U.S. patent application number 13/886936 was filed with the patent office on 2013-09-19 for highly effective anti-cadherin antibody for induction of antibody-dependent cellular cytotoxicity in vivo.
This patent application is currently assigned to PERSEUS PROTEOMICS INC.. The applicant listed for this patent is PERSEUS PROTEOMICS INC., THE UNIVERSITY OF TOKYO. Invention is credited to Hiroyuki ABURATANI, Keisuke ISHII, Keiko KATSUMI, Yoko KAYUKAWA, Katsushi KOUDA, Hiroshi ONISHI, Aya SAKAMOTO, Lilin ZHANG.
Application Number | 20130245232 13/886936 |
Document ID | / |
Family ID | 43032279 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130245232 |
Kind Code |
A1 |
ABURATANI; Hiroyuki ; et
al. |
September 19, 2013 |
HIGHLY EFFECTIVE ANTI-CADHERIN ANTIBODY FOR INDUCTION OF
ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY IN VIVO
Abstract
It is an object of the present invention to provide an
anti-cadherin antibody having high antibody-dependent cellular
cytotoxicity. The present invention provides an anti-cadherin
antibody, which recognizes any one of an upstream region of EC1, a
cadherin domain 4 (EC4) and a cadherin domain 5 (EC5), wherein an
antibody-dependent cellular cytotoxicity at an antibody
concentration of 1 .mu.g/mL is 30% or more.
Inventors: |
ABURATANI; Hiroyuki; (Tokyo,
JP) ; ZHANG; Lilin; (Tokyo, JP) ; ISHII;
Keisuke; (Tokyo, JP) ; KOUDA; Katsushi;
(Tokyo, JP) ; SAKAMOTO; Aya; (Tokyo, JP) ;
KATSUMI; Keiko; (Tokyo, JP) ; ONISHI; Hiroshi;
(Tokyo, JP) ; KAYUKAWA; Yoko; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF TOKYO
PERSEUS PROTEOMICS INC. |
TOKYO
TOKYO |
|
JP
JP |
|
|
Assignee: |
PERSEUS PROTEOMICS INC.
TOKYO
JP
THE UNIVERSITY OF TOKYO
TOKYO
JP
|
Family ID: |
43032279 |
Appl. No.: |
13/886936 |
Filed: |
May 3, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13318422 |
Feb 7, 2012 |
8455249 |
|
|
PCT/JP2010/057694 |
Apr 30, 2010 |
|
|
|
13886936 |
|
|
|
|
Current U.S.
Class: |
530/387.3 ;
435/331; 530/387.9 |
Current CPC
Class: |
C07K 2317/34 20130101;
A61K 2039/505 20130101; C07K 16/28 20130101; A61P 43/00 20180101;
C07K 2317/30 20130101; A61P 35/00 20180101; C07K 16/30 20130101;
C07K 2317/732 20130101 |
Class at
Publication: |
530/387.3 ;
530/387.9; 435/331 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
May 1, 2009 |
JP |
2009-111834 |
Jan 29, 2010 |
JP |
2010-018416 |
Claims
1. An anti-P-cadherin monoclonal antibody, which recognizes any one
of an upstream region of EC1, consisting of amino acids 108-131 of
SEQ ID NO:2, a cadherin domain 4 (EC4), consisting of amino acids
462-550 of SEQ ID NO:2, or a cadherin domain 5 (EC5), consisting of
amino acids 551-654 of SEQ ID NO:2, and having an
antibody-dependent cellular cytotoxicity (ADCC): wherein the ADCC
is no less than 30% at an antibody concentration of 1 .mu.g/mL,
determined by .sup.51Cr release assay using mice bone marrow cells
as effector cells.
2. The antibody according to claim 1, which is an antibody obtained
from an immunized animal, into which a soluble P-cadherin has been
administered as an immunogen.
3. The antibody according to claim 1, wherein the antibody is a
chimeric antibody.
4. The antibody according to claim 1 wherein the antibody is a
humanized antibody.
5. A cytotoxic agent which comprises the antibody of claim 1.
6. A hybridoma, which produces the antibody of claim 1.
Description
[0001] This is a divisional application of U.S. application Ser.
No. 13/318,422 filed on Feb. 7, 2012, which is the national stage
371 application of PCT International Application No.
PCT/JP2010/057694 filed on Apr. 30, 2010, which claims the benefit
of priority of JP 2009-111834, filed on May 1, 2009 and JP
2010-018416 filed on Jan. 29, 2010. The entire contents of each of
the above-identified applications are hereby incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention relates to an anti-cadherin antibody
that recognizes a specific domain of a cadherin and has high
antibody-dependent cellular cytotoxicity.
BACKGROUND ART
[0003] Cancer is a crucial disease that becomes a leading cause of
death, but the therapeutic needs thereof have not yet been
satisfied. In recent years, in order to solve the problem of the
conventional chemotherapy in that it affects even normal cells, a
cancer treatment using a molecular-targeted agent has been
vigorously studied. In this cancer treatment, an agent is designed
to target a specific molecule that is specifically expressed in
cancer cells, and the cancer is treated with the thus designed
agent.
[0004] Cadherin is an example of molecules that can be a target of
a molecular-therapeutic agent in a cancer treatment. Cadherin is a
membrane protein that has been discovered as a molecule that is
calcium-dependently associated with homophilic cell adhesion
(Yoshida and Takeichi, Cell 28: 217-224, 1982). Proteins that have
cadherin repeats (ECs) consisting of approximately 110 amino acid
residues having high homology to one another are referred to as
cadherin superfamily. There
[0005] An increase in the expression of a cadherin in cancer cells
has been reported. With respect to cancer cells in which the
expression level of a cadherin in cancer tissues is higher than
that in normal tissues, the use of an agent prepared by binding an
anticancer agent to an antibody recognizing a cadherin or an
antibody having antibody-dependent cellular cytotoxicity (ADCC) for
the therapy of cancers has been studied (WO2002/097395 and
WO2007/102525).
[0006] Based on the characteristics of their structures, proteins
belonging to the cadherin superfamily can be broadly classified
into (1) classical cadherins, (2) desmosomal cadherins, (3)
protocadherins, and (4) other cadherins. Classical cadherins that
are main members of the cadherin superfamily are highly homologous
to one another (FIG. 1). That is, the classical cadherin is a
single transmembrane protein that seems to form a dimer, and it has
five cadherin domains of EC1-EC5 in the extracellular region
thereof and an intracellular domain. Cell adhesion via such a
classical cadherin is characterized in that it is carried out
between homologous cells. Cells mutually recognize the same species
of cadherin molecules each having different expression status that
is specific to cell species, so that cell adhesion is carried out.
Homologous cells mutually adhere to each other based on a mechanism
whereby an E-cadherin recognizes an E-cadherin and binds thereto
and a P-cadherin recognizes a P-cadherin and binds thereto (FIG.
2).
[0007] Mutual recognition regarding a homologous/heterologous
cadherin is considered to be caused by a cadherin domain 1 (EC1)
located at the N-terminus of an extracellular domain (Nose A. et
al., Cell 61: 147-155, 1990). Klingel et al. have reported that
when the amino acid sequence at positions 1 to 213 of a human
P-cadherin (SEQ ID NO: 2) is substituted with the corresponding
region of a human E-cadherin, the resultant product does not bind
to the E-cadherin but binds to the P-cadherin (Klingel H. et al., J
of Cell Science 113: 2829-36, 2000). Hence, classical cadherins
including an E-cadherin and a P-cadherin as typical examples are
considered to mutually bind to one another by a single same
mechanism.
[0008] In recent years, a large number of antibody drugs for use in
cancer treatments have been actually placed on the market as
molecular-targeted agents, and certain therapeutic effects can be
obtained. Antibody-dependent cellular cytotoxicity (ADCC) is a main
antitumor mechanism of commercially available anticancer agents
such as trastuzumab and rituximab, and the increase of the ADCC
activity leads to the improvement of therapeutic effects, reduction
in side effects, etc. Thus, studies for searching for an antibody
having higher ADCC activity and the development of a technique of
enhancing ADCC activity have been carried out. For example, there
have been developed a technique of removing fucose at the end of a
sugar chain binding to the Fc portion of an antibody (WO00/61739)
and a technique of substituting amino acids in the Fc portion with
other amino acids to enhance affinity for effector cells, so as to
increase ADCC activity (WO2008/121160).
[0009] As described above, a concept of using an antibody having
ADCC activity as a therapeutic agent for cancer is publicly known.
However, although there is a report regarding the association of a
domain structure with the functions of classical cadherins
including a P-cadherin, there are no reports suggesting the
association of the level of ADCC activity with the structures of
classical cadherins.
PRIOR ART DOCUMENTS
Patent Documents
[0010] [Patent Document 1] WO2002/097395 [0011] [Patent Document 2]
WO2007/102525 [0012] [Patent Document 3] WO00/61739 [0013] [Patent
Document 4] WO2008/121160
Non Patent Documents
[0013] [0014] [Non Patent Document 1] Yoshida and Takeichi, Cell
28: 217-224, 1982 [0015] [Non Patent Document 2] Nose A. et al.,
Cell 61: 147-155, 1990 [0016] [Non Patent Document 3] Klingel H. et
al., J of Cell Science 113: 2829-36, 2000
SUMMARY OF INVENTION
Object to be Solved by the Invention
[0017] It is an object of the present invention to provide an
anti-cadherin antibody having high antibody-dependent cellular
cytotoxicity.
Means for Solving the Object
[0018] The present inventor has conducted intensive studies
directed towards achieving the aforementioned object. The inventor
measured the antibody-dependent cellular cytotoxic (ADCC) activity
of P-cadherin antibodies, and as a result, the inventor found that
the P-cadherin antibodies tend to be divided into two groups,
depending on the level of the ADCC activity. Thus, the present
inventor classified the antibodies based on a region recognized by
each antibody. As a result, it was found that an antibody having
high ADCC activity recognizes any one of an upstream region of EC1,
a cadherin domain 4 (EC4) and a cadherin domain 5 (EC5) with high
probability.
[0019] Elements that specify the ADCC activity of an antibody
include: the affinity of the Fc region of an antibody for the Fc
receptor of an effector cell; the affinity of an antibody for an
antigen; and an epitope recognized by an antibody. For exertion of
the ADCC activity, it is essential that an antibody bind to an
antigen, and that the Fc receptor of an effector cell bind to the
Fc region of the antibody. It is assumed that the binding of an
effector cell to the Fc region of an antibody involves a spatial
limitation due to a difference in a CDH3 region to which the
antibody binds, and thus that a difference in the level of the ADCC
activity would be made. The present invention has been completed
based on these findings.
[0020] Specifically, the present invention provides the following.
[0021] (1) An anti-cadherin antibody, which recognizes any one of
an upstream region of EC1, a cadherin domain 4 (EC4) and a cadherin
domain 5 (EC5), wherein an antibody-dependent cellular cytotoxicity
at an antibody concentration of 1 .mu.g/mL is 30% or more. [0022]
(2) The antibody according to (1) above, wherein the cadherin is a
P-cadherin. [0023] (3) The antibody according to (1) or (2) above,
which is an antibody produced by antibody-producing cells obtained
from an immunized animal, into which a soluble P-cadherin has been
administered as an immunogen. [0024] (4) The antibody according to
any one of (1) to (3) above, which is a monoclonal antibody. [0025]
(5) A hybridoma, which produces the antibody according to (4)
above. [0026] (6) A cytotoxic agent which comprises the antibody of
any one of (1) to (4) above. [0027] (7) The cytotoxic agent
according to (6) above, which is administered to cancer cells.
[0028] In the present specification, the term "an upstream region
of EC1" is used to mean a region consisting of 24 amino acid
residues on the side upstream of the EC1 of each of an E-cadherin,
a P-cadherin and an N-cadherin, and the corresponding regions of
other cadherins.
Effect of the Invention
[0029] The anti-cadherin antibody of the present invention is
characterized in that it recognizes any one of a upstream region of
EC1, a cadherin domain 4 (EC4) and a cadherin domain 5 (EC5), and
has high antibody-dependent cellular cytotoxicity. An antibody
capable of exhibiting high antibody-dependent cellular cytotoxicity
is useful as a material for producing a modified antibody or an
engineered antibody. In addition, the anti-cadherin antibody of the
present invention is administered to cancer in which a cadherin is
expressed, so that the present antibody can exhibit anticancer
action having antibody-dependent cytotoxicity as a mode of action.
That is to say, the anti-cadherin antibody of the present invention
is useful as an anticancer agent.
BRIEF DESCRIPTION OF DRAWINGS
[0030] FIG. 1 shows the sequences of mature proteins of an
E-cadherin (CDH1), an N-cadherin (CDH2) and a P-cadherin (CDH3),
wherein the signal and propeptide sequences are excluded.
[0031] FIG. 2 shows the adhesion mechanism of molecules belonging
to the classical cadherin family.
[0032] FIG. 3 shows the results of a flow cytometry, in which a
human CDH3 forced expression cell line was allowed to react with a
commercially available anti-human CDH3 antibody. A: CDH3 forced
expression CHO cells; B: CHO cells; a: 0.01 .mu.g/ml anti-CDH3
antibody; b: 0.1 .mu.g/ml anti-CDH3 antibody; and c: 1 .mu.g/ml
anti-CDH3 antibody.
[0033] FIG. 4 shows the results of a typical flow cytometry
regarding the obtained three antibody cases and each cell line. A:
CDH3 forced expression CHO cells; B: CHO cells; C: lung
cancer-derived cell line NCI-H358; a: 0.01 .mu.g/ml anti-CDH3
antibody; b: 0.1 .mu.g/ml anti-CDH3 antibody; and c: 1 .mu.g/ml
anti-CDH3 antibody.
[0034] FIG. 5 shows the ADCC activity of each antibody.
[0035] FIG. 6 shows the correlation of partial-length CDH3 protein
fragments 1 to 5 with a CDH3 extracellular region.
[0036] FIG. 7 shows the results of expression of partial-length
CDH3 proteins. A: fragment 1; B: fragment 2; C: fragment 3; D:
fragment 4; and E: fragment 5.
[0037] FIG. 8 shows the reaction of partial-length CDH3 proteins
with each antibody according to a Western blot method. A: fragment
1; B: fragment 2; C: fragment 3; D: fragment 4; and E: fragment
5.
