U.S. patent application number 14/307589 was filed with the patent office on 2014-11-06 for radioactive metal-labeled anti-cadherin antibody.
This patent application is currently assigned to Fujifilm RI Pharma Co., LTD. The applicant listed for this patent is Akihiro HINO, Tadasi Matsuura, Katsuyuki Mitomo, Akio Nagano, Fumiko Nomura, Hirokazu Satoh, Masahiko Watanabe. Invention is credited to Akihiro HINO, Tadasi Matsuura, Katsuyuki Mitomo, Akio Nagano, Fumiko Nomura, Hirokazu Satoh, Masahiko Watanabe.
Application Number | 20140328754 14/307589 |
Document ID | / |
Family ID | 44367795 |
Filed Date | 2014-11-06 |
United States Patent
Application |
20140328754 |
Kind Code |
A1 |
HINO; Akihiro ; et
al. |
November 6, 2014 |
RADIOACTIVE METAL-LABELED ANTI-CADHERIN ANTIBODY
Abstract
A radioactive metal-labeled anti-cadherin antibody which is
obtained by binding a radioactive metallic element to an
anti-cadherin antibody via a metal-chelating reagent.
Inventors: |
HINO; Akihiro; (Sammu-shi,
JP) ; Nagano; Akio; (Sammu-shi, JP) ;
Watanabe; Masahiko; (Sammu-shi, JP) ; Matsuura;
Tadasi; (Tokyo, JP) ; Satoh; Hirokazu; (Tokyo,
JP) ; Nomura; Fumiko; (Tokyo, JP) ; Mitomo;
Katsuyuki; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HINO; Akihiro
Nagano; Akio
Watanabe; Masahiko
Matsuura; Tadasi
Satoh; Hirokazu
Nomura; Fumiko
Mitomo; Katsuyuki |
Sammu-shi
Sammu-shi
Sammu-shi
Tokyo
Tokyo
Tokyo
Tokyo |
|
JP
JP
JP
JP
JP
JP
JP |
|
|
Assignee: |
Fujifilm RI Pharma Co., LTD
Tokyo
JP
Perseus Proteomics Inc.
Tokyo
JP
|
Family ID: |
44367795 |
Appl. No.: |
14/307589 |
Filed: |
June 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13578462 |
Aug 10, 2012 |
8815211 |
|
|
PCT/JP2011/052759 |
Feb 9, 2011 |
|
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14307589 |
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Current U.S.
Class: |
424/1.53 ;
530/387.3; 530/387.9 |
Current CPC
Class: |
C07K 16/18 20130101;
A61K 51/1093 20130101; C07K 16/28 20130101; A61P 35/00 20180101;
A61K 51/1096 20130101; C12N 15/02 20130101; C07K 16/46 20130101;
A61K 2039/505 20130101; A61K 51/1027 20130101; C12N 15/85 20130101;
C07K 2317/24 20130101 |
Class at
Publication: |
424/1.53 ;
530/387.9; 530/387.3 |
International
Class: |
A61K 51/10 20060101
A61K051/10 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2010 |
JP |
2010-028028 |
Claims
1: A radioactive metal-labeled anti-cadherin antibody, which is
obtained by binding a radioactive metallic element to an
anti-cadherin antibody via a metal-chelating reagent, wherein a
mole ratio of the anti-p-cadherin antibody to the metal-chelating
reagent is from 1:0.1 to 1:4.5.
2: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the mole ratio of the anti-p-cadherin antibody to the
metal-chelating reagent is from 1:0.5 to 1:3.
3: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the anti-p-cadherin antibody is bonded to a region 1 to
655 of SEQ ID NO:2.
4: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the metal-chelating reagent is selected from the group
consisting of isothiocyanobenzyl DOTA, methylisothiocyanobenzyl
DTPA and cyclohexylisothiocyanobenzyl DTPA.
5: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the anti-p-cadherin antibody is a monoclonal antibody, a
recombinant antibody, a chimeric antibody thereof, a humanized
antibody, or a fragment thereof.
6: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the radioactive metallic element is cytotoxic
radioactive metal suitable for treating a p-cadherin expressing
cancer.
7: The radioactive metal-labeled anti-p-cadherin antibody of claim
6, wherein the cytotoxic radioactive metal is selected from the
group consisting of yttrium-90 (.sup.90Y), rhenium-186
(.sup.186Re), rhenium-188 (.sup.188Re), copper-67 (.sup.67Cu),
iron-59 (.sup.59Fe), strontium-89 (.sup.89Sr), gold-198
(.sup.198Au), dysprosium-165 (.sup.165Dy), ruthenium-103
(.sup.103Ru), holmium-166 (.sup.166Ho), samarium-153 (.sup.153Sm),
and lutetium-177 (.sup.177Lu).
