Arg Thr Ala
Ile Cys Ile Glu Asn Ser Cys Met Val Arg Gly Ser Lys465 470 475
480Gln Gly Arg Asn Gly Ala Ile His Ile Phe Arg Glu Ile Ile Gln Pro
485 490 495Ala Glu Lys Ser Leu His Asp Lys Leu Arg Gln Asp Lys Arg
Phe Ser 500 505 510Ile Phe Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys
Asp Leu Leu Thr 515 520 525Gln Pro Gly Asp Trp Thr Leu Phe Ala Pro
Thr Asn Asp Ala Phe Lys 530 535 540Gly Met Thr Ser Glu Glu Arg Glu
Leu Leu Ile Gly Asp Lys Asn Ala545 550 555 560Leu Gln Asn Ile Ile
Leu Tyr His Leu Thr Pro Gly Val Tyr Ile Gly 565 570 575Lys Gly Phe
Glu Pro Gly Val Thr Asn Ile Leu Lys Thr Thr Gln Gly 580 585 590Ser
Lys Ile Tyr Leu Lys Gly Val Asn Glu Thr Leu Leu Val Asn Glu 595 600
605Leu Lys Ser Lys Glu Ser Asp Ile Met Thr Thr Asn Gly Val Ile His
610 615 620Val Val Asp Lys Leu Leu Tyr Pro Ala Asp Ile Pro Val Gly
Asn Asp625 630 635 640Gln Leu Leu Glu Leu Leu Asn Lys Leu Ile Lys
Tyr Ile Gln Ile Lys 645 650 655Phe Val Arg Gly Ser Thr Phe Lys Glu
Ile Pro Met Thr Val Tyr Arg 660 665 670Pro Ala Met Thr Lys Ile Gln
Ile Glu Gly Asp Pro Asp Phe Arg Leu 675 680 685Ile Lys Glu Gly Glu
Thr Val Thr Glu Val Ile His Gly Glu Pro Val 690 695 700Ile Lys Lys
Tyr Thr Lys Ile Ile Asp Gly Val Pro Val Glu Ile Thr705 710 715
720Glu Lys Gln Thr Arg Glu Glu Arg Ile Ile Thr Gly Pro Glu Ile Lys
725 730 735Tyr Thr Arg Ile Ser Thr Gly Gly Gly Glu Thr Gly Glu Thr
Leu Gln 740 745 750Lys Phe Leu Gln Lys Glu Val Ser Lys Val Thr Lys
Phe Ile Glu Gly 755 760 765Gly Asp Gly His Leu Phe Glu Asp Glu Glu
Ile Lys Arg Leu Leu Gln 770 775 780Gly Asp Thr Pro Ala Lys Lys Ile
Pro Ala Asn Lys Arg Val Gln Gly785 790 795 800Pro Arg Arg Arg Ser
Arg Glu Gly Arg Ser Gln 805 810102436DNAMus musculus 10atggttcctc
tcctgccctt atatgctctg ctgctgctgt tcctgtgtga tattaaccct 60gcaaatgcca
acagttacta tgacaaggtc ctggctcaca gccgcatcag gggtcgggat
120cagggcccaa acgtctgtgc cctccagcaa attctgggca ccaaaaagaa
atacttcagc 180tcctgtaaga actggtatca aggtgctatc tgcgggaaga
aaaccactgt gctatatgaa 240tgctgccctg gctatatgag aatggaaggg
atgaaaggct gccccgcagt gatgcctatt 300gaccatgttt atggcacgct
gggcattgtg ggagccacta ccactcagca ctactccgat 360gtctcgaagc
tgagagaaga gattgaagga aaagggtcat acacgtactt cgcgccgagt
420aacgaggctt gggagaacct ggattctgac attcgcagag gactggagaa
caatgtcaat 480gttgagctac tgaatgcctt acacagccac atggttaata
agagaatgtt aaccaaggac 540ctgaaacacg gcatggttat tccttcaatg
tacaacaatc tggggctttt tattaaccat 600tatcccaatg gggttgtcac
tgtgaactgt gctcgagtca tccatgggaa ccagattgcc 660acaaatggtg
tcgtccatgt cattgaccgt gtcctgacac aaattggtac ctccatccaa
720gacttccttg aagcagaaga cgacctttca tcatttagag cagccgccat
cacctctgac 780ctcttggagt cccttggaag agatggtcac ttcacgctct
ttgctcccac caatgaagct 840ttcgagaaac tgccacgagg tgtcctagaa
aggatcatgg gagacaaagt ggcttctgaa 900gctctcatga agtaccacat
cctaaatacc ctccagtgct ctgaggccat cactggagga 960gccgtgtttg
agaccatgga aggaaacact attgagatag ggtgcgaagg ggacagtatc
1020tccattaacg gaatcaagat ggtgaacaag aaagacattg tgactaagaa
tggtgtcatc 1080cacctgattg atgaagtcct cattcctgat tctgccaaac
aagttattga gctggctgga 1140aaacagcaaa ccactttcac cgacctggta
gcccaattag gcttggcatc ctctctgaag 1200ccagatggag agtacacctt
attagcacct gtgaacaatg cgttctctga tgacactctg 1260agcatggacc