[0038] FIG. 9 shows the results of the epitope analysis of PPMX13
using a peptide array. The numerical value on the X-axis indicates
the number of the peptide array. A: PPMX13; and B: no primary
antibodies.
[0039] FIG. 10 shows the results of the mRNA expression of CDH3 in
various types of tumor tissues. A: normal tissues; B: various types
of cancer tissues; and C: differentiation degree of pancreatic
cancer.
[0040] FIG. 11 shows the results of the expression of CDH3 in
various types of tumor tissues.
[0041] FIG. 12 shows the antitumor effect of PPMX12-producing
antibody in a xenograft into which a human lung cancer-derived cell
line NCI-H351 has been transplanted.
[0042] FIG. 13 shows the antitumor effect of PPMX12-producing
antibody in a xenograft into which a human pancreatic
cancer-derived cell line PK-45P has been transplanted.
[0043] FIG. 14 shows the antitumor effect of PPMX12-producing
antibody in a xenograft into which a human skin cancer-derived cell
line A431 has been transplanted.
EMBODIMENT FOR CARRYING OUT THE INVENTION
[0044] The present invention will be described more in detail
below.
[0045] The antibody of the present invention is: an anti-cadherin
antibody, which recognizes any one of an upstream region of EC1, a
cadherin domain 4 (EC4) and a cadherin domain 5 (EC5), wherein an
antibody-dependent cellular cytotoxicity at an antibody
concentration of 1 .mu.g/mL is 30% or more; an anti-cadherin
antibody, which recognizes any one of an upstream region of EC1, a
cadherin domain 4 (EC4) and a cadherin domain 5 (EC5), wherein an
antibody-dependent cellular cytotoxicity at an antibody
concentration of 0.1 .mu.g/mL is 25% or more (which is, for
example, stronger than the activity of PPMX5); or an anti-cadherin
antibody, which recognizes any one of an upstream region of EC1, a
cadherin domain 4 (EC4) and a cadherin domain 5 (EC5), wherein the
maximum ADCC activity is 35% or more (which is, for example,
stronger than the activity of PPMX6). Herein, the term "maximum
ADCC activity" is used to mean ADCC activity obtained when an
antibody concentration is increased and an increase in the ADCC
activity reaches plateau.
[0046] In the present specification, the upstream region of EC1,
the cadherin domain 1 (EC1), the cadherin domain 2 (EC2), the
cadherin domain 3 (EC3), the cadherin domain 4 (EC4) and the
cadherin domain 5 (EC5) of each of a P-cadherin, an E-cadherin and
an N-cadherin are as described below. Moreover, the corresponding
regions of other cadherins can be determined by making a comparison
among the sequences of known cadherin proteins obtained from
Genbank and the like. The sequences can be compared using a known
program such as Clustal W2 (Thompson J D et al., Nucleic Acids
Research 22 (22): 3673-3680, 1994) or Clustal X2 (Thompson J D et
al., Nucleic Acids Research 25 (24): 4876-4882, 1997).
P-cadherin (CDH3)
[0047] Upstream region of EC1: amino acids at positions 108-131 of
the amino acid sequence shown in SEQ ID NO: 2 [0048] Cadherin
domain 1 (EC1): amino acids at positions 132-236 of the amino acid
sequence shown in SEQ ID NO: 2 [0049] Cadherin domain 2 (EC2):
amino acids at positions 237-348 of the amino acid sequence shown
in SEQ ID NO: 2 [0050] Cadherin domain 3 (EC3): amino acids at
positions 349-461 of the amino acid sequence shown in SEQ ID NO: 2
[0051] Cadherin domain 4 (EC4): amino acids at positions 462-550 of
the amino acid sequence shown in SEQ ID NO: 2 [0052] Cadherin
domain 5 (EC5): amino acids at positions 551-654 of the amino acid
sequence shown in SEQ ID NO: 2
E-cadherin (CDH1)
[0052] [0053] Upstream region of EC1: amino acids at positions
155-178 of the amino acid sequence shown in SEQ ID NO: 4 [0054]
Cadherin domain 1 (EC1): amino acids at positions 179-283 of the
amino acid sequence shown in SEQ ID NO: 4 [0055] Cadherin domain 2
(EC2): amino acids at positions 284-395 of the amino acid sequence
shown in SEQ ID NO: 4 [0056] Cadherin domain 3 (EC3): amino acids
at positions 396-507 of the amino acid sequence shown in SEQ ID NO:
4 [0057] Cadherin domain 4 (EC4): amino acids at positions 508-597
of the amino acid sequence shown in SEQ ID NO: 4 [0058] Cadherin
domain 5 (EC5): amino acids at positions 598-704 of the amino acid
sequence shown in SEQ ID NO: 4
N-cadherin (CDH2)
[0058] [0059] Upstream region of EC1: amino acids at positions
160-183 of the amino acid sequence shown in SEQ ID NO: 6 [0060]
Cadherin domain 1 (EC1): amino acids at positions 184-288 of the
amino acid sequence shown in SEQ ID NO: 6 [0061] Cadherin domain 2
(EC2): amino acids at positions 289-402 of the amino acid sequence
shown in SEQ ID NO: 6 [0062] Cadherin domain 3 (EC3): amino acids
at positions 403-518 of the amino acid sequence shown in SEQ ID NO:
6 [0063] Cadherin domain 4 (EC4): amino acids at positions 519-607
of the amino acid sequence shown in SEQ ID NO: 6 [0064] Cadherin
domain 5 (EC5): amino acids at positions 608-719 of the amino acid
sequence shown in SEQ ID NO: 6
[0065] The antibody-dependent cellular cytotoxicity (ADCC activity)
can be measured by a known method. The numerical value of the ADCC
activity of the present specification means antibody-dependent
cellular cytotoxicity measured under the same measurement
conditions as those in Example 4. Specifically, the ADCC activity
can be measured as follows.
(1) Preparation of Effector Cells
[0066] Bone-marrow cells were collected from the femur of a C3H/HeJ
Jcl mouse (8-week-old, male, CLEA Japan, Inc.), and the cells were
then prepared to a concentration of 2.times.10.sup.6 cells/mL in a
10% FBS-containing RPMI1640 medium. Thereafter, the cells were
cultured for 6 days in the presence of 50 ng/mL human IL-2
(PEPROTECH) and 10 ng/mL mouse GM-CSF (PEPROTECH). On the day of
measurement, the cells were recovered and were then washed with a
10% FBS-containing HAM medium, so as to prepare an effector cell
solution.
(2) Preparation of Target Cells
[0067] As target cells, full-length CDH3-expressing CHO cells
(EXZ1501) were used. The cells were removed from a plate, and were
then suspended in a 10% FBS-containing HAM medium to a
concentration of 1.times.10.sup.7 cells/mL. Then, .sup.51Cr was
added to the suspension to a final concentration of 150 .mu.Ci. The
thus obtained mixture was cultured in a 5% CO.sub.2 incubator at
37.degree. C. for 1.5 hours. The resultant cells were washed with a
medium twice, and a 10% FBS-containing HAM medium was then added
thereto. Thereafter, the cells were inoculated on a 96-well
U-bottom plate (NUNC) to a concentration of 1.times.10.sup.4
cells/mL, so as to prepare target cells.
(3) Measurement of ADCC Activity
[0068] An antibody solution, which had been prepared to have a
concentration of each of 0.001, 0.01, 0.1 and 1 .mu.g/mL, was
dispensed in 50 .mu.L/well into the target cells. The obtained
mixture was incubated at room temperature for 15 minutes.
Thereafter, 100 .mu.L of the effector cells (1.times.10.sup.5
cells/well) was dispensed therein, and the obtained mixture was
then cultured in a CO.sub.2 incubator for 4 hours. Thereafter, a
culture supernatant was recovered, and radioactivity in 100 .mu.L
of the culture supernatant was measured with a scintillation
counter. Cytotoxicity can be obtained by the following formula.
Cytotoxicity (%)=(A-C)/(B-C).times.100 [0069] A: the radioactivity
value (cpm) of each antibody-added well [0070] B: the radioactivity
value (cpm) of a well, in which 100 .mu.L of a 2% NP40 solution and
50 .mu.L of a 10% FBS-containing RPMI medium have been added to the
target cells [0071] C: the radioactivity value (cpm) of a well, in
which 150 .mu.L of a 10% FBS-containing medium comprising the
effector cells has been added to the target cells
[0072] The type of a cadherin recognized by the antibody of the
present invention is desirably a classical cadherin. Examples of
the classical cadherin include, but are limited to, an E-cadherin,
an N-cadherin, and P-cadherin.
[0073] As an antigen used to produce the antibody of the present
invention, a cadherin or a partial peptide thereof can be used. An
example of such an antigen that can be used herein is a soluble
CDH3 protein. However, examples are not limited thereto.
[0074] The antibody of the present invention may be either a
polyclonal antibody or a monoclonal antibody. The antibody of the
present invention (a polyclonal antibody and a monoclonal antibody)
can be produced by any one of various methods. Methods for
producing such antibodies are well known in the present technical
field [see, for example, Sambrook, J et al., Molecular Cloning,
Cold Spring Harbor Laboratory Press (1989)].
(a) Preparation of Polyclonal Antibody
[0075] In order to produce a polyclonal antibody, a cadherin or a
partial peptide thereof (which is preferably any one of an upstream
region of EC1, a cadherin domain 4 (EC4) and a cadherin domain 5
(EC5)) is used as an antigen, and a mammal such as a rat, a mouse
or a rabbit is immunized with this antigen. The applied dose of the
antigen per animal is 0.1 to 100 mg, when no adjuvant is used. It
is 1 to 100 .mu.g, when an adjuvant is used. Examples of the
adjuvant include a Freund's complete adjuvant (FCA), a Freund's
incomplete adjuvant (FIA), and an aluminum hydroxide adjuvant.
Immunization is mainly carried out by injection into the vein,
subcutis, abdominal cavity, etc. In addition, immunization interval
is not particularly limited, and immunization is carried out at
intervals of several days to several weeks, and preferably of 2 to
5 weeks, 1 to 10 times, and preferably 2 to 5 times. Then, six to
sixty days after the final immunization, antibody titer is measured
according to enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), radioimmunoassay (RIA), etc. Blood is collected
on the day when the maximum antibody titer is obtained, and
antiserum is then obtained. When an antibody needs to be purified
from the antiserum, it can be purified by selecting an appropriate
method from known methods such as ammonium sulfate precipitation
method, ion exchange chromatography, gel filtration, and affinity
chromatography, or by combining such known methods.
(b) Preparation of Monoclonal Antibody
[0076] In order to produce a monoclonal antibody, first, a cadherin
or a partial peptide thereof (which is preferably any one of an
upstream region of EC1, a cadherin domain 4 (EC4) and a cadherin
domain 5 (EC5)) is used as an antigen, and a mammal such as a rat,
a mouse or a rabbit is immunized with this antigen. The applied
dose of the antigen per animal is 0.1 to 100 mg, when no adjuvant
is used. It is 1 to 100 .mu.g, when an adjuvant is used. Examples
of the adjuvant include a Freund's complete adjuvant (FCA), a
Freund's incomplete adjuvant (FIA), and an aluminum hydroxide
adjuvant. Immunization is mainly carried out by injection into the
vein, subcutis, abdominal cavity, etc. In addition, immunization
interval is not particularly limited, and immunization is carried
out at intervals of several days to several weeks, and preferably
of 2 to 5 weeks, 1 to 10 times, and preferably 2 to 5 times. Then,
one to sixty days, and preferably one to fourteen days after the
final immunization, antibody-producing cells are collected.
Examples of antibody-producing cells include splenic cells, lymph
node cells, and peripheral blood cells. Of these, splenic cells and
local lymph node cells are preferable.
[0077] To obtain cell fusion hybridomas, antibody-producing cells
are fused with myeloma cells. As myeloma cells to be fused with
antibody-producing cells, commonly available established cells from
an animal such as a mouse can be used. Preferably, the used cell
line has drug selectivity, cannot survive in a HAT selective medium
(containing hypoxanthine, aminopterin and thymidine) when it is in
an unfused state, and can survive only in a state in which it is
fused with antibody-producing cells. Examples of myeloma cells
include a mouse myeloma cell line such as P3X63-Ag.8.U1 (P3U1) and
NS-1.
[0078] Subsequently, the aforementioned myeloma cells are fused
with the aforementioned antibody-producing cells. For cell fusion,
antibody-producing cells (1.times.10.sup.6 to 1.times.10.sup.7
cells/ml) are mixed with myeloma cells (2.times.10.sup.5 to
2.times.10.sup.6 cells/ml) in a medium used for culture of animal
cells, such as DMEM or RPMI-1640 medium containing no serum
(wherein the ratio between the antibody-producing cells and the
myeloma cells is preferably 5:1), and fusion is then carried out in
the presence of a cell fusion promoter. As a cell fusion promoter,
polyethylene glycol having an average molecular weight of 1000 to
6000 Daltons, or the like can be used. Alternatively, the
antibody-producing cells can also be fused with the myeloma cells
using a commercially available cell fusion apparatus that utilizes
electrical stimulation (for example, electroporation).
[0079] Hybridomas of interest are selected from the cells after
completion of the cell fusion treatment. As a selection method, a
cell suspension is appropriately diluted with a fetal bovine
serum-containing RPMI-1640 medium, for example, and the thus
diluted solution is then inoculated on a microtiter plate to a
concentration of approximately 3.times.10.sup.5 cells/well.
Thereafter, a selective medium is added to each well, and the
obtained mixture is then cultured, while appropriately exchanging
the medium with a fresh selective medium. As a result, cells that
grow approximately 14 days after initiation of the culture in the
selective medium can be obtained as hybridomas.
[0080] Subsequently, whether or not an antibody of interest is
present in a culture supernatant of the growing hybridomas is
screened. The screening of the hybridomas may be carried out
according to an ordinary method, and thus the screening method is
not particularly limited. For example, an aliquot of the culture
supernatant contained in the well in which the hybridomas have
grown is collected, and thereafter, a hybridoma that produces an
antibody binding to the upstream region of EC1, the EC4 domain or
the EC5 domain of a cadherin can be screened. The cloning of the
fused cells is carried out by a limiting dilution method or the
like, and a hybridoma that is a monoclonal antibody-producing cell
can be finally established.