8: The radioactive metal-labeled anti-p-cadherin antibody of claim
6, wherein the cytotoxic radioactive metal is yttrium-90
(.sup.90Y).
9: The radioactive metal-labeled anti-p-cadherin antibody of claim
1, wherein the radioactive metallic element is non-cytotoxic
radioactive metal suitable for diagnosing a p-cadherin expressing
cancer.
10: The radioactive metal-labeled anti-p-cadherin antibody of claim
9, wherein the non-cytotoxic radioactive metal is selected from the
group consisting of technetium-99m (.sup.99m-Tc), indium-111
(.sup.111In), indium-113m (.sup.113mIn), gallium-67 (.sup.67Ga),
gallium-68 (.sup.68Ga), thallium-201 (.sup.201Tl), cobalt-57
(.sup.57Co), strontium-85 (.sup.85Sr), and copper-64
(.sup.64Cu).
11: The radioactive metal-labeled anti-p-cadherin antibody of claim
9, the non-cytotoxic radioactive metal is indium-111 (.sup.111In)
or copper-64 (.sup.64Cu).
12: A p-cadherin expressing cancer therapeutic agent comprising as
an active ingredient the radioactive metal-labeled anti-p-cadherin
antibody of claim 6.
13: A p-cadherin expressing cancer diagnosing agent comprising as
an active ingredient the radioactive metal-labeled anti-p-cadherin
antibody of claim 9.
14: A metal-chelating reagent-binding anti-p-cadherin antibody,
obtained by a process comprising binding a metal chelating reagent
to an anti-p-cadherin antibody, wherein a mole ratio of the
anti-p-cadherin antibody to the metal-chelating reagent is from
1:0.1 to 1:4.5.
15: The metal chelating reagent-binding anti-p-cadherin antibody of
claim 14, wherein the mole ratio of the anti-p-cadherin antibody to
the metal-chelating reagent is from 1:0.5 to 1:3.
16: The metal chelating reagent-binding anti-p-cadherin antibody of
claim 14, wherein the anti-p-cadherin antibody is bonded to a
region 1 to 655 of SEQ ID NO:2
17: The metal chelating reagent-binding anti-p-cadherin antibody of
claim 14, wherein the metal-chelating reagent is selected from the
group consisting of isothiocyanobenzyl DOTA,
methylisothiocyanobenzyl DTPA and cyclohexylisothiocyanobenzyl
DTPA.
18: The metal chelating reagent-binding anti-p-cadherin antibody of
claim 14, wherein the anti-p-cadherin antibody is a monoclonal
antibody, a recombinant antibody, a chimeric antibody, a humanized
antibody, or a fragment thereof.
19: A kit suitable for preparing a radioactive metal-labeled
anti-p-cadherin antibody, comprising a metal chelating
reagent-binding anti-p-cadherin antibody of claim 14.
20: The kit of claim 19, wherein the anti-p-cadherin antibody is a
monoclonal antibody produced by an antibody-producing cell of an
accession number of NITE BP-897, NITE BP-898, NITE BP-899, NITE
BP-1040, NITE BP-1044, NITE BP-1048, NITE BP-1049 or NITE BP-1050,
or a recombinant antibody thereof or a chimeric antibody thereof or
a humanized antibody thereof, or a fragment of any of these
antibodies.
21: A method for producing a metal-chelating reagent-binding
anti-p-cadherin antibody; comprising adding an anti-p-cadherin
antibody and a metal-chelating reagent at a ratio of 1:0.1 to
1:less than 5 to react.
22: The method of claim 21, wherein the anti-p-cadherin antibody
and the metal chelating reagent are added at a ratio of 1:1 to
1:less than 3 to react.
23: A method for treating a p-cadherin expressing cancer,
comprising: administering an effective amount of the radioactive
metal-labeled anti-p-cadherin antibody of claim 6 to a subject in
need thereof.
24: A method for diagnosing a p-cadherin expressing cancer,
comprising: administering an effective amount of the radioactive
metal-labeled anti-p-cadherin antibody of claim 9 to a subject in
need thereof; and diagnosing the p-cadherin expressing cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
13/578,462 filed Aug. 10, 2012, allowed and incorporated herein by
reference, which is a National Stage of PCT/JP11/052,759 filed Feb.
9, 2011 and claims the benefit of JP 2010-028028 filed Feb. 10,
2010.