aacgccttct taagctaatt ctgcaaaatc acatattgaa agtaaaagtt
1320ggccttagcg acctctacaa tggacagata ctggaaacca ttggaggcaa
acaactccga 1380gtctttgtgt atcggacggc tatctgcata gaaaactcat
gcatggtgag aggaagcaag 1440cagggaagga atggtgccat tcacatattc
cgagaaatca tccaaccagc agagaaatcc 1500ctgcacgaca agctgcggca
agacaagcgc tttagcatct tcctcagcct ccttgaagct 1560gcagatttga
aagatctcct gacacagccc ggagattgga ccttgtttgc accaaccaat
1620gatgccttca agggaatgac tagcgaagaa agggagcttc tgattgggga
taaaaatgct 1680ctccaaaaca tcattcttta tcacctgacc ccaggggttt
atattggaaa gggattcgaa 1740cccggagtca ctaatatcct gaagaccaca
cagggaagca aaatctatct gaaaggagta 1800aacgaaacgc ttctagtgaa
tgagttgaag tccaaagaat ctgacatcat gacgacaaat 1860ggtgtcatcc
acgtcgtgga caaactcctc tatccagcag atattccagt tggaaatgat
1920cagctcttgg aattactgaa caaactgata aaatacatcc aaatcaagtt
tgttcgtggc 1980agcaccttca aagaaatccc catgactgtc tatagacctg
caatgacgaa gatccaaatt 2040gaaggtgatc ccgacttcag gctgattaaa
gaaggcgaaa cggtgacaga agtgatccac 2100ggagagccag tcattaaaaa
gtacaccaaa atcatagatg gagttcctgt tgaaataact 2160gaaaaacaga
ctcgggaaga acgaatcatt acaggtcctg agataaaata taccaggatt
2220tccacaggag gtggagaaac aggagagacc ttgcagaaat tcttgcaaaa
agaggtctcc 2280aaggtcacaa agttcattga aggtggcgat ggtcacttat
ttgaagatga ggagattaaa 2340agactgcttc agggagacac acctgcaaag
aagataccag ccaacaaaag ggttcaaggg 2400cctagaagac gatcaagaga
aggccgttct cagtga 243611783PRTMus musculus 11Met Val Pro Leu Leu
Pro
Leu Tyr Ala Leu Leu Leu Leu Phe Leu Cys1 5 10 15Asp Ile Asn Pro Ala
Asn Ala Asn Ser Tyr Tyr Asp Lys Val Leu Ala 20 25 30His Ser Arg Ile
Arg Gly Arg Asp Gln Gly Pro Asn Val Cys Ala Leu 35 40 45Gln Gln Ile
Leu Gly Thr Lys Lys Lys Tyr Phe Ser Ser Cys Lys Asn 50 55 60Trp Tyr
Gln Gly Ala Ile Cys Gly Lys Lys Thr Thr Val Leu Tyr Glu65 70 75
80Cys Cys Pro Gly Tyr Met Arg Met Glu Gly Met Lys Gly Cys Pro Ala
85 90 95Val Met Pro Ile Asp His Val Tyr Gly Thr Leu Gly Ile Val Gly
Ala 100 105 110Thr Thr Thr Gln His Tyr Ser Asp Val Ser Lys Leu Arg
Glu Glu Ile 115 120 125Glu Gly Lys Gly Ser Tyr Thr Tyr Phe Ala Pro
Ser Asn Glu Ala Trp 130 135 140Glu Asn Leu Asp Ser Asp Ile Arg Arg
Gly Leu Glu Asn Asn Val Asn145 150 155 160Val Glu Leu Leu Asn Ala
Leu His Ser His Met Val Asn Lys Arg Met 165 170 175Leu Thr Lys Asp
Leu Lys His Gly Met Val Ile Pro Ser Met Tyr Asn 180 185 190Asn Leu
Gly Leu Phe Ile Asn His Tyr Pro Asn Gly Val Val Thr Val 195 200
205Asn Cys Ala Arg Val Ile His Gly Asn Gln Ile Ala Thr Asn Gly Val
210 215 220Val His Val Ile Asp Arg Val Leu Thr Gln Ile Gly Thr Ser
Ile Gln225 230 235 240Asp Phe Leu Glu Ala Glu Asp Asp Leu Ser Ser
Phe Arg Ala Ala Ala 245 250 255Ile Thr Ser Asp Leu Leu Glu Ser Leu
Gly Arg Asp Gly His Phe Thr 260 265 270Leu Phe Ala Pro Thr Asn Glu
Ala Phe Glu Lys Leu Pro Arg Gly Val 275 280 285Leu Glu Arg Ile Met
Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys 290 295 300Tyr His Ile
Leu Asn Thr Leu Gln Cys Ser Glu Ala Ile Thr Gly Gly305 310 315
320Ala Val Phe Glu Thr Met Glu Gly Asn Thr Ile Glu Ile Gly Cys Glu
325 330 335Gly Asp Ser Ile Ser Ile Asn Gly Ile Lys Met Val Asn Lys
Lys Asp 340 345 350Ile Val Thr Lys Asn Gly Val Ile His Leu Ile Asp
Glu Val Leu Ile 355 360 365Pro Asp Ser Ala Lys Gln Val Ile Glu Leu