[0081] As a method of collecting a monoclonal antibody from the
thus established hybridomas, a common cell culture method, an
ascites collection method or the like can be adopted. In the case
of the cell culture method, hybridomas are cultured in an animal
cell culture medium such as a 10% fetal bovine serum-containing
RPMI-1640 medium, MEM medium or a serum-free medium under ordinary
culture conditions (for example, at 37.degree. C. in a 5% CO.sub.2
concentration) for 7 to 14 days, and an antibody is then obtained
from the culture supernatant.
[0082] In the case of the ascites collection method, hybridomas
(approximately 1.times.10.sup.7 cells) are administered into the
abdominal cavity of an animal of the same species as a mammal, from
which myeloma cells are derived, so that large quantities of
hybridomas are allowed to grow. Then, one to two weeks later, the
ascites is collected. When purification of an antibody is necessary
in the above-described antibody collection method, the antibody can
be purified by selecting an appropriate method from known methods
such as ammonium sulfate precipitation method, ion exchange
chromatography, gel filtration, and affinity chromatography, or by
combining such known methods.
[0083] The type of the antibody of the present invention is not
particularly limited. Any of a mouse antibody, a human antibody, a
rat antibody, a rabbit antibody, a sheep antibody, a camel
antibody, an avian antibody and the like, and a genetically
recombinant antibody that is artificially modified for the purpose
of reduction in heterogenetic antigenecity against human, such as a
chimeric antibody and a humanized antibody, may be used. A
genetically recombinant antibody can be produced by a previously
known method. A chimeric antibody is an antibody consisting of the
variable regions of heavy and light chains of a mammalian antibody
other than a human antibody, such as a mouse antibody, and the
constant regions of heavy and light chains of a human antibody.
Such a chimeric antibody can be obtained by ligating DNA encoding
the variable region of a mouse antibody to DNA encoding the
constant region of a human antibody, then incorporating the thus
ligated DNA into an expression vector, and then introducing the
expression vector into a host, so as to produce an antibody of
interest. A humanized antibody is prepared by transplanting the
complementarity determining region (CDR) of a mammalian antibody
other than a human antibody, for example, the CDR of a mouse
antibody, into the CDR of a human antibody. A common genetic
recombination method therefor has been known. Specifically, a DNA
sequence designed such that the CDR of a mouse antibody is ligated
to the framework region (FR) of a human antibody is synthesized by
PCR method from several oligonucleotides produced such that they
have some overlapping portions at the termini thereof. The obtained
DNA is ligated to DNA encoding the constant region of a human
antibody, and the thus ligated DNA is then incorporated into an
expression vector. This expression vector is introduced into a
host, so that the host generates a humanized antibody (EP239400,
International Publication WO96/02576, etc.).
[0084] Also, a method of obtaining a human antibody has been known.
For example, human lymphocytes are sensitized in vitro with a
desired antigen or cells that express a desired antigen, and the
thus sensitized lymphocytes are then fused with human myeloma cells
such as U266, so as to obtain a desired human antibody having
binding activity to an antigen (see JP Patent Publication (Kokoku)
No. 1-59878 B (1989)). Alternatively, a transgenic animal having
all repertories of human antibody genes is immunized with a desired
antigen, so as to obtain a desired human antibody (see WO93/12227,
WO92/03918, WO94/02602, WO94/25585, WO96/34096, and WO96/33735).
Moreover, a technique of obtaining a human antibody by panning of a
human antibody library has also been known. For example, the
variable region of a human antibody is allowed to express as a
single-stranded antibody (scFv) on the surface of a phage according
to a phage display method, and a phage binding to an antigen can be
selected. Then, by analyzing the gene of the selected phage, the
sequence of DNA encoding the variable region of a human antibody
binding to an antigen can be determined. If the DNA sequence of the
scFv binding to the antigen is determined, a suitable expression
vector is prepared from the sequence, and a human antibody can be
then obtained. These methods have already been publicly known, and
WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172,
WO95/01438 and WO95/15388 can be used as references.
[0085] These antibodies may be any of monovalent antibodies,
divalent antibodies and polyvalent antibodies, unless they lose
their characteristics in that they are: antibodies which recognize
any one of an upstream region of EC1, a cadherin domain 4 (EC4) and
a cadherin domain 5 (EC5), wherein an antibody-dependent cellular
cytotoxicity at an antibody concentration of 1 .mu.g/mL is 30% or
more; antibodies which recognize any one of an upstream region of
EC1, a cadherin domain 4 (EC4) and a cadherin domain 5 (EC5),
wherein an antibody-dependent cellular cytotoxicity at an antibody
concentration of 0.1 .mu.g/mL is 25% or more (which is, for
example, stronger than the activity of PPMX5); or antibodies which
recognize any one of an upstream region of EC1, a cadherin domain 4
(EC4) and a cadherin domain 5 (EC5) wherein a maximum ADCC activity
is 35% or more (which is, for example, stronger than the activity
of PPMX6). Moreover, the antibodies may also be low molecular
weight antibodies such as antibody fragments, modified products of
antibodies, and the like. Furthermore, an antibody, which is
prepared by fusing a Fc portion with an antibody fragment or a low
molecular weight antibody, such as Fab, Fab', F(ab').sub.2, Fv,
scFv (single chain Fv), or Diabody, so as to acquire ADCC activity,
may also be used. In order to obtain such an antibody, a gene
encoding such an antibody may be constructed, the gene may be then
introduced into an expression vector, and the gene may be then
allowed to express in a suitable host cell.
[0086] As a modified product of antibody, an antibody that is bound
to various types of molecules such as polyethylene glycol (PEG) may
be used. Moreover, it may also be possible to bind a radioisotope,
a chemotherapeutic agent or the like to an antibody. A radiolabeled
antibody is particularly useful. Such a modified product of
antibody can be obtained by performing chemical modification on the
obtained antibody. It is to be noted that a method of modifying
antibodies is known to a person skilled in the art.
[0087] Since the antibody of the present invention exhibits high
antibody-dependent cellular cytotoxicity, it can be used as a
cytotoxic agent. The cytotoxic agent of the present invention may
cause damage on, for example, cancer cells that express cadherin,
by allowing it to come into contact with the cancer cells.
[0088] The cytotoxic agent of the present invention may comprise,
as appropriate, a pharmaceutically acceptable carrier, an
excipient, a diluent and other additives as well as the antibody of
the present invention, as necessary. The cytotoxic agent of the
present invention can be formulated in the form of an injection,
for example. The applied dose of the cytotoxic agent of the present
invention depends on the degree of symptoms, age and body weight of
a patient, an administration method, and the like. The applied dose
is generally within the range from approximately 10 ng/kg of body
weight to approximately 100 mg/kg of body weight, in terms of the
weight of an antibody as an active ingredient.
[0089] The present invention will be more specifically described in
the following examples. However, these examples are not intended to
limit the scope of the present invention.
EXAMPLES
Example 1
Establishment of CDH3-Expressing CHO Cell Line
[0090] In order to obtain a cell line used in the screening of an
anti-CDH3 antibody, CHO cells that expressed full-length CDH3 were
established.
(1) Preparation of Expression Vector for CDH3 Gene
[0091] In order to insert full-length human CDH3 DNA shown in SEQ
ID NO: 1 into a mammalian expression vector pEF4/myc-HisB
(Invitrogen), the DNA was treated with two types of restriction
enzymes KpnI (Takara Bio Inc.) and XbaI (Takara Bio Inc.) at
37.degree. C. for 1 hour, and it was then inserted into
pEF4/myc-HisB treated with the same KpnI and XbaI according to an
ordinary method using T4 DNA ligase (Promega), so as to obtain an
expression vector pEF4-CDH3-myc-His.
(2) Achievement of CDH3 Stably Expressing Cell Line
[0092] In accordance with the protocols of FuGENE (registered
trademark) 6 transfection reagent (Roche Diagnostics), on the day
before transfection, CHO cells (8.times.10.sup.5 cells) were
inoculated on a dish with a diameter of 10 cm, and they were then
cultured overnight. Thereafter, 8 .mu.g of the expression vector
pEF4-CDH3-myc-His and 16 .mu.L of the FuGENE 6 reagent were mixed
into 400 .mu.L of a serum-free RPMI 1640 medium (SIGMA-ALDRICH),
and the mixture was then left at room temperature for 15 minutes.
Thereafter, the mixture was added to the cell culture solution, so
as to carry out transfection. Two days after the transfection,
using a selective reagent (Zeocin (registered trademark)), cloning
was carried out according to a limiting dilution method.
[0093] Clones of full-length CDH3-expressing CHO cells were
selected according to a Western blot method using an anti-c-Myc
monoclonal antibody (SANTA CRUZ BIOTECHNOLOGY). As a result, there
was obtained a full-length CDH3-expressing CHO cell line (EXZ1501)
which provided a high expression level of CDH3 and was able to grow
favorably. The measurement results of a flow cytometry, in which
the above-mentioned cell line was allowed to react with a
commercially available anti-CDH3 antibody (R & D SYSTEMS), are
shown in FIG. 3.
Example 2
Preparation of Soluble CDH3 Antigen
[0094] A soluble CDH3 (sCDH3) protein, in which its C-terminal
transmembrane region and the subsequent regions were deleted, was
prepared to be used as an immunogen in the production of an
anti-CDH3 antibody.
(1) Preparation of Expression Vector for Soluble CDH3 Antigen
[0095] Using full-length CDH3 cDNA as a template, a PCR reaction
was carried out employing a forward primer (SEQ ID NO. 7:
CGCGGTACCATGGGGCTCCCTCGT (hCDH3 Full FW)) and a reverse primer (SEQ
ID NO. 8: CCGTCTAGATAACCTCCCTTCCAGGGTCC (hCDH3 Solb RV)) that had
been designed to amplify a region corresponding to a CDH3
extracellular region (which corresponds to 1-654 of SEQ ID NO: 2;
hereinafter referred to as sCDH3 cDNA). KOD-Plus (Toyobo Co., Ltd.)
was used in the reaction, and the reaction was carried out under
reaction conditions consisting of 30 cycles of 94.degree. C.-15
seconds, 55.degree. C.-30 seconds and 68.degree. C.-90 seconds.
[0096] Thereafter, a gel fragment containing an approximately 2.0
kbp band that was a size of interest was cut out in agarose gel
electrophoresis, and using QIA (registered trademark) quick Gel
Extraction Kit (QIAGEN), sCDH3 cDNA of interest was obtained.
[0097] In order to insert this sCDH3 cDNA into an expression vector
pEF4/myc-HisB, the DNA was treated with two types of restriction
enzymes KpnI and XbaI, and it was then inserted into pEF4/myc-HisB
treated with the same KpnI and XbaI according to an ordinary method
using T4 DNA ligase, so as to obtain an expression vector
pEF4-sCDH3-myc-His.
(2) Expression of Soluble CDH3 Protein
[0098] In accordance with the protocols of the FuGENE 6
transfection reagent, on the day before transfection, CHO cells
(8.times.10.sup.5 cells) were inoculated on a dish with a diameter
of 10 cm, and they were then cultured overnight. Thereafter, 8
.mu.g of the expression vector pEF4-sCDH3-myc-His and 16 .mu.L of
the FuGENE 6 reagent were mixed into 400 .mu.L of a serum-free RPMI
1640 medium, and the mixture was then left at room temperature for
15 minutes. Thereafter, the mixture was added to the cell culture
solution, so as to carry out transfection. Two days after the
transfection, using a selective reagent (Zeocin), cloning was
carried out according to a limiting dilution method.
[0099] Soluble CDH3-expressing CHO cells were selected according to
a Western blot method using an anti-c-Myc monoclonal antibody
(SANTA CRUZ BIOTECHNOLOGY). It was attempted to select a cell line,
which secreted a large amount of soluble CDH3 into the culture
supernatant and which was able to grow favorably. As a result, a
soluble CDH3-expressing CHO cell line (EXZ1702) was obtained. Using
three roller bottles each having a culture area of 1,500 cm.sup.2,
the selected soluble CDH3-expressing CHO cell line (EXZ1702) was
cultured for 72 hours in 333 mL of a serum-free medium CHO-S-SFM-II
(Invitrogen) per roller bottle. Thereafter, a culture supernatant
was recovered. A soluble CDH3 protein was obtained from the
recovered culture supernatant according to affinity chromatography
using HisTrap (registered trademark) HP column (GE Healthcare
Biosciences) and gel filtration chromatography using Superdex
(registered trademark) 200 pg column (GE Healthcare
Biosciences).
Example 3
Production of Anti-CDH3 Monoclonal Antibody
(1) Preparation of Monoclonal Antibody Using Soluble CDH3 Protein
as Immunogen
[0100] 50 .mu.g of a soluble CDH3 protein dissolved in a normal
saline and Titer-MAX Gold (registered trademark) (TiterMax) were
mixed at equal volume. The obtained mixture was injected into the
abdominal cavity and subcutis of an MRL/lpr mouse (Japan SLC, Inc.)
so as to carry out initial immunization. The second immunization
and the subsequent immunizations were carried out by mixing a
soluble CDH3 protein (protein amount: 25 .mu.g) that had been
prepared in the same manner as described above with Titer-MAX gold
and then injecting the obtained mixture into the abdominal cavity
and subcutis of the mouse. Three days after the final immunization,
splenic cells were aseptically prepared from the mouse, and the
splenic cells were then fused with mouse myeloma cells SP2/O-Ag14
or P3-X63-Ag8.653 according to an ordinary method (polyethylene
glycol method).
(2) Selection of Anti-CDH3 Antibody-Producing Hybridomas
[0101] An anti-CDH3 antibody was selected by flow cytometry using a
CHO cell line (EXZ1501) expressing full-length CDH3.