TECHNICAL FIELD
[0002] The present invention relates to a radioactive metal-labeled
anti-cadherin antibody which highly specifically accumulates in
cancer cells, and to a cancer therapeutic agent and a cancer
diagnostic agent each containing the antibody.
BACKGROUND ART
[0003] There is keen demand for new cancer therapy for the
treatment of cancer, which is now the leading cause of death.
Currently, cancer therapies such as surgical therapy, radiotherapy,
and chemotherapy (by use of an anti-cancer agent) are employed.
Even after surgery, an anti-cancer agent is employed in
postoperative therapy.
[0004] Currently employed anti-cancer agents include an alkylating
agent, an antimetabolite, an alkaloide anti-cancer agent, an
antibiotic anti-cancer agent, and a platinum agent. The treatment
effects of these agents are not completely satisfactory. Some
agents are not cancer cell-specific and frequently cause adverse
side effects, which is problematic. Under such circumstances, there
is demand for development of more effective anti-cancer agents.
[0005] Meanwhile, cadherin is a Ca.sup.2+-dependent adhesion
molecule which is expressed on the cell surface. Examples of known
cadherin species include classic cadherins such as E cadherin, N
cadherin, and P cadherin (CDH3); as well as protocadherin, and
desmosomal cadherin. These cadherins are known to bind
homophylicly, to form an adherence junction, and to link to the
cytoskeletal system (actin filaments) via intracellular catenin and
are considered to control cell adhesion by such a mechanism.
[0006] In addition to cell adhesion, cadherin is thought to relate
to embryogenesis, morphogenesis, synaptogenesis, synaptic
plasticity, and infiltration and metastasis of cancer. Thus, an
anti-cadherin antibody is reported to be useful for cancer therapy
(Patent Documents 1 to 3).
PRIOR ART DOCUMENTS
Patent Documents
Patent Document 1: Japanese Kohyo (PCT) Patent Publication No.
2005-522982
Patent Document 2: Japanese Kohyo (PCT) Patent Publication No.
2008-538909
Patent Document 3: Japanese Kohyo (PCT) Patent Publication No.
2009-528257
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0007] However, the anti-cancer effect of the anti-cadherin
antibody is not satisfactory, and there has been demand for
development of a more potent cancer therapeutic agent.
[0008] Thus, an object of the present invention is to provide a
radioactive metal-labeled anti-cadherin antibody which can be
highly accumulated in cancer tissue. Another object is to provide a
cancer therapeutic agent which contains the antibody as an active
ingredient and which exhibits high anti-cancer effect. Still
another object is to provide a cancer diagnostic agent which can
predict the efficacy of a cancer therapeutic agent and confirm the
therapeutic effect thereof.
Means for Solving the Problems
[0009] The present inventors have conducted extensive studies to
attain the aforementioned objects, and have found that a
radioactive metal-labeled anti-cadherin antibody in which a
radioactive metallic element is bound to a anti-cadherin antibody
via a metal-chelating reagent is accumulated specifically in the
cancer tissue of a cancer-bearing animal, and that the anti-cancer
effect thereof is particularly remarkably enhanced as compared to
the unlabeled anti-cadherin antibody-administration group. The
present invention has been accomplished on the basis of these
findings.
[0010] Accordingly, the present invention provides a radioactive
metal-labeled anti-cadherin antibody which is obtained by binding a
radioactive metallic element to an anti-cadherin antibody via a
metal-chelating reagent, and a cancer therapeutic agent and a
cancer diagnostic agent each containing, as an active ingredient,
the radioactive metal-labeled anti-cadherin antibody.
[0011] The present invention also provides the radioactive
metal-labeled anti-cadherin antibody for use in the treatment or
diagnosis of cancer.
[0012] The present invention also provides use of the radioactive
metal-labeled anti-cadherin antibody for producing a cancer
therapeutic agent or a cancer diagnostic agent.
[0013] The present invention also provides a method for the
treatment or diagnosis of cancer, containing administering an
effective amount of the radioactive metal-labeled anti-cadherin
antibody to a subject in need thereof.
Effects of the Invention
[0014] The cancer therapeutic agent containing, as an active
ingredient, the radioactive metal-labeled anti-cadherin antibody of
the present invention is highly accumulated in cancer tissue and
exhibits high cancer tissue-shrinking effect. Therefore, by use of
the cancer therapeutic agent, cancer therapy can be effectively
performed without causing adverse side effects. Also, by use of the
cancer diagnostic agent of the present invention, the efficacy of
the cancer therapeutic agent of the present invention can be
predicted, and the therapeutic effect thereof can be confirmed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 Graphs showing the result of the affinity of
antibodies evaluated by flow cytometry.