Ala Gly Lys Gln Gln Thr 370 375 380Thr Phe Thr Asp Leu Val Ala Gln
Leu Gly Leu Ala Ser Ser Leu Lys385 390 395 400Pro Asp Gly Glu Tyr
Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser 405 410 415Asp Asp Thr
Leu Ser Met Asp Gln Arg Leu Leu Lys Leu Ile Leu Gln 420 425 430Asn
His Ile Leu Lys Val Lys Val Gly Leu Ser Asp Leu Tyr Asn Gly 435 440
445Gln Ile Leu Glu Thr Ile Gly Gly Lys Gln Leu Arg Val Phe Val Tyr
450 455 460Arg Thr Ala Ile Cys Ile Glu Asn Ser Cys Met Val Arg Gly
Ser Lys465 470 475 480Gln Gly Arg Asn Gly Ala Ile His Ile Phe Arg
Glu Ile Ile Gln Pro 485 490 495Ala Glu Lys Ser Leu His Asp Lys Leu
Arg Gln Asp Lys Arg Phe Ser 500 505 510Ile Phe Leu Ser Leu Leu Glu
Ala Ala Asp Leu Lys Asp Leu Leu Thr 515 520 525Gln Pro Gly Asp Trp
Thr Leu Phe Ala Pro Thr Asn Asp Ala Phe Lys 530 535 540Gly Met Thr
Ser Glu Glu Arg Glu Leu Leu Ile Gly Asp Lys Asn Ala545 550 555
560Leu Gln Asn Ile Ile Leu Tyr His Leu Thr Pro Gly Val Tyr Ile Gly
565 570 575Lys Gly Phe Glu Pro Gly Val Thr Asn Ile Leu Lys Thr Thr
Gln Gly 580 585 590Ser Lys Ile Tyr Leu Lys Gly Val Asn Glu Thr Leu
Leu Val Asn Glu 595 600 605Leu Lys Ser Lys Glu Ser Asp Ile Met Thr
Thr Asn Gly Val Ile His 610 615 620Val Val Asp Lys Leu Leu Tyr Pro
Ala Asp Ile Pro Val Gly Asn Asp625 630 635 640Gln Leu Leu Glu Leu
Leu Asn Lys Leu Ile Lys Tyr Ile Gln Ile Lys 645 650 655Phe Val Arg
Gly Ser Thr Phe Lys Glu Ile Pro Met Thr Val Tyr Arg 660 665 670Pro
Ala Met Thr Lys Ile Gln Ile Glu Gly Asp Pro Asp Phe Arg Leu 675 680
685Ile Lys Glu Gly Glu Thr Val Thr Glu Val Ile His Gly Glu Pro Val
690 695 700Ile Lys Lys Tyr Thr Lys Ile Ile Asp Gly Val Pro Val Glu
Ile Thr705 710 715 720Glu Lys Gln Thr Arg Glu Glu Arg Ile Ile Thr
Gly Pro Glu Ile Lys 725 730 735Tyr Thr Arg Ile Ser Thr Gly Gly Gly
Glu Thr Gly Glu Thr Leu Gln 740 745 750Lys Phe Leu Gln Lys Asp Thr
Pro Ala Lys Lys Ile Pro Ala Asn Lys 755 760 765Arg Val Gln Gly Pro
Arg Arg Arg Ser Arg Glu Gly Arg Ser Gln 770 775 780122352DNAMus
musculus 12atggttcctc tcctgccctt atatgctctg ctgctgctgt tcctgtgtga
tattaaccct 60gcaaatgcca acagttacta tgacaaggtc ctggctcaca gccgcatcag
gggtcgggat 120cagggcccaa acgtctgtgc cctccagcaa attctgggca
ccaaaaagaa atacttcagc 180tcctgtaaga actggtatca aggtgctatc
tgcgggaaga aaaccactgt gctatatgaa 240tgctgccctg gctatatgag
aatggaaggg atgaaaggct gccccgcagt gatgcctatt 300gaccatgttt
atggcacgct gggcattgtg ggagccacta ccactcagca ctactccgat
360gtctcgaagc tgagagaaga gattgaagga aaagggtcat acacgtactt
cgcgccgagt 420aacgaggctt gggagaacct ggattctgac attcgcagag
gactggagaa caatgtcaat 480gttgagctac tgaatgcctt acacagccac
atggttaata agagaatgtt aaccaaggac 540ctgaaacacg gcatggttat
tccttcaatg tacaacaatc tggggctttt tattaaccat 600tatcccaatg
gggttgtcac tgtgaactgt gctcgagtca tccatgggaa ccagattgcc
660acaaatggtg tcgtccatgt cattgaccgt gtcctgacac aaattggtac
ctccatccaa 720gacttccttg aagcagaaga cgacctttca tcatttagag
cagccgccat cacctctgac 780ctcttggagt cccttggaag agatggtcac
ttcacgctct ttgctcccac caatgaagct 840ttcgagaaac tgccacgagg
tgtcctagaa aggatcatgg gagacaaagt ggcttctgaa 900gctctcatga
agtaccacat cctaaatacc ctccagtgct ctgaggccat cactggagga
960gccgtgtttg agaccatgga aggaaacact attgagatag ggtgcgaagg
ggacagtatc 1020tccattaacg gaatcaagat ggtgaacaag aaagacattg