[0102] Specifically, the CHO cell line (EXZ1501) that expressed
full-length CDH3 was treated with 2 mM EDTA-PBS, so that it was
removed from the culture plate. Thereafter, the cells were
suspended in a FACS solution to a concentration of 1.times.10.sup.6
cells/mL. This cell suspension was inoculated on a 96-well plate to
a concentration of 50 .mu.L/well, and a culture supernatant of
hybridomas was then added thereto, so that they were reacted at
4.degree. C. for 60 minutes. Thereafter, the reaction solution was
washed with a FACS solution (200 .mu.L/well) two times, and
AlexaFluor 488-labeled anti-mouse IgG-goat F(ab').sub.2
(Invitrogen) was added to the resultant. Then, the mixture was
reacted at 4.degree. C. for 30 minutes. Thereafter, the reaction
solution was washed with a FACS solution two times, and it was then
subjected to flow cytometry, so as to select hybridomas that were
strongly reacted with the CDH3-expressing CHO cells.
[0103] The results of typical reactions of the antibody obtained
from the aforementioned hybridomas with CDH3-expressing CHO cells
(EXZ1501), with CHO cells as a parent cell line, and with cancer
cells NCI-H358 that had been confirmed to express CDH3 at a high
level, are shown in FIG. 4. All of the selected hybridomas were
confirmed to react with the CDH3-expressing CHO cells (EXZ1501) and
with the NCI-H358, but not to react with the CHO cells.
Example 4
Measurement of Antibody-Dependent Cellular Cytotoxic (ADCC)
Activity of Anti-CDH3 Antibody
[0104] ADCC activity was measured by a method comprising allowing
an antibody to act on radiolabeled target cells in the presence of
effector cells and then measuring the released radioactivity.
(1) Preparation of Effector Cells
[0105] Bone-marrow cells were collected from the femur of a C3H/HeJ
Jcl mouse (8-week-old, male, CLEA Japan, Inc.), and the cells were
then prepared to a concentration of 2.times.10.sup.6 cells/mL in a
10% FBS-containing RPMI1640 medium. Thereafter, the cells were
cultured for 6 days in the presence of 50 ng/mL human IL-2
(PEPROTECH) and 10 ng/mL mouse GM-CSF (PEPROTECH). On the day of
measurement, the cells were recovered and were then washed with a
10% FBS-containing HAM medium, so as to prepare an effector cell
solution.
(2) Preparation of Target Cells
[0106] As target cells, full-length CDH3-expressing CHO cells
(EXZ1501) were used. The cells were removed from a plate, and were
then suspended in a 10% FBS-containing HAM medium to a
concentration of 1.times.10.sup.7 cells/mL. Then, .sup.51Cr was
added to the suspension to a final concentration of 150 .mu.Ci. The
thus obtained mixture was cultured in a 5% CO.sub.2 incubator at
37.degree. C. for 1.5 hours. The resultant cells were washed with a
medium twice, and a 10% FBS-containing HAM medium was then added
thereto. Thereafter, the cells were inoculated on a 96-well
U-bottom plate (NUNC) to a concentration of 1.times.10.sup.4
cells/mL, so as to prepare target cells.
(3) Measurement of ADCC Activity
[0107] An antibody solution, which had been prepared to have a
concentration of each of 0.001, 0.01, 0.1 and 1 .mu.g/mL, was
dispensed in 50 .mu.L/well into the target cells. The obtained
mixture was incubated at room temperature for 15 minutes.
Thereafter, 100 .mu.L of the effector cells (1.times.10.sup.5
cells/well) was dispensed therein, and the obtained mixture was
then cultured in a CO.sub.2 incubator for 4 hours. Thereafter, a
culture supernatant was recovered, and radioactivity in 100 .mu.L
of the culture supernatant was measured with a scintillation
counter.
[0108] Cytotoxicity was obtained by the following formula.
Cytotoxicity (%)=(A-C)/(B-C).times.100 [0109] A: the radioactivity
value (cpm) of each antibody-added well [0110] B: the radioactivity
value (cpm) of a well, in which 100 .mu.L of a 2% NP40 solution and
50 .mu.L of a 10% FBS-containing RPMI medium have been added to the
target cells [0111] C: the radioactivity value (cpm) of a well, in
which 150 .mu.L of a 10% FBS-containing medium comprising the
effector cells has been added to the target cells
[0112] The test was carried out by measuring ADCC activity
according to a triplicate assay, and cytotoxicity (%) was
calculated based on the obtained mean value.
[0113] The test results are shown in Table 1 and FIG. 5. An
antibody group having particularly strong ADCC activity was found
among antibodies having ADCC activity. Antibodies wherein ADCC
activity at an antibody concentration of 1 .mu.g/mL was 30% or
more, were defined as a high ADCC activity group. Antibodies
wherein ADCC activity at the same antibody concentration was less
than 30%, were defined as a low ADCC activity group.
TABLE-US-00001 TABLE 1 Antibody concentration (.mu.g/mL) Antibody
Subtype 0.001 0.01 0.1 1 Evaluation* PPMX3 IgG1 6% 9% 17% 22% W
PPMX9 IgG1 16% 17% 17% 19% W PPMX11 IgG1 11% 15% 18% 23% W PPMX15
IgG1 12% 16% 16% 17% W R&D-104805 IgG1 10% 12% 16% 19% W
BD-610227 IgG1 11% 11% 10% 8% W PPMX1 IgG1 8% 11% 10% 16% W PPMX10
IgG1 11% 27% 40% 40% S PPMX13 IgG1 11% 19% 43% 45% S PPMX18 IgG1
13% 21% 43% 49% S PPMX14 IgG1 7% 20% 45% 51% S PPMX4 IgG1 8% 14%
35% 46% S PPMX5 IgG1 12% 13% 28% 40% S PPMX6 IgG1 10% 16% 35% 37% S
PPMX16 IgG1 8% 16% 35% 42% S PPMX17 IgG1 13% 31% 48% 45% S PPMX2
IgG2a 7% 8% 9% 11% W PPMX21 IgG2a 10% 11% 11% 14% W PPMX7 IgG2a 9%
11% 16% 18% W PPMX8 IgG2a 10% 14% 18% 26% W PPMX20 IgG2a 9% 6% 9%
10% W PPMX23 IgG2a 10% 10% 9% 12% W PPMX22 IgG2a 12% 11% 12% 17% W
PPMX12 IgG2a 11% 17% 36% 41% S PPMX19 IgG2b 8% 11% 15% 33% S
Negative Ab1 IgG1 10% 10% 9% 8% -- Negative Ab2 IgG2a 12% 13% 13%
11% -- R & D-104805 indicates a commercially available CDH3
antibody (R & D SYSTEMS). BD-610227 indicates another
commercially available CDH3 antibody (BD BIOSCIENCES). Negative Ab1
and Ab2 indicate antibodies that recognize antigens irrelevant to
CDH3. *S: high ADCC activity (30% or more at an antibody
concentration of 1 (.mu.g/mL) W: low ADCC activity (less than 30%
at an antibody concentration of 1 .mu.g/mL)
[0114] Hybridoma PPMX12 that produces antibody PPMX12 was deposited
under the terms of the Budapest Treaty with the International
Patent Organism Depositary, the National Institute of Technology
and Evaluation, an Independent Administrative Institution under the
Ministry of Economy, Trade and Industry (2-5-8, Kazusa Kamatari,
Kisarazu-shi, Chiba-ken, Japan, postal code: 292-0818), under
accession No. NITE BP-865 on Jan. 20, 2010.
Example 5
Classification of Epitopes of Anti-CDH3 Monoclonal Antibody with
Use of Partial-Length CDH3-Expressing Protein
[0115] The obtained anti-CDH3 antibody epitopes were classified by
a Western blot method with CDH3 partial sequence expression
products. As such CDH3 partial sequence expression products,
fragments 1 to 5 were designed, so that the sequences of the
fragments could be sufficiently overlapped (FIG. 6).
(1) Production of Expression Vector for Partial-Length CDH3
Protein
[0116] Using the full-length CDH3 cDNA of Example 1 as a template,
a PCR reaction was carried out employing the after-mentioned primer
sets. Using iProof High Fidelity DNA Polymerase (Bio-Rad), the
reaction was carried out under reaction conditions consisting of 35
cycles of 98.degree. C.-10 seconds, 60.degree. C.-10 seconds, and
72.degree. C.-30 seconds. Thereafter, gel containing a band with a
size near the size of interest was cut out in agarose gel
electrophoresis, and using QIA (registered trademark) quick Gel
Extraction Kit, a CDH3 cDNA fragment of interest was obtained.
[0117] In order to insert this CDH3 cDNA fragment into an
Escherichia coli expression vector pCold (registered trademark) TF
(Takara Bio Inc.), the fragment was treated with two types of
restriction enzymes KpnI and XbaI, and it was then inserted into
pCold TF treated with the same KpnI and XbaI according to an
ordinary method using T4 DNA ligase, so as to obtain an expression
vector for expressing each fragment.
[0118] Using the following primer sets, PCR reactions were carried
out, so as to obtain each fragment.
TABLE-US-00002 Fragment 1 (positions 108-236 of SEQ ID NO: 2)
Forward primer: (SEQ ID NO: 9)
TATGGAGCTCGGTACCGATTGGGTGGTTGCTCCAATATCTG Reverse primer: (SEQ ID
NO: 10) AGATTACCTATCTAGACTACTGCATCACAGAAGTACCTGGTAGG Fragment 2
(positions 132-348 of SEQ ID NO: 2) Forward primer: (SEQ ID NO: 11)
TATGGAGCTCGGTACCAAGTCTAATAAAGATAGAGACACCAAG Reverse primer: (SEQ ID
NO: 12) AGATTACCTATCTAGACTACCTCTGCACCTCATGGCCCACTGCATTCTCA Fragment
3 (positions 237-461 of SEQ ID NO: 2) Forward primer: (SEQ ID NO:
13) TATGGAGCTCGGTACCGTGACAGCCACGGATGAGGATGATG Reverse primer: (SEQ
ID NO: 14) AGATTACCTATCTAGACTAGACACACACAGGCTCCCCAGTG Fragment 4
(positions 349-550 of SEQ ID NO: 2) Forward primer: (SEQ ID NO: 15)
TATGGAGCTCGGTACCCTGACGGTCACTGATCTGGACG Reverse primer: (SEQ ID NO:
16) AGATTACCTATCTAGACTAGGGCTCAGGGACTGGGCCATGGTCATTG Fragment 5
(positions 462-654 of SEQ ID NO: 2) Forward primer: (SEQ ID NO: 17)
TATGGAGCTCGGTACCTACACTGCAGAAGACCCTGACAAGG Reverse primer: (SEQ ID
NO: 18) AGATTACCTATCTAGACTAACCTCCCTTCCAGGGTCCAGGGCAGGTTTCG
(2) Expression of Partial-Length CDH3 Protein
[0119] Using the expression vector of the CDH3 fragment described
in (1) above, Escherichia coli Rossetta (registered trademark) 2
(Merck) was transformed according to an ordinary method, and the
transformant was then cultured in a LB medium. When the absorbance
at 600 nm became 0.4, the culture product was cooled on ice for 30
minutes. Then, the concentration of
isopropyl-.beta.-thiogalactopyranoside (IPTG) was set at 0.5 mM,
and the cells was cultured at 20.degree. C. for 18 hours, and the
resultant was then recovered.
[0120] The expression of a partial-length CDH3 protein was
confirmed by electrophoresing the culture solution of the
Escherichia coli, subjecting the resultant to a Western blot method
using an anti-Penta-His antibody (QIAGEN), and then confirming the
presence of a band in a predicted position.
[0121] Specifically, an electrophorestic buffer was added to the
above-described Escherichia coli culture solution in an amount of
1/10 of the culture solution, and the thus mixed solution was then
charged to 5%-20% gradient gel (Bio-Rad) under reductive
conditions, followed by performing electrophoresis. Thereafter, the
resultant was transferred on Immobilon (registered trademark) P
membrane (Millipore). The transfer membrane was lightly washed with
TBS-T (0.05% Tween (registered trademark) 20, TBS), and it was then
shaken in 40% BSA-containing TBS for 1 hour. Thereafter, each
anti-CDH3 antibody that had been diluted with TBS-T containing 10%
Block Ace (registered trademark) (Snow Brand Milk Products Co.,
Ltd.) was added to the resultant, and the obtained mixture was then
shaken for 1 hour. Thereafter, the reaction product was washed with
BS-T, and a HRP-anti-mouse IgG antibody (GE Healthcare Biosciences)
diluted with 10% Block Ace-containing TBS-T was added thereto,
followed by shaking the obtained mixture for 1 hour. Subsequently,
the reaction product was washed with TBS-T. Using ECL (registered
trademark)-Plus (GE Healthcare Biosciences), color development was
detected with X-ray film RX-u (Fujifilm Corporation) in accordance
with the instructions provided by the manufacturer. The obtained
results are shown in FIG. 7.
(3) Classification of Antibody Epitopes Using CDH3 Partial Sequence
Expression Products
[0122] The above-described Escherichia coli lysate, in which each
partial-length CDH3 protein had been expressed, was charged to
5%-20% gradient gel (Bio-Rad) under reductive conditions, followed
by performing electrophoresis. Thereafter, using a blotting device
(Bio-Rad), the resultant was transferred on Immobilon P membrane
(Millipore). The transfer membrane was lightly washed with TBS-T
(0.05% Tween 20, TBS), and it was then shaken in 40% BSA-containing
TBS for 1 hour. Thereafter, the membrane was cut at equal intervals
in the form of straps, and each anti-CDH3 antibody that had been
diluted with 10% Block Ace-containing TBS-T was added thereto. The
obtained mixture was shaken for 1 hour. Thereafter, the reaction
product was washed with TBS-T, and a HRP-anti-mouse IgG antibody
(GE Healthcare Biosciences) diluted with 10% Block Ace-containing
TBS-T was added thereto, followed by shaking the obtained mixture
for 1 hour. Subsequently, the reaction product was washed with
TBS-T. Using ECL (registered trademark)-Plus (GE Healthcare
Biosciences), color development was detected with X-ray film RX-u
(Fujifilm Corporation) in accordance with the instructions provided
by the manufacturer. The obtained results are shown in FIG. 8.
[0123] Regions recognized by individual antibodies were determined
based on the reactivity with each partial-length CDH3 protein
(Table 2).