[0016] FIG. 2 Bio-distribution of .sup.67Ga-DOTA-PPMX2016 antibody
(adding ratio of 1:10) 96 hours after administration thereof.
[0017] FIG. 3 Bio-distribution of .sup.67Ga-DOTA-PPMX2016 antibody
(adding ratio of 1:3) 96 hours after administration thereof.
[0018] FIG. 4 Bio-distribution of .sup.67Ga-DOTA-PPMX2029 antibody
(adding ratio of 1:10) 96 hours after administration thereof.
[0019] FIG. 5 Bio-distribution of .sup.67Ga-DOTA-PPMX2029 antibody
(adding ratio of 1:3) 96 hours after administration thereof.
[0020] FIG. 6 Bio-distribution of .sup.67Ga-DOTA-PPMX2025 antibody
(adding ratio of 1:10) 96 hours after administration thereof.
[0021] FIG. 7 Bio-distribution of .sup.67Ga-DOTA-PPMX2025 antibody
(adding ratio of 1:3) 96 hours after administration thereof.
[0022] FIG. 8 Bio-distribution of .sup.67Ga-DOTA-PPMX2025 antibody
in case of the added antibody-DOTA ratios of 1:1, 1:3, and 1:10 96
hours after administration thereof.
[0023] FIG. 9 Bio-distribution of .sup.111In-DOTA-PPAT-052-27c
antibody (adding ratio of 1:3) 96 hours after administration
thereof.
[0024] FIG. 10 Bio-distribution of .sup.111In-DOTA-PPAT-052-27c
antibody (adding ratio of 1:3) 48 hours after administration
thereof.
[0025] FIG. 11 Bio-distribution of .sup.111In-DOTA-PPAT-052-28c
antibody (adding ratio of 1:3) 48 hours and 96 hours after
administration thereof.
[0026] FIG. 12 Anti-tumor effect of .sup.90Y-DOTA-PPMX2029 antibody
(adding ratio of 1:3) in xenograft model.
[0027] FIG. 13 Anti-tumor effect of .sup.90Y-DOTA-PPAT-052-27c
antibody (adding ratio of 1:3) in xenograft model.
[0028] FIG. 14 Photoimages showing the results of
immunohistochemical staining for confirming CDH3 protein
expression.
MODES FOR CARRYING OUT THE INVENTION
[0029] The radioactive metal-labeled anti-cadherin antibody of the
present invention is a labeled anti-cadherin antibody to which a
radioactive metallic element is bound to via a metal-chelating
reagent. The cancer therapeutic agent or the cancer diagnostic
agent contains the radioactive metal-labeled anti-cadherin
antibody.
[0030] The anti-cadherin antibody is not particularly limited, so
long as the antibody specifically binds to cadherin. Examples of
cadherin include E cadherin, N cadherin, and P cadherin. Of these,
P cadherin is more preferred.
[0031] The anti-cadherin antibody encompasses a monoclonal
antibody, a polyclonal antibody, an antibody maintaining ability of
binding specifically to an antigenic determinant group and variants
and derivatives of an antibody such as T-cell receptor
fragment.
[0032] The type of the anti-cadherin antibody is not particularly
limited, and there may be appropriately employed antibodies such as
a mouse antibody, a human antibody, a rat antibody, a rabbit
antibody, a sheep antibody, a camel antibody, and a chicken
antibody; and gene recombinant antibodies which are intentionally
modified so as to reduce the hetero-antigenicity to human such as a
chimeric antibody and a humanized antibody. The recombinant
antibody may be produced through a known method. The chimeric
antibody is an antibody formed of variable regions of a heavy chain
and a light chain of a mammalian antibody other than human
antibody, for example mouse antibody and constant regions of a
heavy chain and a light chain of a human antibody and may be
produced by linking a DNA fragment encoding the variable region of
the mouse antibody to a DNA fragment encoding the constant region
of the human antibody, incorporating the resultant fragment into an
expression vector, and incorporating the vector into host cells
(see, for example, Cabilly S. et al., Proc. Natl. Acad. Sci. USA,
1984, 81(11) 3273-7; Morrison et al., Proc. Natl. Acad. Sci. USA,
1984, 81(21), 6851-5; and European Patent Application Laid-Open No.