tgactaagaa tggtgtcatc 1080cacctgattg atgaagtcct cattcctgat
tctgccaaac aagttattga gctggctgga 1140aaacagcaaa ccactttcac
cgacctggta gcccaattag gcttggcatc ctctctgaag 1200ccagatggag
agtacacctt attagcacct gtgaacaatg cgttctctga tgacactctg
1260agcatggacc aacgccttct taagctaatt ctgcaaaatc acatattgaa
agtaaaagtt 1320ggccttagcg acctctacaa tggacagata ctggaaacca
ttggaggcaa acaactccga 1380gtctttgtgt atcggacggc tatctgcata
gaaaactcat gcatggtgag aggaagcaag 1440cagggaagga atggtgccat
tcacatattc cgagaaatca tccaaccagc agagaaatcc 1500ctgcacgaca
agctgcggca agacaagcgc tttagcatct tcctcagcct ccttgaagct
1560gcagatttga aagatctcct gacacagccc ggagattgga ccttgtttgc
accaaccaat 1620gatgccttca agggaatgac tagcgaagaa agggagcttc
tgattgggga taaaaatgct 1680ctccaaaaca tcattcttta tcacctgacc
ccaggggttt atattggaaa gggattcgaa 1740cccggagtca ctaatatcct
gaagaccaca cagggaagca aaatctatct gaaaggagta 1800aacgaaacgc
ttctagtgaa tgagttgaag tccaaagaat ctgacatcat gacgacaaat
1860ggtgtcatcc acgtcgtgga caaactcctc tatccagcag atattccagt
tggaaatgat 1920cagctcttgg aattactgaa caaactgata aaatacatcc
aaatcaagtt tgttcgtggc 1980agcaccttca aagaaatccc catgactgtc
tatagacctg caatgacgaa gatccaaatt 2040gaaggtgatc ccgacttcag
gctgattaaa gaaggcgaaa cggtgacaga agtgatccac 2100ggagagccag
tcattaaaaa gtacaccaaa atcatagatg gagttcctgt tgaaataact
2160gaaaaacaga ctcgggaaga acgaatcatt acaggtcctg agataaaata
taccaggatt 2220tccacaggag gtggagaaac aggagagacc ttgcagaaat
tcttgcaaaa agacacacct 2280gcaaagaaga taccagccaa caaaagggtt
caagggccta gaagacgatc aagagaaggc 2340cgttctcagt ga 235213809PRTHomo
sapiens 13Met Ile Pro Phe Leu Pro Met Phe Ser Leu Leu Leu Leu Leu
Ile Val1 5 10 15Asn Pro Ile Asn Ala Asn Asn His Tyr Asp Lys Ile Leu
Ala His Ser 20 25 30Arg Ile Arg Gly Arg Asp Gln Gly Pro Asn Val Cys
Ala Leu Gln Gln 35 40 45Ile Leu Gly Thr Lys Lys Lys Tyr Phe Ser Thr
Cys Lys Asn Trp Tyr 50 55 60Lys Lys Ser Ile Cys Gly Gln Lys Thr Thr
Val Leu Tyr Glu Cys Cys65 70 75 80Pro Gly Tyr Met Arg Met Glu Gly
Met Lys Gly Cys Pro Ala Val Leu 85 90 95Pro Ile Asp His Val Tyr Gly
Thr Leu Gly Ile Val Gly Ala Thr Thr 100 105 110Thr Gln Arg Tyr Ser
Asp Ala Ser Lys Leu Arg Glu Glu Ile Glu Gly 115 120 125Lys Gly Ser
Phe Thr Tyr Phe Ala Pro Ser Asn Glu Ala Trp Asp Asn 130 135 140Leu
Asp Ser Asp Ile Arg Arg Gly Leu Glu Ser Asn Val Asn Val Glu145 150
155 160Leu Leu Asn Ala Leu His Ser His Met Ile Asn Lys Arg Met Leu
Thr 165 170 175Lys Asp Leu Lys Asn Gly Met Ile Ile Pro Ser Met Tyr
Asn Asn Leu 180 185 190Gly Leu Phe Ile Asn His Tyr Pro Asn Gly Val
Val Thr Val Asn Cys 195 200 205Ala Arg Ile Ile His Gly Asn Gln Ile
Ala Thr Asn Gly Val Val His 210 215 220Val Ile Asp Arg Val Leu Thr
Gln Ile Gly Thr Ser Ile Gln Asp Phe225 230 235 240Ile Glu Ala Glu
Asp Asp Leu Ser Ser Phe Arg Ala Ala Ala Ile Thr 245 250 255Ser Asp
Ile Leu Glu Ala Leu Gly Arg Asp Gly His Phe Thr Leu Phe 260 265
270Ala Pro Thr Asn Glu Ala Phe Glu Lys Leu Pro Arg Gly Val Leu Glu
275 280 285Arg Ile Met Gly Asp Lys Val Ala Ser Glu Ala Leu Met Lys
Tyr His 290 295 300Ile Leu Asn Thr Leu Gln Cys Ser Glu Ser Ile Met
Gly Gly Ala Val305 310 315 320Phe Glu Thr Leu Glu Gly Asn Thr Ile
Glu Ile Gly Cys Asp Gly Asp 325 330 335Ser Ile Thr Val Asn Gly Ile