[0124] Correspondence relation with regions on the CDH3 sequence
shown in SEQ ID NO: 2 that are recognized by individual antibodies
is shown below. [0125] Upstream region of EC1: positions 108-131
[0126] EC1: positions 132-236 [0127] EC2: positions 237-348 [0128]
EC3: positions 349-461 [0129] EC4: positions 462-550 [0130] ECS:
positions 551-654
TABLE-US-00003 [0130] TABLE 2 Antibody concentration (.mu.g/mL)
Antibody Subtype 0.001 0.01 0.1 1 Evaluation* Recognized region
PPMX3 IgG1 6% 9% 17% 22% W Region upstream of EC1 PPMX9 IgG1 16%
17% 17% 19% W Region upstream of EC1 PPMX11 IgG1 11% 15% 18% 23% W
EC1 PPMX15 IgG1 12% 16% 16% 17% W Region upstream of EC1
R&D-104805 IgG1 10% 12% 16% 19% W Region upstream of EC1
BD-610227 IgG1 11% 11% 10% 8% W EC1 PPMX1 IgG1 8% 11% 10% 16% W EC3
PPMX10 IgG1 11% 27% 40% 40% S Region upstream of EC1 PPMX13 IgG1
11% 19% 43% 45% S Border between EC3 and EC4 PPMX18 IgG1 13% 21%
43% 49% S EC4 PPMX14 IgG1 7% 20% 45% 51% S EC5 PPMX4 IgG1 8% 14%
35% 46% S Region upstream of EC1 PPMX5 IgG1 12% 13% 28% 40% S
Region upstream of EC1 PPMX6 IgG1 10% 16% 35% 37% S Region upstream
of EC1, or EC5 PPMX16 IgG1 8% 16% 35% 42% S EC5 PPMX17 IgG1 13% 31%
48% 45% S EC5 PPMX2 IgG2a 7% 8% 9% 11% W Region upstream of EC1
PPMX21 IgG2a 10% 11% 11% 14% W Region upstream of EC1 PPMX7 IgG2a
9% 11% 16% 18% W EC1 PPMX8 IgG2a 10% 14% 18% 26% W EC1 PPMX20 IgG2a
9% 6% 9% 10% W EC2 PPMX23 IgG2a 10% 10% 9% 12% W Region upstream of
EC1, or EC3 PPMX22 IgG2a 12% 11% 12% 17% W EC4 PPMX12 IgG2a 11% 17%
36% 41% S Region upstream of EC1 PPMX19 IgG2b 8% 11% 15% 33% S EC4
Negative Ab1 IgG1 10% 10% 9% 8% -- -- Negative Ab2 IgG2a 12% 13%
13% 11% -- -- R & D-104805 indicates a commercially available
CDH3 antibody (R & D SYSTEMS). BD-610227 indicates another
commercially available CDH3 antibody (BD BIOSCIENCES). Negative Ab1
and Ab2 indicate antibodies that recognize antigens irrelevant to
CDH3. *S: high ADCC activity (30% or more at an antibody
concentration of 1 .mu.g/mL) W: low ADCC activity (less than 30% at
an antibody concentration of 1 .mu.g/mL)
Example 6
Determination of Anti-CDH3 Monoclonal Antibody Epitopes Using
Peptide Array
[0131] A peptide array (Replitope; manufactured by JPT Peptide
Technologies) was applied to antibody PPMX13 that was considered to
correspond to a boundary region in the above-described epitope
determination using a partial-length CDH3-expressing protein, so
that epitope determination was carried out more in detail.
[0132] Specifically, a region corresponding to the extracellular
region of CDH3 (which corresponds to positions 108-563 of SEQ ID
NO: 2) was shifted by every 13 residues from the N-terminus, while
each initial residue was shifted by every two amino acid residues
(that is, positions 108-120, 110-122, . . . and 551-563), so as to
synthesize peptides. The thus synthesized peptides were immobilized
on a glass slide, and were then blocked by SuperBlock (PIERCE). The
thus prepared product was used as a primary antibody, and it was
allowed to react with antibodies as targets of epitope searching.
The reaction product was washed with TBS-T three times, and
detection was then carried out using an anti-mouse antibody
(PIERCE) that had been fluorescently labeled with DyLight649. A
primary antibody that had not been allowed to react with the
antibody as a epitope searching target was used as a negative
control in the measurement. The measurement results are shown in
FIG. 9. Strong signals were observed in regions corresponding to
positions 446-472 and 490-504 of the amino acid sequence of CDH3
shown in SEQ ID NO: 2, and these were assumed to be epitopes of the
present antibody.
[0133] The correlation of ADCC activity in view of the results
regarding the regions recognized by the antibodies, which were
determined in Examples 5 and 6, was analyzed. As a result, it was
found that antibodies having high ADCC activity were concentrated
in an upstream region of EC1, the EC4 region and the EC5
region.
Example 7
Expression of CDH3 mRNA in Normal Tissues and Cancer Tissues
[0134] Samples were recovered from normal human tissues and various
types of cancer tissues according to a Lase Capture Microdissection
method. Total RNA was prepared from the recovered samples according
to a common method using ISOGEN (Nippon Gene Co., Ltd.). 10 ng each
of RNA was subjected to the analysis of gene expression using
GeneChip U-133B (Affimetrix) in accordance with Expression Analysis
Technical Manual (Affimetrix). The mean value of the expression
scores of all genes was set at 100, and genes whose expression was
increased in cancer cells were then searched. As a result, it was
found that CDH3 was highly expressed in lung cancer, colon cancer,
and pancreatic cancer (FIG. 10B). In addition, the expression of
CDH3 mRNA in various types of pancreatic cancer tissues having
different degrees of differentiation was studied. As a result,
there were found tissues in which CDH3 was highly expressed,
regardless of degree of differentiation (FIG. 10C).
Example 8
Expression of CDH3 Protein in Cancer Tissues by Immunohistochemical
Staining
[0135] In order to confirm the expression of a CDH3 protein in
cancer clinical samples, immunostaining was carried out using
cancer sample tissue arrays.
[0136] As such cancer sample tissue arrays, pancreatic cancer
(adenocarcinoma), lung cancer (adenocarcinoma), lung cancer
(squamous-cell carcinoma) and colon cancer (adenocarcinoma), which
were manufactured by Shanghai Outdo Biotech Co., Ltd.), were
used.
[0137] Each tissue array slide was deparaffinized, and it was then
activated with 10 mM Tris-1 mM EDTA (pH 9.0) at 95.degree. C. for
40 minutes. Using a blocking reagent included with ENVISION+ Kit
(Dako), endogenous peroxidase was inactivated, and the resultant
was then reacted with anti-CDH3 antibody 610227 (BD BIOSCIENCES)
and anti-HBs antibody Hyb-3423 used as a negative control, in a
concentration of 5 .mu.g/mL at 4.degree. C. overnight. After the
antibody solution had been washed out, the resultant was reacted
with a polymer secondary antibody reagent included with ENVISION+
Kit at room temperature for 30 minutes. Thereafter, color
development was carried out using a coloring reagent included with
ENVISION+ Kit, and nuclear staining was then carried out with a
hematoxylin eosin solution.
[0138] The results are shown in FIG. 11. Cancer cells were stained
with the anti-CDH3 antibody, and normal cells were not stained
therewith.
Example 9
Antitumor Effects in Xenograft Models
[0139] The antitumor effect of an anti-CDH3 antibody was confirmed
using xenografts, into which a human lung cancer-derived cell line
NCI-H358, a human skin cancer-derived cell line A431, and a human
pancreatic cancer-derived cell line PK-45P had been
transplanted.
[0140] NCI-H358 and PK-45P were cultured in a 10% FBS-containing
RPMI 1640 medium, whereas A431 was cultured in a 10% FBS-containing
DMEM medium. Thereafter, each cell line was transplanted into the
subcutis of the right ventral portion of each SCID mouse (female,
7-week-old, CLEA Japan), resulting in a concentration of
5.times.10.sup.6 cells/mouse.
[0141] NCI-H358-transplanted mice were divided into six groups
(n=8). A PPMX12-producing antibody was administered in a
concentration of 0.01 mg/kg, 0.06 mg/kg, 0.3 mg, kg, or 1.5 mg/kg
into the caudal vein of each mouse in each group. A RCB
1205-producing antibody (anti-pertussis toxin mouse IgG antibody)
used as a control was administered in a concentration of 7.5 mg/kg
into the caudal vein of each mouse in each group. Administration
was initiated at the time point in which the mean tumor diameter
became 90 mm.sup.3, and the aforementioned antibody was
administered twice a week (every 3 or 4 days) eight times in
total.
[0142] PK-45P-transplanted mice were divided into two groups (n=8).
A PPMX12-producing antibody was administered in a concentration of
7.5 mg/kg into the caudal vein of each mouse in each group. A RCB
1205-producing antibody (anti-pertussis toxin mouse IgG antibody)
used as a control was administered in a concentration of 7.5 mg/kg
into the caudal vein of each mouse in each group. Administration
was initiated at the time point in which the mean tumor diameter
became 120 mm.sup.3, and the aforementioned antibody was
administered twice a week (every 3 or 4 days) seven times in
total.
[0143] A431-transplanted mice were divided into two groups (n=8). A
PPMX12-producing antibody was administered in a concentration of
7.5 mg/kg into the caudal vein of each mouse in each group. A RCB
1205-producing antibody (anti-pertussis toxin mouse IgG antibody)
used as a control was administered in a concentration of 7.5 mg/kg
into the caudal vein of each mouse in each group. Administration
was initiated at the time point in which the mean tumor diameter
became 110 mm.sup.3, and the aforementioned antibody was
administered twice a week (every 3 or 4 days) six times in
total.
[0144] On the day of administration, a tumor size and a body weight
were measured. After completion of the final administration, the
mice were further observed for one week, and a body weight, a tumor
size, and a tumor weight were measured. The results of each type of
mouse were shown in FIGS. 12-14. The PPMX12-producing antibody
exhibited antitumor activity in all of the tests. In addition, it
was confirmed that antitumor effect was enhanced in a
dose-dependent manner in the test using the NCI-H358-transplanted
mice.
Sequence CWU 1
1
1812490DNAhumanCDS(1)..(2490) 1atg ggg ctc cct cgt gga cct ctc gcg
tct ctc ctc ctt ctc cag gtt 48Met Gly Leu Pro Arg Gly Pro Leu Ala