Lys Met Val Asn Lys Lys Asp Ile Val 340 345 350Thr Asn Asn Gly Val
Ile His Leu Ile Asp Gln Val Leu Ile Pro Asp 355 360 365Ser Ala Lys
Gln Val Ile Glu Leu Ala Gly Lys Gln Gln Thr Thr Phe 370 375 380Thr
Asp Leu Val Ala Gln Leu Gly Leu Ala Ser Ala Leu Arg Pro Asp385 390
395 400Gly Glu Tyr Thr Leu Leu Ala Pro Val Asn Asn Ala Phe Ser Asp
Asp 405 410 415Thr Leu Ser Met Asp Gln Arg Leu Leu Lys Leu Ile Leu
Gln Asn His 420 425 430Ile Leu Lys Val Lys Val Gly Leu Asn Glu Leu
Tyr Asn Gly Gln Ile 435 440 445Leu Glu Thr Ile Gly Gly Lys Gln Leu
Arg Val Phe Val Tyr Arg Thr 450 455 460Ala Val Cys Ile Glu Asn Ser
Cys Met Glu Lys Gly Ser Lys Gln Gly465 470 475 480Arg Asn Gly Ala
Ile His Ile Phe Arg Glu Ile Ile Lys Pro Ala Glu 485 490 495Lys Ser
Leu His Glu Lys Leu Lys Gln Asp Lys Arg Phe Ser Thr Phe 500 505
510Leu Ser Leu Leu Glu Ala Ala Asp Leu Lys Glu Leu Leu Thr Gln Pro
515 520 525Gly Asp Trp Thr Leu Phe Val Pro Thr Asn Asp Ala Phe Lys
Gly Met 530 535 540Thr Ser Glu Glu Lys Glu Ile Leu Ile Arg Asp Lys
Asn Ala Leu Gln545 550 555 560Asn Ile Ile Leu Tyr His Leu Thr Pro
Gly Val Phe Ile Gly Lys Gly 565 570 575Phe Glu Pro Gly Val Thr Asn
Ile Leu Lys Thr Thr Gln Gly Ser Lys 580 585 590Ile Phe Leu Lys Glu
Val Asn Asp Thr Leu Leu Val Asn Glu Leu Lys 595 600 605Ser Lys Glu
Ser Asp Ile Met Thr Thr Asn Gly Val Ile His Val Val 610 615 620Asp
Lys Leu Leu Tyr Pro Ala Asp Thr Pro Val Gly Asn Asp Gln Leu625 630
635 640Leu Glu Ile Leu Asn Lys Leu Ile Lys Tyr Ile Gln Ile Lys Phe
Val 645 650 655Arg Gly Ser Thr Phe Lys Glu Ile Pro Val Thr Val Tyr
Arg Pro Thr 660 665 670Leu Thr Lys Val Lys Ile Glu Gly Glu Pro Glu
Phe Arg Leu Ile Lys 675 680 685Glu Gly Glu Thr Ile Thr Glu Val Ile
His Gly Glu Pro Ile Ile Lys 690 695 700Lys Tyr Thr Lys Ile Ile Asp
Gly Val Pro Val Glu Ile Thr Glu Lys705 710 715 720Glu Thr Arg Glu
Glu Arg Ile Ile Thr Gly Pro Glu Ile Lys Tyr Thr 725 730 735Arg Ile
Ser Thr Gly Gly Gly Glu Thr Glu Glu Thr Leu Lys Lys Leu 740 745
750Leu Gln Glu Glu Val Thr Lys Val Thr Lys Phe Ile Glu Gly Gly Asp
755 760 765Gly His Leu Phe Glu Asp Glu Glu Ile Lys Arg Leu Leu Gln
Gly Asp 770 775 780Thr Pro Val Arg Lys Leu Gln Ala Asn Lys Lys Val
Gln Gly Ser Arg785 790 795 800Arg Arg Leu Arg Glu Gly Arg Ser Gln
805142430DNAHomo sapiens 14atgattccct ttttacccat gttttctcta
ctattgctgc ttattgttaa ccctataaac 60gccaacaatc attatgacaa gatcttggct
catagtcgta tcaggggtcg ggaccaaggc 120ccaaatgtct gtgcccttca
acagattttg ggcaccaaaa agaaatactt cagcacttgt 180aagaactggt
ataaaaagtc catctgtgga cagaaaacga ctgtgttata tgaatgttgc
240cctggttata tgagaatgga aggaatgaaa ggctgcccag cagttttgcc
cattgaccat 300gtttatggca ctctgggcat cgtgggagcc accacaacgc
agcgctattc tgacgcctca 360aaactgaggg aggagatcga gggaaaggga
tccttcactt actttgcacc gagtaatgag 420gcttgggaca acttggattc
tgatatccgt agaggtttgg agagcaacgt gaatgttgaa 480ttactgaatg
ctttacatag tcacatgatt aataagagaa tgttgaccaa ggacttaaaa
540aatggcatga ttattccttc aatgtataac aatttggggc ttttcattaa
ccattatcct 600aatggggttg tcactgttaa ttgtgctcga atcatccatg
ggaaccagat tgcaacaaat 660ggtgttgtcc atgtcattga ccgtgtgctt
acacaaattg gtacctcaat tcaagacttc 720attgaagcag aagatgacct
ttcatctttt agagcagctg ccatcacatc ggacatattg 780gaggcccttg
gaagagacgg tcacttcaca ctctttgctc ccaccaatga ggcttttgag
840aaacttccac gaggtgtcct agaaaggatc atgggagaca aagtggcttc
cgaagctctt 900atgaagtacc acatcttaaa tactctccag tgttctgagt
ctattatggg aggagcagtc 960tttgagacgc tggaaggaaa tacaattgag
ataggatgtg acggtgacag tataacagta 1020aatggaatca aaatggtgaa
caaaaaggat attgtgacaa ataatggtgt gatccatttg 1080attgatcagg
tcctaattcc tgattctgcc aaacaagtta ttgagctggc tggaaaacag
1140caaaccacct tcacggatct tgtggcccaa ttaggcttgg catctgctct
gaggccagat 1200ggagaataca ctttgctggc acctgtgaat aatgcatttt
ctgatgatac tctcagcatg 1260gatcagcgcc tccttaaatt aattctgcag
aatcacatat tgaaagtaaa agttggcctt 1320aatgagcttt acaacgggca
aatactggaa accatcggag gcaaacagct cagagtcttc 1380gtatatcgta
cagctgtctg cattgaaaat tcatgcatgg agaaagggag taagcaaggg
1440agaaacggtg cgattcacat attccgcgag atcatcaagc cagcagagaa
atccctccat 1500gaaaagttaa aacaagataa gcgctttagc accttcctca
gcctacttga agctgcagac 1560ttgaaagagc tcctgacaca acctggagac
tggacattat ttgtgccaac caatgatgct 1620tttaagggaa tgactagtga
agaaaaagaa attctgatac gggacaaaaa tgctcttcaa 1680aacatcattc
tttatcacct gacaccagga gttttcattg gaaaaggatt tgaacctggt
1740gttactaaca ttttaaagac cacacaagga agcaaaatct ttctgaaaga
agtaaatgat 1800acacttctgg tgaatgaatt gaaatcaaaa gaatctgaca
tcatgacaac aaatggtgta 1860attcatgttg tagataaact cctctatcca
gcagacacac ctgttggaaa tgatcaactg 1920ctggaaatac ttaataaatt
aatcaaatac atccaaatta agtttgttcg tggtagcacc 1980ttcaaagaaa
tccccgtgac tgtctataga cccacactaa caaaagtcaa aattgaaggt
2040gaacctgaat tcagactgat taaagaaggt gaaacaataa ctgaagtgat
ccatggagag 2100ccaattatta aaaaatacac caaaatcatt gatggagtgc
ctgtggaaat aactgaaaaa 2160gagacacgag aagaacgaat cattacaggt
cctgaaataa aatacactag gatttctact 2220ggaggtggag aaacagaaga
aactctgaag aaattgttac aagaagaggt caccaaggtc 2280accaaattca
ttgaaggtgg tgatggtcat ttatttgaag atgaagaaat taaaagactg
2340cttcagggag acacacccgt gaggaagttg caagccaaca aaaaagttca
aggatctaga 2400agacgattaa gggaaggtcg ttctcagtga 243015781PRTHomo
sapiens 15Met Ile Pro Phe Leu Pro Met Phe Ser Leu Leu Leu Leu Leu
Ile Val1 5 10 15Asn Pro Ile Asn Ala Asn Asn His Tyr Asp Lys Ile Leu
Ala His Ser 20 25 30Arg Ile Arg Gly Arg Asp Gln Gly Pro Asn Val Cys
Ala Leu Gln Gln 35 40 45Ile Leu Gly Thr Lys Lys Lys Tyr Phe Ser Thr
Cys Lys Asn Trp Tyr 50 55 60Lys Lys Ser Ile Cys Gly Gln Lys Thr Thr
Val Leu Tyr Glu Cys Cys65 70
75 80Pro Gly Tyr Met Arg Met Glu Gly Met Lys Gly Cys Pro Ala Val
Leu 85 90 95Pro Ile Asp His Val Tyr Gly Thr Leu Gly Ile Val Gly Ala
Thr Thr 100 105 110Thr Gln Arg Tyr Ser Asp Ala Ser Lys Leu Arg Glu
Glu Ile Glu Gly 115 120 125Lys Gly Ser Phe Thr Tyr Phe Ala Pro Ser
Asn Glu Ala Trp Asp Asn 130 135 140Leu Asp Ser Asp Ile Arg Arg Gly
Leu Glu Ser Asn Val Asn Val Glu145 150 155 160Leu Leu Asn Ala Leu
His Ser His Met Ile Asn Lys Arg Met Leu Thr 165 170 175Lys Asp Leu
Lys Asn Gly Met Ile Ile Pro Ser Met Tyr Asn Asn Leu 180 185 190Gly
Leu Phe Ile Asn His Tyr Pro Asn Gly Val Val Thr Val Asn Cys 195 200
205Ala Arg Ile Ile His Gly Asn Gln Ile Ala Thr Asn Gly Val Val His
210 215 220Val Ile Asp Arg Val Leu Thr Gln Ile Gly Thr Ser Ile Gln
Asp Phe225 230 235 240Ile Glu Ala Glu Asp Asp Leu Ser Ser Phe Arg
Ala Ala Ala Ile Thr 245 250 255Ser Asp Ile Leu Glu Ala Leu Gly Arg
Asp Gly His Phe Thr Leu Phe 260 265 270Ala Pro Thr Asn Glu Ala Phe