Ser Leu Leu Leu Leu Gln Val 1 5 10 15 tgc tgg ctg cag tgc gcg gcc
tcc gag ccg tgc cgg gcg gtc ttc agg 96Cys Trp Leu Gln Cys Ala Ala
Ser Glu Pro Cys Arg Ala Val Phe Arg 20 25 30 gag gct gaa gtg acc
ttg gag gcg gga ggc gcg gag cag gag ccc ggc 144Glu Ala Glu Val Thr
Leu Glu Ala Gly Gly Ala Glu Gln Glu Pro Gly 35 40 45 cag gcg ctg
ggg aaa gta ttc atg ggc tgc cct ggg caa gag cca gct 192Gln Ala Leu
Gly Lys Val Phe Met Gly Cys Pro Gly Gln Glu Pro Ala 50 55 60 ctg
ttt agc act gat aat gat gac ttc act gtg cgg aat ggc gag aca 240Leu
Phe Ser Thr Asp Asn Asp Asp Phe Thr Val Arg Asn Gly Glu Thr 65 70
75 80 gtc cag gaa aga agg tca ctg aag gaa agg aat cca ttg aag atc
ttc 288Val Gln Glu Arg Arg Ser Leu Lys Glu Arg Asn Pro Leu Lys Ile
Phe 85 90 95 cca tcc aaa cgt atc tta cga aga cac aag aga gat tgg
gtg gtt gct 336Pro Ser Lys Arg Ile Leu Arg Arg His Lys Arg Asp Trp
Val Val Ala 100 105 110 cca ata tct gtc cct gaa aat ggc aag ggt ccc
ttc ccc cag aga ctg 384Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro
Phe Pro Gln Arg Leu 115 120 125 aat cag ctc aag tct aat aaa gat aga
gac acc aag att ttc tac agc 432Asn Gln Leu Lys Ser Asn Lys Asp Arg
Asp Thr Lys Ile Phe Tyr Ser 130 135 140 atc acg ggg ccg ggg gca gac
agc ccc cct gag ggt gtc ttc gct gta 480Ile Thr Gly Pro Gly Ala Asp
Ser Pro Pro Glu Gly Val Phe Ala Val 145 150 155 160 gag aag gag aca
ggc tgg ttg ttg ttg aat aag cca ctg gac cgg gag 528Glu Lys Glu Thr
Gly Trp Leu Leu Leu Asn Lys Pro Leu Asp Arg Glu 165 170 175 gag att
gcc aag tat gag ctc ttt ggc cac gct gtg tca gag aat ggt 576Glu Ile
Ala Lys Tyr Glu Leu Phe Gly His Ala Val Ser Glu Asn Gly 180 185 190
gcc tca gtg gag gac ccc atg aac atc tcc atc atc gtg acc gac cag
624Ala Ser Val Glu Asp Pro Met Asn Ile Ser Ile Ile Val Thr Asp Gln
195 200 205 aat gac cac aag ccc aag ttt acc cag gac acc ttc cga ggg
agt gtc 672Asn Asp His Lys Pro Lys Phe Thr Gln Asp Thr Phe Arg Gly
Ser Val 210 215 220 tta gag gga gtc cta cca ggt act tct gtg atg cag
gtg aca gcc acg 720Leu Glu Gly Val Leu Pro Gly Thr Ser Val Met Gln
Val Thr Ala Thr 225 230 235 240 gat gag gat gat gcc atc tac acc tac
aat ggg gtg gtt gct tac tcc 768Asp Glu Asp Asp Ala Ile Tyr Thr Tyr
Asn Gly Val Val Ala Tyr Ser 245 250 255 atc cat agc caa gaa cca aag
gac cca cac gac ctc atg ttc acc att 816Ile His Ser Gln Glu Pro Lys
Asp Pro His Asp Leu Met Phe Thr Ile 260 265 270 cac cgg agc aca ggc
acc atc agc gtc atc tcc agt ggc ctg gac cgg 864His Arg Ser Thr Gly
Thr Ile Ser Val Ile Ser Ser Gly Leu Asp Arg 275 280 285 gaa aaa gtc
cct gag tac aca ctg acc atc cag gcc aca gac atg gat 912Glu Lys Val
Pro Glu Tyr Thr Leu Thr Ile Gln Ala Thr Asp Met Asp 290 295 300 ggg
gac ggc tcc acc acc acg gca gtg gca gta gtg gag atc ctt gat 960Gly
Asp Gly Ser Thr Thr Thr Ala Val Ala Val Val Glu Ile Leu Asp 305 310
315 320 gcc aat gac aat gct ccc atg ttt gac ccc cag aag tac gag gcc
cat 1008Ala Asn Asp Asn Ala Pro Met Phe Asp Pro Gln Lys Tyr Glu Ala
His 325 330 335 gtg cct gag aat gca gtg ggc cat gag gtg cag agg ctg
acg gtc act 1056Val Pro Glu Asn Ala Val Gly His Glu Val Gln Arg Leu
Thr Val Thr 340 345 350 gat ctg gac gcc ccc aac tca cca gcg tgg cgt
gcc acc tac ctt atc 1104Asp Leu Asp Ala Pro Asn Ser Pro Ala Trp Arg
Ala Thr Tyr Leu Ile 355 360 365 atg ggc ggt gac gac ggg gac cat ttt
acc atc acc acc cac cct gag 1152Met Gly Gly Asp Asp Gly Asp His Phe
Thr Ile Thr Thr His Pro Glu 370 375 380 agc aac cag ggc atc ctg aca
acc agg aag ggt ttg gat ttt gag gcc 1200Ser Asn Gln Gly Ile Leu Thr
Thr Arg Lys Gly Leu Asp Phe Glu Ala 385 390 395 400 aaa aac cag cac
acc ctg tac gtt gaa gtg acc aac gag gcc cct ttt 1248Lys Asn Gln His
Thr Leu Tyr Val Glu Val Thr Asn Glu Ala Pro Phe 405 410 415 gtg ctg
aag ctc cca acc tcc aca gcc acc ata gtg gtc cac gtg gag 1296Val Leu
Lys Leu Pro Thr Ser Thr Ala Thr Ile Val Val His Val Glu 420 425 430
gat gtg aat gag gca cct gtg ttt gtc cca ccc tcc aaa gtc gtt gag
1344Asp Val Asn Glu Ala Pro Val Phe Val Pro Pro Ser Lys Val Val Glu
435 440 445 gtc cag gag ggc atc ccc act ggg gag cct gtg tgt gtc tac
act gca 1392Val Gln Glu Gly Ile Pro Thr Gly Glu Pro Val Cys Val Tyr
Thr Ala 450 455 460 gaa gac cct gac aag gag aat caa aag atc agc tac
cgc atc ctg aga 1440Glu Asp Pro Asp Lys Glu Asn Gln Lys Ile Ser Tyr
Arg Ile Leu Arg 465 470 475 480 gac cca gca ggg tgg cta gcc atg gac
cca gac agt ggg cag gtc aca 1488Asp Pro Ala Gly Trp Leu Ala Met Asp
Pro Asp Ser Gly Gln Val Thr 485 490 495 gct gtg ggc acc ctc gac cgt
gag gat gag cag ttt gtg agg aac aac 1536Ala Val Gly Thr Leu Asp Arg
Glu Asp Glu Gln Phe Val Arg Asn Asn 500 505 510 atc tat gaa gtc atg
gtc ttg gcc atg gac aat gga agc cct ccc acc 1584Ile Tyr Glu Val Met
Val Leu Ala Met Asp Asn Gly Ser Pro Pro Thr 515 520 525 act ggc acg
gga acc ctt ctg cta aca ctg att gat gtc aat gac cat 1632Thr Gly Thr
Gly Thr Leu Leu Leu Thr Leu Ile Asp Val Asn Asp His 530 535 540 ggc
cca gtc cct gag ccc cgt cag atc acc atc tgc aac caa agc cct 1680Gly
Pro Val Pro Glu Pro Arg Gln Ile Thr Ile Cys Asn Gln Ser Pro 545 550
555 560 gtg cgc cag gtg ctg aac atc acg gac aag gac ctg tct ccc cac
acc 1728Val Arg Gln Val Leu Asn Ile Thr Asp Lys Asp Leu Ser Pro His
Thr 565 570 575 tcc cct ttc cag gcc cag ctc aca gat gac tca gac atc
tac tgg acg 1776Ser Pro Phe Gln Ala Gln Leu Thr Asp Asp Ser Asp Ile
Tyr Trp Thr 580 585 590 gca gag gtc aac gag gaa ggt gac aca gtg gtc
ttg tcc ctg aag aag 1824Ala Glu Val Asn Glu Glu Gly Asp Thr Val Val
Leu Ser Leu Lys Lys 595 600 605 ttc ctg aag cag gat aca tat gac gtg
cac ctt tct ctg tct gac cat 1872Phe Leu Lys Gln Asp Thr Tyr Asp Val
His Leu Ser Leu Ser Asp His 610 615 620 ggc aac aaa gag cag ctg acg
gtg atc agg gcc act gtg tgc gac tgc 1920Gly Asn Lys Glu Gln Leu Thr
Val Ile Arg Ala Thr Val Cys Asp Cys 625 630 635 640 cat ggc cat gtc
gaa acc tgc cct gga ccc tgg aag gga ggt ttc atc 1968His Gly His Val
Glu Thr Cys Pro Gly Pro Trp Lys Gly Gly Phe Ile 645 650 655 ctc cct
gtg ctg ggg gct gtc ctg gct ctg ctg ttc ctc ctg ctg gtg 2016Leu Pro
Val Leu Gly Ala Val Leu Ala Leu Leu Phe Leu Leu Leu Val 660 665 670
ctg ctt ttg ttg gtg aga aag aag cgg aag atc aag gag ccc ctc cta
2064Leu Leu Leu Leu Val Arg Lys Lys Arg Lys Ile Lys Glu Pro Leu Leu
675 680 685 ctc cca gaa gat gac acc cgt gac aac gtc ttc tac tat ggc
gaa gag 2112Leu Pro Glu Asp Asp Thr Arg Asp Asn Val Phe Tyr Tyr Gly
Glu Glu 690 695 700 ggg ggt ggc gaa gag gac cag gac tat gac atc acc
cag ctc cac cga 2160Gly Gly Gly Glu Glu Asp Gln Asp Tyr Asp Ile Thr
Gln Leu His Arg 705 710 715 720 ggt ctg gag gcc agg ccg gag gtg gtt
ctc cgc aat gac gtg gca cca 2208Gly Leu Glu Ala Arg Pro Glu Val Val
Leu Arg Asn Asp Val Ala Pro 725 730 735 acc atc atc ccg aca ccc atg
tac cgt cct cgg cca gcc aac cca gat 2256Thr Ile Ile Pro Thr Pro Met
Tyr Arg Pro Arg Pro Ala Asn Pro Asp 740 745 750 gaa atc ggc aac ttt
ata att gag aac ctg aag gcg gct aac aca gac 2304Glu Ile Gly Asn Phe
Ile Ile Glu Asn Leu Lys Ala Ala Asn Thr Asp 755 760 765 ccc aca gcc
ccg ccc tac gac acc ctc ttg gtg ttc gac tat gag ggc 2352Pro Thr Ala
Pro Pro Tyr Asp Thr Leu Leu Val Phe Asp Tyr Glu Gly 770 775 780 agc
ggc tcc gac gcc gcg tcc ctg agc tcc ctc acc tcc tcc gcc tcc 2400Ser
Gly Ser Asp Ala Ala Ser Leu Ser Ser Leu Thr Ser Ser Ala Ser 785 790
795 800 gac caa gac caa gat tac gat tat ctg aac gag tgg ggc agc cgc
ttc 2448Asp Gln Asp Gln Asp Tyr Asp Tyr Leu Asn Glu Trp Gly Ser Arg
Phe 805 810 815 aag aag ctg gca gac atg tac ggt ggc ggg gag gac gac
tag 2490Lys Lys Leu Ala Asp Met Tyr Gly Gly Gly Glu Asp Asp 820 825
2829PRThuman 2Met Gly Leu Pro Arg Gly Pro Leu Ala Ser Leu Leu Leu
Leu Gln Val 1 5 10 15 Cys Trp Leu Gln Cys Ala Ala Ser Glu Pro Cys
Arg Ala Val Phe Arg 20 25 30 Glu Ala Glu Val Thr Leu Glu Ala Gly
Gly Ala Glu Gln Glu Pro Gly 35 40 45 Gln Ala Leu Gly Lys Val Phe
Met Gly Cys Pro Gly Gln Glu Pro Ala 50 55 60 Leu Phe Ser Thr Asp
Asn Asp Asp Phe Thr Val Arg Asn Gly Glu Thr 65 70 75 80 Val Gln Glu
Arg Arg Ser Leu Lys Glu Arg Asn Pro Leu Lys Ile Phe 85 90 95 Pro
Ser Lys Arg Ile Leu Arg Arg His Lys Arg Asp Trp Val Val Ala 100 105
110 Pro Ile Ser Val Pro Glu Asn Gly Lys Gly Pro Phe Pro Gln Arg Leu
115 120 125 Asn Gln Leu Lys Ser Asn Lys Asp Arg Asp Thr Lys Ile Phe
Tyr Ser 130 135 140 Ile Thr Gly Pro Gly Ala Asp Ser Pro Pro Glu Gly
Val Phe Ala Val 145 150 155 160 Glu Lys Glu Thr Gly Trp Leu Leu Leu
Asn Lys Pro Leu Asp Arg Glu 165 170 175 Glu Ile Ala Lys Tyr Glu Leu
Phe Gly His Ala Val Ser Glu Asn Gly 180 185 190 Ala Ser Val Glu Asp
Pro Met Asn Ile Ser Ile Ile Val Thr Asp Gln 195 200 205 Asn Asp His
Lys Pro Lys Phe Thr Gln Asp Thr Phe Arg Gly Ser Val 210 215 220 Leu
Glu Gly Val Leu Pro Gly Thr Ser Val Met Gln Val Thr Ala Thr 225 230
235 240 Asp Glu Asp Asp Ala Ile Tyr Thr Tyr Asn Gly Val Val Ala Tyr
Ser 245 250 255 Ile His Ser Gln Glu Pro Lys Asp Pro His Asp Leu Met
Phe Thr Ile 260 265 270 His Arg Ser Thr Gly Thr Ile Ser Val Ile Ser
Ser Gly Leu Asp Arg 275 280 285 Glu Lys Val Pro Glu Tyr Thr Leu Thr
Ile Gln Ala Thr Asp Met Asp 290 295 300 Gly Asp Gly Ser Thr Thr Thr
Ala Val Ala Val Val Glu Ile Leu Asp 305 310 315 320 Ala Asn Asp Asn
Ala Pro Met Phe Asp Pro Gln Lys Tyr Glu Ala His 325 330 335 Val Pro
Glu Asn Ala Val Gly His Glu Val Gln Arg Leu Thr Val Thr 340 345 350
Asp Leu Asp Ala Pro Asn Ser Pro Ala Trp Arg Ala Thr Tyr Leu Ile 355
360 365 Met Gly Gly Asp Asp Gly Asp His Phe Thr Ile Thr Thr His Pro
Glu 370 375 380 Ser Asn Gln Gly Ile Leu Thr Thr Arg Lys Gly Leu Asp
Phe Glu Ala 385 390 395 400 Lys Asn Gln His Thr Leu Tyr Val Glu Val
Thr Asn Glu Ala Pro Phe 405 410 415 Val Leu Lys Leu Pro Thr Ser Thr
Ala Thr Ile Val Val His Val Glu 420 425 430 Asp Val Asn Glu Ala Pro
Val Phe Val Pro Pro Ser Lys Val Val Glu 435 440 445 Val Gln Glu Gly
Ile Pro Thr Gly Glu Pro Val Cys Val Tyr Thr Ala 450 455 460 Glu Asp
Pro Asp Lys Glu Asn Gln Lys Ile Ser Tyr Arg Ile Leu Arg 465 470 475
480 Asp Pro Ala Gly Trp Leu Ala Met Asp Pro Asp Ser Gly Gln Val Thr
485 490 495 Ala Val Gly Thr Leu Asp Arg Glu Asp Glu Gln Phe Val Arg
Asn Asn 500 505 510 Ile Tyr Glu Val Met Val Leu Ala Met Asp Asn Gly
Ser Pro Pro Thr 515 520 525 Thr Gly Thr Gly Thr Leu Leu Leu Thr Leu
Ile Asp Val Asn Asp His 530 535 540 Gly Pro Val Pro Glu Pro Arg Gln
Ile Thr Ile Cys Asn Gln Ser Pro 545 550 555 560 Val Arg Gln Val Leu
Asn Ile Thr Asp Lys Asp Leu Ser Pro His Thr 565 570 575 Ser Pro Phe
Gln Ala Gln Leu Thr Asp Asp Ser Asp Ile Tyr Trp Thr 580 585 590 Ala
Glu Val Asn Glu Glu Gly Asp Thr Val Val Leu Ser Leu Lys Lys 595 600
605 Phe Leu Lys Gln Asp Thr Tyr Asp Val His Leu Ser Leu Ser Asp His
610 615 620 Gly Asn Lys Glu Gln Leu Thr Val Ile Arg Ala Thr Val Cys
Asp Cys 625 630 635 640 His Gly His Val Glu Thr Cys Pro Gly Pro Trp
Lys Gly Gly Phe Ile 645 650 655 Leu Pro Val Leu Gly Ala Val Leu Ala
Leu Leu Phe Leu Leu Leu Val 660 665 670 Leu Leu Leu Leu Val Arg Lys
Lys Arg Lys Ile Lys Glu Pro Leu Leu 675 680 685 Leu Pro Glu Asp Asp
Thr Arg Asp Asn Val Phe Tyr Tyr Gly Glu Glu 690 695 700 Gly Gly Gly
Glu Glu Asp Gln Asp Tyr Asp Ile Thr Gln Leu His Arg 705 710 715 720
Gly Leu Glu Ala Arg Pro Glu Val Val Leu Arg Asn Asp Val Ala Pro 725
730 735 Thr Ile Ile Pro Thr Pro Met Tyr Arg Pro Arg Pro Ala Asn Pro
Asp 740 745 750 Glu Ile Gly Asn Phe Ile Ile Glu Asn Leu Lys Ala Ala
Asn Thr Asp 755 760 765 Pro Thr Ala Pro Pro Tyr Asp Thr Leu Leu Val
Phe Asp Tyr Glu Gly 770 775 780 Ser Gly Ser Asp Ala Ala Ser Leu Ser
Ser Leu Thr Ser Ser Ala Ser 785 790 795 800 Asp Gln Asp Gln Asp Tyr
Asp Tyr Leu Asn Glu Trp Gly Ser Arg Phe 805 810 815 Lys Lys Leu Ala
Asp Met Tyr Gly Gly Gly Glu Asp Asp 820 825 32040DNAhuman
3agtggcgtcg gaactgcaaa gcacctgtga gcttgcggaa gtcagttcag actccagccc
60gctccagccc ggcccgaccc gaccgcaccc ggcgcctgcc ctcgctcggc gtccccggcc
120agccatgggc ccttggagcc gcagcctctc ggcgctgctg ctgctgctgc
aggtctcctc 180ttggctctgc caggagccgg agccctgcca ccctggcttt
gacgccgaga
gctacacgtt 240cacggtgccc cggcgccacc tggagagagg ccgcgtcctg
ggcagagtga attttgaaga 300ttgcaccggt cgacaaagga cagcctattt
ttccctcgac acccgattca aagtgggcac 360agatggtgtg attacagtca
aaaggcctct acggtttcat aacccacaga tccatttctt 420ggtctacgcc
tgggactcca cctacagaaa gttttccacc aaagtcacgc tgaatacagt
480ggggcaccac caccgccccc cgccccatca ggcctccgtt tctggaatcc
aagcagaatt 540gctcacattt cccaactcct ctcctggcct cagaagacag
aagagagact gggttattcc 600tcccatcagc tgcccagaaa atgaaaaagg
cccatttcct aaaaacctgg ttcagatcaa 660atccaacaaa gacaaagaag
gcaaggtttt ctacagcatc actggccaag gagctgacac 720accccctgtt
ggtgtcttta ttattgaaag agaaacagga tggctgaagg tgacagagcc
780tctggataga gaacgcattg ccacatacac tctcttctct cacgctgtgt
catccaacgg 840gaatgcagtt gaggatccaa tggagatttt gatcacggta
accgatcaga atgacaacaa 900gcccgaattc acccaggagg tctttaaggg
gtctgtcatg gaaggtgctc ttccaggaac 960ctctgtgatg gaggtcacag
ccacagacgc ggacgatgat gtgaacacct acaatgccgc 1020catcgcttac
accatcctca gccaagatcc tgagctccct gacaaaaata tgttcaccat
1080taacaggaac acaggagtca tcagtgtggt caccactggg ctggaccgag
agagtttccc 1140tacgtatacc ctggtggttc aagctgctga ccttcaaggt
gaggggttaa gcacaacagc 1200aacagctgtg atcacagtca ctgacaccaa
cgataatcct ccgatcttca atcccaccac 1260gtacaagggt caggtgcctg
agaacgaggc taacgtcgta atcaccacac tgaaagtgac 1320tgatgctgat
gcccccaata ccccagcgtg ggaggctgta tacaccatat tgaatgatga
1380tggtggacaa tttgtcgtca ccacaaatcc agtgaacaac gatggcattt
tgaaaacagc 1440aaagggcttg gattttgagg ccaagcagca gtacattcta
cacgtagcag tgacgaatgt 1500ggtacctttt gaggtctctc tcaccacctc
cacagccacc gtcaccgtgg atgtgctgga 1560tgtgaatgaa gcccccatct
ttgtgcctcc tgaaaagaga gtggaagtgt ccgaggactt 1620tggcgtgggc
caggaaatca catcctacac tgcccaggag ccagacacat ttatggaaca
1680gaaaataaca tatcggattt ggagagacac tgccaactgg ctggagatta
atccggacac 1740tggtgccatt tccactcggg ctgagctgga cagggaggat
tttgagcacg tgaagaacag 1800cacgtacaca gccctaatca tagctacaga
caatggttct ccagttgcta ctggaacagg 1860gacacttctg ctgatcctgt
ctgatgtgaa tgacaacgcc cccataccag aacctcgaac 1920tatattcttc
tgtgagagga atccaaagcc tcaggtcata aacatcattg atgcagacct
1980tcctcccaat acatctccct tcacagcaga actaacacac ggggcgagtg
ccaactggac 20404882PRThuman 4Met Gly Pro Trp Ser Arg Ser Leu Ser
Ala Leu Leu Leu Leu Leu Gln 1 5 10 15 Val Ser Ser Trp Leu Cys Gln
Glu Pro Glu Pro Cys His Pro Gly Phe 20 25 30 Asp Ala Glu Ser Tyr
Thr Phe Thr Val Pro Arg Arg His Leu Glu Arg 35 40 45 Gly Arg Val
Leu Gly Arg Val Asn Phe Glu Asp Cys Thr Gly Arg Gln 50 55 60 Arg
Thr Ala Tyr Phe Ser Leu Asp Thr Arg Phe Lys Val Gly Thr Asp 65 70
75 80 Gly Val Ile Thr Val Lys Arg Pro Leu Arg Phe His Asn Pro Gln
Ile 85 90 95 His Phe Leu Val Tyr Ala Trp Asp Ser Thr Tyr Arg Lys
Phe Ser Thr 100 105 110 Lys Val Thr Leu Asn Thr Val Gly His His His
Arg Pro Pro Pro His 115 120 125 Gln Ala Ser Val Ser Gly Ile Gln Ala
Glu Leu Leu Thr Phe Pro Asn 130 135 140 Ser Ser Pro Gly Leu Arg Arg
Gln Lys Arg Asp Trp Val Ile Pro Pro 145 150 155 160 Ile Ser Cys Pro
Glu Asn Glu Lys Gly Pro Phe Pro Lys Asn Leu Val 165 170 175 Gln Ile
Lys Ser Asn Lys Asp Lys Glu Gly Lys Val Phe Tyr Ser Ile 180 185 190
Thr Gly Gln Gly Ala Asp Thr Pro Pro Val Gly Val Phe Ile Ile Glu 195
200 205 Arg Glu Thr Gly Trp Leu Lys Val Thr Glu Pro Leu Asp Arg Glu
Arg 210 215 220 Ile Ala Thr Tyr Thr Leu Phe Ser His Ala Val Ser Ser
Asn Gly Asn 225 230 235 240 Ala Val Glu Asp Pro Met Glu Ile Leu Ile
Thr Val Thr Asp Gln Asn 245 250 255 Asp Asn Lys Pro Glu Phe Thr Gln
Glu Val Phe Lys Gly Ser Val Met 260 265 270 Glu Gly Ala Leu Pro Gly
Thr Ser Val Met Glu Val Thr Ala Thr Asp 275 280 285 Ala Asp Asp Asp
Val Asn Thr Tyr Asn Ala Ala Ile Ala Tyr Thr Ile 290 295 300 Leu Ser
Gln Asp Pro Glu Leu Pro Asp Lys Asn Met Phe Thr Ile Asn 305 310 315
320 Arg Asn Thr Gly Val Ile Ser Val Val Thr Thr Gly Leu Asp Arg Glu
325 330 335 Ser Phe Pro Thr Tyr Thr Leu Val Val Gln Ala Ala Asp Leu
Gln Gly 340 345 350 Glu Gly Leu Ser Thr Thr Ala Thr Ala Val Ile Thr
Val Thr Asp Thr 355 360 365 Asn Asp Asn Pro Pro Ile Phe Asn Pro Thr
Thr Tyr Lys Gly Gln Val 370 375 380 Pro Glu Asn Glu Ala Asn Val Val
Ile Thr Thr Leu Lys Val Thr Asp 385 390 395 400 Ala Asp Ala Pro Asn
Thr Pro Ala Trp Glu Ala Val Tyr Thr Ile Leu 405 410 415 Asn Asp Asp
Gly Gly Gln Phe Val Val Thr Thr Asn Pro Val Asn Asn 420 425 430 Asp
Gly Ile Leu Lys Thr Ala Lys Gly Leu Asp Phe Glu Ala Lys Gln 435 440
445 Gln Tyr Ile Leu His Val Ala Val Thr Asn Val Val Pro Phe Glu Val
450 455 460 Ser Leu Thr Thr Ser Thr Ala Thr Val Thr Val Asp Val Leu
Asp Val 465 470 475 480 Asn Glu Ala Pro Ile Phe Val Pro Pro Glu Lys
Arg Val Glu Val Ser 485 490 495 Glu Asp Phe Gly Val Gly Gln Glu Ile
Thr Ser Tyr Thr Ala Gln Glu 500 505 510 Pro Asp Thr Phe Met Glu Gln
Lys Ile Thr Tyr Arg Ile Trp Arg Asp 515 520 525 Thr Ala Asn Trp Leu
Glu Ile Asn Pro Asp Thr Gly Ala Ile Ser Thr 530 535 540 Arg Ala Glu
Leu Asp Arg Glu Asp Phe Glu His Val Lys Asn Ser Thr 545 550 555 560
Tyr Thr Ala Leu Ile Ile Ala Thr Asp Asn Gly Ser Pro Val Ala Thr 565
570 575 Gly Thr Gly Thr Leu Leu Leu Ile Leu Ser Asp Val Asn Asp Asn
Ala 580 585 590 Pro Ile Pro Glu Pro Arg Thr Ile Phe Phe Cys Glu Arg
Asn Pro Lys 595 600 605 Pro Gln Val Ile Asn Ile Ile Asp Ala Asp Leu
Pro Pro Asn Thr Ser 610 615 620 Pro Phe Thr Ala Glu Leu Thr His Gly
Ala Ser Ala Asn Trp Thr Ile 625 630 635 640 Gln Tyr Asn Asp Pro Thr
Gln Glu Ser Ile Ile Leu Lys Pro Lys Met 645 650 655 Ala Leu Glu Val
Gly Asp Tyr Lys Ile Asn Leu Lys Leu Met Asp Asn 660 665 670 Gln Asn
Lys Asp Gln Val Thr Thr Leu Glu Val Ser Val Cys Asp Cys 675 680 685
Glu Gly Ala Ala Gly Val Cys Arg Lys Ala Gln Pro Val Glu Ala Gly 690
695 700 Leu Gln Ile Pro Ala Ile Leu Gly Ile Leu Gly Gly Ile Leu Ala
Leu 705 710 715 720 Leu Ile Leu Ile Leu Leu Leu Leu Leu Phe Leu Arg
Arg Arg Ala Val 725 730 735 Val Lys Glu Pro Leu Leu Pro Pro Glu Asp
Asp Thr Arg Asp Asn Val 740 745 750 Tyr Tyr Tyr Asp Glu Glu Gly Gly
Gly Glu Glu Asp Gln Asp Phe Asp 755 760 765 Leu Ser Gln Leu His Arg
Gly Leu Asp Ala Arg Pro Glu Val Thr Arg 770 775 780 Asn Asp Val Ala
Pro Thr Leu Met Ser Val Pro Arg Tyr Leu Pro Arg 785 790 795 800 Pro
Ala Asn Pro Asp Glu Ile Gly Asn Phe Ile Asp Glu Asn Leu Lys 805 810
815 Ala Ala Asp Thr Asp Pro Thr Ala Pro Pro Tyr Asp Ser Leu Leu Val
820 825 830 Phe Asp Tyr Glu Gly Ser Gly Ser Glu Ala Ala Ser Leu Ser
Ser Leu 835 840 845 Asn Ser Ser Glu Ser Asp Lys Asp Gln Asp Tyr Asp
Tyr Leu Asn Glu 850 855 860 Trp Gly Asn Arg Phe Lys Lys Leu Ala Asp
Met Tyr Gly Gly Gly Glu 865 870 875 880 Asp Asp 54380DNAhuman
5ggggagcgcc atccgctcca cttccacctc cacatcctcc accggccaag gtccccgccg
60ctgcatccct cgcggcttcc gctgcgctcc gggccggagc cgagccgcct gcgctgccac
120agcagccgcc tccacacact cgcagacgct cacacgctct ccctccctgt
tcccccgccc 180cctccccagc tccttgatct ctgggtctgt tttattactc
ctggtgcgag tcccgcggac 240tccgcggccc gctatttgtc atcagctcgc
tctccattgg cggggagcgg agagcagcga 300agaagggggt ggggagggga
ggggaaggga agggggtgga aactgcctgg agccgtttct 360ccgcgccgct
gttggtgctg ccgctgcctc ctcctcctcc gccgccgccg ccgccgccgc
420cgcctcctcc ggctcttcgc tcggcccctc tccgcctcca tgtgccggat
agcgggagcg 480ctgcggaccc tgctgccgct gctggcggcc ctgcttcagg
cgtctgtaga ggcttctggt 540gaaatcgcat tatgcaagac tggatttcct
gaagatgttt acagtgcagt cttatcgaag 600gatgtgcatg aaggacagcc
tcttctcaat gtgaagttta gcaactgcaa tggaaaaaga 660aaagtacaat
atgagagcag tgagcctgca gattttaagg tggatgaaga tggcatggtg
720tatgccgtga gaagctttcc actctcttct gagcatgcca agttcctgat
atatgcccaa 780gacaaagaga cccaggaaaa gtggcaagtg gcagtaaaat
tgagcctgaa gccaacctta 840actgaggagt cagtgaagga gtcagcagaa
gttgaagaaa tagtgttccc aagacaattc 900agtaagcaca gtggccacct
acaaaggcag aagagagact gggtcatccc tccaatcaac 960ttgccagaaa
actccagggg accttttcct caagagcttg tcaggatcag gtctgataga
1020gataaaaacc tttcactgcg gtacagtgta actgggccag gagctgacca