Glu Lys Leu Pro Arg Gly Val Leu Glu 275 280 285Arg Ile Met Gly Asp
Lys Val Ala Ser Glu Ala Leu Met Lys Tyr His 290 295 300Ile Leu Asn
Thr Leu Gln Cys Ser Glu Ser Ile Met Gly Gly Ala Val305 310 315
320Phe Glu Thr Leu Glu Gly Asn Thr Ile Glu Ile Gly Cys Asp Gly Asp
325 330 335Ser Ile Thr Val Asn Gly Ile Lys Met Val Asn Lys Lys Asp
Ile Val 340 345 350Thr Asn Asn Gly Val Ile His Leu Ile Asp Gln Val
Leu Ile Pro Asp 355 360 365Ser Ala Lys Gln Val Ile Glu Leu Ala Gly
Lys Gln Gln Thr Thr Phe 370 375 380Thr Asp Leu Val Ala Gln Leu Gly
Leu Ala Ser Ala Leu Arg Pro Asp385 390 395 400Gly Glu Tyr Thr Leu
Leu Ala Pro Val Asn Asn Ala Phe Ser Asp Asp 405 410 415Thr Leu Ser
Met Asp Gln Arg Leu Leu Lys Leu Ile Leu Gln Asn His 420 425 430Ile
Leu Lys Val Lys Val Gly Leu Asn Glu Leu Tyr Asn Gly Gln Ile 435 440
445Leu Glu Thr Ile Gly Gly Lys Gln Leu Arg Val Phe Val Tyr Arg Thr
450 455 460Ala Val Cys Ile Glu Asn Ser Cys Met Glu Lys Gly Ser Lys
Gln Gly465 470 475 480Arg Asn Gly Ala Ile His Ile Phe Arg Glu Ile
Ile Lys Pro Ala Glu 485 490 495Lys Ser Leu His Glu Lys Leu Lys Gln
Asp Lys Arg Phe Ser Thr Phe 500 505 510Leu Ser Leu Leu Glu Ala Ala
Asp Leu Lys Glu Leu Leu Thr Gln Pro 515 520 525Gly Asp Trp Thr Leu
Phe Val Pro Thr Asn Asp Ala Phe Lys Gly Met 530 535 540Thr Ser Glu
Glu Lys Glu Ile Leu Ile Arg Asp Lys Asn Ala Leu Gln545 550 555
560Asn Ile Ile Leu Tyr His Leu Thr Pro Gly Val Phe Ile Gly Lys Gly
565 570 575Phe Glu Pro Gly Val Thr Asn Ile Leu Lys Thr Thr Gln Gly
Ser Lys 580 585 590Ile Phe Leu Lys Glu Val Asn Asp Thr Leu Leu Val
Asn Glu Leu Lys 595 600 605Ser Lys Glu Ser Asp Ile Met Thr Thr Asn
Gly Val Ile His Val Val 610 615 620Asp Lys Leu Leu Tyr Pro Ala Asp
Thr Pro Val Gly Asn Asp Gln Leu625 630 635 640Leu Glu Ile Leu Asn
Lys Leu Ile Lys Tyr Ile Gln Ile Lys Phe Val 645 650 655Arg Gly Ser
Thr Phe Lys Glu Ile Pro Val Thr Val Tyr Arg Pro Thr 660 665 670Leu
Thr Lys Val Lys Ile Glu Gly Glu Pro Glu Phe Arg Leu Ile Lys 675 680
685Glu Gly Glu Thr Ile Thr Glu Val Ile His Gly Glu Pro Ile Ile Lys
690 695 700Lys Tyr Thr Lys Ile Ile Asp Gly Val Pro Val Glu Ile Thr
Glu Lys705 710 715 720Glu Thr Arg Glu Glu Arg Ile Ile Thr Gly Pro
Glu Ile Lys Tyr Thr 725 730 735Arg Ile Ser Thr Gly Gly Gly Glu Thr
Glu Glu Thr Leu Lys Lys Leu 740 745 750Leu Gln Glu Asp Thr Pro Val
Arg Lys Leu Gln Ala Asn Lys Lys Val 755 760 765Gln Gly Ser Arg Arg
Arg Leu Arg Glu Gly Arg Ser Gln 770 775 780162346DNAHomo sapiens
16atgattccct ttttacccat gttttctcta ctattgctgc ttattgttaa ccctataaac
60gccaacaatc attatgacaa gatcttggct catagtcgta tcaggggtcg ggaccaaggc
120ccaaatgtct gtgcccttca acagattttg ggcaccaaaa agaaatactt
cagcacttgt 180aagaactggt ataaaaagtc catctgtgga cagaaaacga
ctgtgttata tgaatgttgc 240cctggttata tgagaatgga aggaatgaaa
ggctgcccag cagttttgcc cattgaccat 300gtttatggca ctctgggcat
cgtgggagcc accacaacgc agcgctattc tgacgcctca 360aaactgaggg
aggagatcga gggaaaggga tccttcactt actttgcacc gagtaatgag
420gcttgggaca acttggattc tgatatccgt agaggtttgg agagcaacgt
gaatgttgaa 480ttactgaatg ctttacatag tcacatgatt aataagagaa
tgttgaccaa ggacttaaaa 540aatggcatga ttattccttc aatgtataac
aatttggggc ttttcattaa ccattatcct 600aatggggttg tcactgttaa
ttgtgctcga atcatccatg ggaaccagat tgcaacaaat 660ggtgttgtcc
atgtcattga ccgtgtgctt acacaaattg gtacctcaat tcaagacttc
720attgaagcag aagatgacct ttcatctttt agagcagctg ccatcacatc
ggacatattg 780gaggcccttg gaagagacgg tcacttcaca ctctttgctc
ccaccaatga ggcttttgag 840aaacttccac gaggtgtcct agaaaggatc
atgggagaca aagtggcttc cgaagctctt 900atgaagtacc acatcttaaa
tactctccag tgttctgagt ctattatggg aggagcagtc 960tttgagacgc
tggaaggaaa tacaattgag ataggatgtg acggtgacag tataacagta
1020aatggaatca aaatggtgaa caaaaaggat attgtgacaa ataatggtgt
gatccatttg 1080attgatcagg tcctaattcc tgattctgcc aaacaagtta
ttgagctggc tggaaaacag 1140caaaccacct tcacggatct tgtggcccaa
ttaggcttgg catctgctct gaggccagat 1200ggagaataca ctttgctggc
acctgtgaat aatgcatttt ctgatgatac tctcagcatg 1260gatcagcgcc
tccttaaatt aattctgcag aatcacatat tgaaagtaaa agttggcctt
1320aatgagcttt acaacgggca aatactggaa accatcggag gcaaacagct
cagagtcttc 1380gtatatcgta cagctgtctg cattgaaaat tcatgcatgg
agaaagggag taagcaaggg 1440agaaacggtg cgattcacat attccgcgag
atcatcaagc cagcagagaa atccctccat 1500gaaaagttaa aacaagataa
gcgctttagc accttcctca gcctacttga agctgcagac 1560ttgaaagagc
tcctgacaca acctggagac tggacattat ttgtgccaac caatgatgct
1620tttaagggaa tgactagtga agaaaaagaa attctgatac gggacaaaaa
tgctcttcaa 1680aacatcattc tttatcacct gacaccagga gttttcattg
gaaaaggatt tgaacctggt 1740gttactaaca ttttaaagac cacacaagga
agcaaaatct ttctgaaaga agtaaatgat 1800acacttctgg tgaatgaatt
gaaatcaaaa gaatctgaca tcatgacaac aaatggtgta 1860attcatgttg
tagataaact cctctatcca gcagacacac ctgttggaaa tgatcaactg
1920ctggaaatac ttaataaatt aatcaaatac atccaaatta agtttgttcg
tggtagcacc 1980ttcaaagaaa tccccgtgac tgtctataga cccacactaa
caaaagtcaa aattgaaggt 2040gaacctgaat tcagactgat taaagaaggt
gaaacaataa ctgaagtgat ccatggagag 2100ccaattatta aaaaatacac
caaaatcatt gatggagtgc ctgtggaaat aactgaaaaa 2160gagacacgag
aagaacgaat cattacaggt cctgaaataa aatacactag gatttctact
2220ggaggtggag aaacagaaga aactctgaag aaattgttac aagaagacac
acccgtgagg 2280aagttgcaag ccaacaaaaa agttcaagga tctagaagac
gattaaggga aggtcgttct 2340cagtga 23461728PRTMus musculus 17Glu Val
Ser Lys Val Thr Lys Phe Ile Glu Gly Gly Asp Gly His Leu1 5 10 15Phe
Glu Asp Glu Glu Ile Lys Arg Leu Leu Gln Gly 20 251828PRTHomo
sapiens 18Glu Val Thr Lys Val Thr Lys Phe Ile Glu Gly Gly Asp Gly
His Leu1 5 10 15Phe Glu Asp Glu Glu Ile Lys Arg Leu Leu Gln Gly 20
25196PRTHomo sapiens 19Val Thr Lys Val Thr Lys1 5207PRTHomo sapiens
20Phe Glu Asp Glu Glu Ile Lys1 5215PRTHomo sapiens 21Thr Lys Val
Thr Lys1 5228PRTHomo sapiens 22Gln Gly Asp Thr Pro Val Arg Lys1
52323DNAArtificialPrimer 1 23gttcattgaa ggtggcgatg gtc
232424DNAArtificialPrimer 2 24gagataaaat ccctgcatgg tcct
242525DNAArtificialPrimer 3 25cacggtcgat gacatggaca acacc
252622DNAArtificialPrimer 4 26acggagctca gggctgaaga tg
222731DNAArtificialPrimer 5 27aagctagcga agatggttcc tctcctgccc t
312820DNAArtificialPrimer 6 28ctttgggttt ttccagcctc
202922DNAArtificialPrimer 7 29ccccatgact gtctatagac ct
223022DNAArtificialPrimer 8 30atttccctta aaaatcagat tg
223129PRTArtificialantigen peptide of example 6 31Cys Glu Val Thr
Lys Val Thr Lys Phe Ile Glu Gly Gly Asp Gly His1 5 10 15Leu Phe Glu
Asp Glu Glu Ile Lys Arg Leu Leu Gln Gly 20 25
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References