gcctccaact 1080ggtatcttca ttatcaaccc catctcgggt cagctgtcgg
tgacaaagcc cctggatcgc 1140gagcagatag cccggtttca tttgagggca
catgcagtag atattaatgg aaatcaagtg 1200gagaacccca ttgacattgt
catcaatgtt attgacatga atgacaacag acctgagttc 1260ttacaccagg
tttggaatgg gacagttcct gagggatcaa agcctggaac atatgtgatg
1320accgtaacag caattgatgc tgacgatccc aatgccctca atgggatgtt
gaggtacaga 1380atcgtgtctc aggctccaag caccccttca cccaacatgt
ttacaatcaa caatgagact 1440ggtgacatca tcacagtggc agctggactt
gatcgagaaa aagtgcaaca gtatacgtta 1500ataattcaag ctacagacat
ggaaggcaat cccacatatg gcctttcaaa cacagccacg 1560gccgtcatca
cagtgacaga tgtcaatgac aatcctccag agtttactgc catgacgttt
1620tatggtgaag ttcctgagaa cagggtagac atcatagtag ctaatctaac
tgtgaccgat 1680aaggatcaac cccatacacc agcctggaac gcagtgtaca
gaatcagtgg cggagatcct 1740actggacggt tcgccatcca gaccgaccca
aacagcaacg acgggttagt caccgtggtc 1800aaaccaatcg actttgaaac
aaataggatg tttgtcctta ctgttgctgc agaaaatcaa 1860gtgccattag
ccaagggaat tcagcacccg cctcagtcaa ctgcaaccgt gtctgttaca
1920gttattgacg taaatgaaaa cccttatttt gcccccaatc ctaagatcat
tcgccaagaa 1980gaagggcttc atgccggtac catgttgaca acattcactg
ctcaggaccc agatcgatat 2040atgcagcaaa atattagata cactaaatta
tctgatcctg ccaattggct aaaaatagat 2100cctgtgaatg gacaaataac
tacaattgct gttttggacc gagaatcacc aaatgtgaaa 2160aacaatatat
ataatgctac tttccttgct tctgacaatg gaattcctcc tatgagtgga
2220acaggaacgc tgcagatcta tttacttgat attaatgaca atgcccctca
agtgttacct 2280caagaggcag agacttgcga aactccagac cccaattcaa
ttaatattac agcacttgat 2340tatgacattg atccaaatgc tggaccattt
gcttttgatc ttcctttatc tccagtgact 2400attaagagaa attggaccat
cactcggctt aatggtgatt ttgctcagct taatttaaag 2460ataaaatttc
ttgaagctgg tatctatgaa gttcccatca taatcacaga ttcgggtaat
2520cctcccaaat caaatatttc catcctgcgc gtgaaggttt gccagtgtga
ctccaacggg 2580gactgcacag atgtggacag gattgtgggt gcggggcttg
gcaccggtgc catcattgcc 2640atcctgctct gcatcatcat cctgcttatc
cttgtgctga tgtttgtggt atggatgaaa 2700cgccgggata aagaacgcca
ggccaaacaa cttttaattg atccagaaga tgatgtaaga 2760gataatattt
taaaatatga tgaagaaggt ggaggagaag aagaccagga ctatgacttg
2820agccagctgc agcagcctga cactgtggag cctgatgcca tcaagcctgt
gggaatccga 2880cgaatggatg aaagacccat ccacgccgag ccccagtatc
cggtccgatc tgcagcccca 2940caccctggag acattgggga cttcattaat
gagggcctta aagcggctga caatgacccc 3000acagctccac catatgactc
cctgttagtg tttgactatg aaggcagtgg ctccactgct 3060gggtccttga
gctcccttaa ttcctcaagt agtggtggtg agcaggacta tgattacctg
3120aacgactggg ggccacggtt caagaaactt gctgacatgt atggtggagg
tgatgactga 3180acttcagggt gaacttggtt tttggacaag tacaaacaat
ttcaactgat attcccaaaa 3240agcattcaga agctaggctt taactttgta
gtctactagc acagtgcttg ctggaggctt 3300tggcataggc tgcaaaccaa
tttgggctca gagggaatat cagtgatcca tactgtttgg 3360aaaaacactg
agctcagtta cacttgaatt ttacagtaca gaagcactgg gattttatgt
3420gcctttttgt acctttttca gattggaatt agttttctgt ttaaggcttt
aatggtactg 3480atttctgaaa cgataagtaa aagacaaaat attttgtggt
gggagcagta agttaaacca 3540tgatatgctt caacacgctt ttgttacatt
gcatttgctt ttattaaaat acaaaattaa 3600acaaacaaaa aaactcatgg
agcgatttta ttatcttggg ggatgagacc atgagattgg 3660aaaatgtaca
ttacttctag ttttagactt tagtttgttt tttttttttt cactaaaatc
3720ttaaaactta ctcagctggt tgcaaataaa gggagttttc atatcaccaa
tttgtagcaa 3780aattgaattt tttcataaac tagaatgtta gacacatttt
ggtcttaatc catgtacact 3840tttttatttc tgtatttttc cacttcactg
taaaaatagt atgtgtacat aatgttttat 3900tggcatagtc tatggagaag
tgcagaaact tcagaacatg tgtatgtatt atttggacta 3960tggattcagg
ttttttgcat gtttatatct ttcgttatgg ataaagtatt tacaaaacag
4020tgacatttga ttcaattgtt gagctgtagt tagaatactc aatttttaat
ttttttaatt 4080tttttatttt ttattttctt tttggtttgg ggagggagaa
aagttcttag cacaaatgtt 4140ttacataatt tgtaccaaaa aaaaaaaaaa
aggaaaggaa agaaaggggt ggcctgacac 4200tggtggcact actaagtgtg
tgttttttta aaaaaaaaat ggaaaaaaaa aagcttttaa 4260actggagaga
cttctgacaa cagctttgcc tctgtattgt gtaccagaat ataaatgata
4320cacctctgac cccagcgttc tgaataaaat gctaattttg gatctggaaa
aaaaaaaaaa 43806906PRThuman 6Met Cys Arg Ile Ala Gly Ala Leu Arg
Thr Leu Leu Pro Leu Leu Ala 1 5 10 15 Ala Leu Leu Gln Ala Ser Val
Glu Ala Ser Gly Glu Ile Ala Leu Cys 20 25 30 Lys Thr Gly Phe Pro
Glu Asp Val Tyr Ser Ala Val Leu Ser Lys Asp 35 40 45 Val His Glu
Gly Gln Pro Leu Leu Asn Val Lys Phe Ser Asn Cys Asn 50 55 60 Gly
Lys Arg Lys Val Gln Tyr Glu Ser Ser Glu Pro Ala Asp Phe Lys 65 70
75 80 Val Asp Glu Asp Gly Met Val Tyr Ala Val Arg Ser Phe Pro Leu
Ser 85 90 95 Ser Glu His Ala Lys Phe Leu Ile Tyr Ala Gln Asp Lys
Glu Thr Gln 100 105 110 Glu Lys Trp Gln Val Ala Val Lys Leu Ser Leu
Lys Pro Thr Leu Thr 115 120 125 Glu Glu Ser Val Lys Glu Ser Ala Glu
Val Glu Glu Ile Val Phe Pro 130 135 140 Arg Gln Phe Ser Lys His Ser
Gly His Leu Gln Arg Gln Lys Arg Asp 145 150 155 160 Trp Val Ile Pro
Pro Ile Asn Leu Pro Glu Asn Ser Arg Gly Pro Phe 165 170 175 Pro Gln
Glu Leu Val Arg Ile Arg Ser Asp Arg Asp Lys Asn Leu Ser 180 185 190
Leu Arg Tyr Ser Val Thr Gly Pro Gly Ala Asp Gln Pro Pro Thr Gly 195
200 205 Ile Phe Ile Ile Asn Pro Ile Ser Gly Gln Leu Ser Val Thr Lys
Pro 210 215 220 Leu Asp Arg Glu Gln Ile Ala Arg Phe His Leu Arg Ala
His Ala Val 225 230 235 240 Asp Ile Asn Gly Asn Gln Val Glu Asn Pro
Ile Asp Ile Val Ile Asn 245 250 255 Val Ile Asp Met Asn Asp Asn Arg
Pro Glu Phe Leu His Gln Val Trp 260 265 270 Asn Gly Thr Val Pro Glu
Gly Ser Lys Pro Gly Thr Tyr Val Met Thr 275 280 285 Val Thr Ala Ile
Asp Ala Asp Asp Pro Asn Ala Leu Asn Gly Met Leu 290 295 300 Arg Tyr
Arg Ile Val Ser Gln Ala Pro Ser Thr Pro Ser Pro Asn Met 305 310 315
320 Phe Thr Ile Asn Asn Glu Thr Gly Asp Ile Ile Thr Val Ala Ala Gly
325 330 335 Leu Asp Arg Glu Lys Val Gln Gln Tyr Thr Leu Ile Ile Gln
Ala Thr 340 345 350 Asp Met Glu Gly Asn Pro Thr Tyr Gly Leu Ser Asn
Thr Ala Thr Ala 355 360 365 Val Ile Thr Val Thr Asp Val Asn Asp Asn
Pro Pro Glu Phe Thr Ala 370 375 380 Met Thr Phe Tyr Gly Glu Val Pro
Glu Asn Arg Val Asp Ile Ile Val 385 390 395 400 Ala Asn Leu Thr Val
Thr Asp Lys Asp Gln Pro His Thr Pro Ala Trp 405 410 415 Asn Ala Val
Tyr Arg Ile Ser Gly Gly Asp Pro Thr Gly Arg Phe Ala 420 425 430 Ile
Gln Thr Asp Pro Asn Ser Asn Asp Gly Leu Val Thr Val Val Lys 435 440
445 Pro Ile Asp Phe Glu Thr Asn Arg Met Phe Val Leu Thr Val Ala Ala
450 455 460 Glu Asn Gln Val Pro Leu Ala Lys Gly Ile Gln His Pro Pro
Gln Ser 465
470 475 480 Thr Ala Thr Val Ser Val Thr Val Ile Asp Val Asn Glu Asn
Pro Tyr 485 490 495 Phe Ala Pro Asn Pro Lys Ile Ile Arg Gln Glu Glu
Gly Leu His Ala 500 505 510 Gly Thr Met Leu Thr Thr Phe Thr Ala Gln
Asp Pro Asp Arg Tyr Met 515 520 525 Gln Gln Asn Ile Arg Tyr Thr Lys
Leu Ser Asp Pro Ala Asn Trp Leu 530 535 540 Lys Ile Asp Pro Val Asn
Gly Gln Ile Thr Thr Ile Ala Val Leu Asp 545 550 555 560 Arg Glu Ser
Pro Asn Val Lys Asn Asn Ile Tyr Asn Ala Thr Phe Leu 565 570 575 Ala
Ser Asp Asn Gly Ile Pro Pro Met Ser Gly Thr Gly Thr Leu Gln 580 585
590 Ile Tyr Leu Leu Asp Ile Asn Asp Asn Ala Pro Gln Val Leu Pro Gln
595 600 605 Glu Ala Glu Thr Cys Glu Thr Pro Asp Pro Asn Ser Ile Asn
Ile Thr 610 615 620 Ala Leu Asp Tyr Asp Ile Asp Pro Asn Ala Gly Pro
Phe Ala Phe Asp 625 630 635 640 Leu Pro Leu Ser Pro Val Thr Ile Lys
Arg Asn Trp Thr Ile Thr Arg 645 650 655 Leu Asn Gly Asp Phe Ala Gln
Leu Asn Leu Lys Ile Lys Phe Leu Glu 660 665 670 Ala Gly Ile Tyr Glu
Val Pro Ile Ile Ile Thr Asp Ser Gly Asn Pro 675 680 685 Pro Lys Ser
Asn Ile Ser Ile Leu Arg Val Lys Val Cys Gln Cys Asp 690 695 700 Ser
Asn Gly Asp Cys Thr Asp Val Asp Arg Ile Val Gly Ala Gly Leu 705 710
715 720 Gly Thr Gly Ala Ile Ile Ala Ile Leu Leu Cys Ile Ile Ile Leu
Leu 725 730 735 Ile Leu Val Leu Met Phe Val Val Trp Met Lys Arg Arg
Asp Lys Glu 740 745 750 Arg Gln Ala Lys Gln Leu Leu Ile Asp Pro Glu
Asp Asp Val Arg Asp 755 760 765 Asn Ile Leu Lys Tyr Asp Glu Glu Gly
Gly Gly Glu Glu Asp Gln Asp 770 775 780 Tyr Asp Leu Ser Gln Leu Gln
Gln Pro Asp Thr Val Glu Pro Asp Ala 785 790 795 800 Ile Lys Pro Val
Gly Ile Arg Arg Met Asp Glu Arg Pro Ile His Ala 805 810 815 Glu Pro
Gln Tyr Pro Val Arg Ser Ala Ala Pro His Pro Gly Asp Ile 820 825 830
Gly Asp Phe Ile Asn Glu Gly Leu Lys Ala Ala Asp Asn Asp Pro Thr 835
840 845 Ala Pro Pro Tyr Asp Ser Leu Leu Val Phe Asp Tyr Glu Gly Ser
Gly 850 855 860 Ser Thr Ala Gly Ser Leu Ser Ser Leu Asn Ser Ser Ser
Ser Gly Gly 865 870 875 880 Glu Gln Asp Tyr Asp Tyr Leu Asn Asp Trp
Gly Pro Arg Phe Lys Lys 885 890 895 Leu Ala Asp Met Tyr Gly Gly Gly
Asp Asp 900 905 724DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 7cgcggtacca tggggctccc tcgt
24829DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 8ccgtctagat aacctccctt ccagggtcc 29941DNAArtificial
SequenceDescription of Artificial Sequence Synthetic DNA
9tatggagctc ggtaccgatt gggtggttgc tccaatatct g 411044DNAArtificial
SequenceDescription of Artificial Sequence Synthetic DNA
10agattaccta tctagactac tgcatcacag aagtacctgg tagg
441143DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 11tatggagctc ggtaccaagt ctaataaaga tagagacacc aag
431250DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 12agattaccta tctagactac ctctgcacct catggcccac
tgcattctca 501341DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 13tatggagctc ggtaccgtga cagccacgga
tgaggatgat g 411441DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 14agattaccta tctagactag acacacacag
gctccccagt g 411538DNAArtificial SequenceDescription of Artificial
Sequence Synthetic DNA 15tatggagctc ggtaccctga cggtcactga tctggacg
381647DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 16agattaccta tctagactag ggctcaggga ctgggccatg gtcattg
471741DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 17tatggagctc ggtacctaca ctgcagaaga ccctgacaag g
411850DNAArtificial SequenceDescription of Artificial Sequence
Synthetic DNA 18agattaccta tctagactaa cctcccttcc agggtccagg
gcaggtttcg 50
* * * * *