171496). The humanized antibody, which is also called a reshaped
antibody, is an antibody produced through transplantation of a
complementarity determining region (CDR) of a mammalian antibody
other than human antibody, e.g., a mouse antibody into a CDR of a
human antibody, and gene recombination techniques therefor are
generally known. Specifically, a DNA sequence including a CDR of a
mouse antibody linked to a framework region (FR) of a human
antibody is synthesized through PCR using several oligonucleotides
which are produced to have overlapped part at an end thereof. The
thus-obtained DNA fragment is linked to a DNA fragment encoding the
constant region of the human antibody, subsequently the resultant
fragment is incorporated into an expression vector and the vector
is incorporated into host cells to thereby produce the humanized
antibody (see EP239400 A and WO 96/02576 A). The FR of the human
antibody linked via the CDR is selected from FRs having a CDR which
forms a suitable antigen-binding site. If needed, an amino acid in
the FR of the variable region of the antibody may be substituted
such that a CDR of the reshaped antibody forms an appropriate
antigen-binding site (Sato, K. et al., Cancer Res., 1993, 53,
851-856).
[0033] The amino acid sequence of the chimeric antibody or
humanized antibody preferably has an identity of 1005 to that of
the Vh or Vl region of cDNA expressing a deposited hybridoma. Due
to genetic modification, an antibody having an identity in amino
acid sequence of 90% or higher is also preferred. In the process of
humanization or chimerization, there has been conventionally
carried out such controlled residue substitution for improving
binding to an antigen. Such an antibody having a partially modified
sequence is essentially considered to be an antibody originating
from the original hybridoma.
[0034] Methods for producing a chimeric antibody and a humanized
antibody based on a genetic engineering technique have been already
known. Specifically, the Vh and VL sequences of a monoclonal
antibody serving as a confirmed group is genetically modified, and
then chimerization or humanization is performed through a routine
technique.
[0035] The method for recovering a human antibody is also known. In
one procedure, human lymphocytes are sensitized in vitro with an
antigen of interest or with cells expressing the antigen, and the
thus-sensitized lymphocytes are fused with human myeloma cells,
e.g., U266, to thereby produce a human antibody of interest having
a binding activity to the antigen (see JP-B-1989-59878).
Alternatively, a human antibody of interest may be recovered
through immunization, with an antigen of interest, of a transgenic
animal having a complete repertory of the human antibody gene (see
WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096, and
WO96/33735). Also known is a technique for recovering a human
antibody through panning by use of a human antibody library. In one
procedure, a variable region of a human antibody is expressed as a
single-chain antibody (scFv) on the phage surface through the phage
display method, and a phage which binds the antigen can be
selected. Through gene analysis of the thus-selected phage, a DNA
sequence encoding the variable region of the human antibody which
binds to the antigen can be determined. When the DNA sequence of
the scFv which binds the antigen is elucidated, an appropriate
expression vector can be produced from the sequence, whereby a
human antibody of interest can be recovered. These methods are
widely known (see WO 92/01047, WO 92/20791, WO 93/06213, WO
93/11236, WO 93/19172, WO 95/01438, and WO 95/15388).
[0036] These anti-cadherin antibodies may be a low molecule
antibody such as an antibody fragment, a modified antibody or the
like, so long as the ability of recognizing the entire or a part of
the protein encoded by the cadherin gene is maintained. Examples of
the antibody fragment include Fab, Fab', F(ab')2, Fv, and Diabody.
Such an antibody fragment may be produced by constructing a gene
encoding the antibody fragment, incorporating the gene into an
expression vector, and expressing the vector in appropriate host
cells (see, for example, Co, M. S. et al., J. Immunol. (1994) 152,
2968-2976; Better, M. and Horwitz, A. H., Methods Enzymol. (1989)
178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989)
178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663;
Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; and
Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,
132-137).
[0037] As a modified antibody, an antibody which is bound to any of
various molecules such as polyethylene glycol (PEG) may be used.
Such a modified antibody may be produced through chemical
modification of the obtained antibody. The antibody modification
technique has already been established in the art.
[0038] In the present invention, there may be also employed a sugar
chain modified antibody for potentiating cytotoxic activity.
Techniques of modifying the sugar chain in an antibody have already
been known (e.g., WO 00/61739 and WO 02/31140).
[0039] The anti-cadherin antibody of the present invention also
encompasses a multi-specific antibody having specificity to two or
more different antigens. A typical example of such a molecule may
be one which can bind two antigens (i.e., a bi-specific antibody).
The "multi-specific antibody" of the present invention includes an
antibody having a specificity to two or more (e.g., three)
antigens. The multi-specific antibody may be a full-length antibody
or a fragment of such an antibody (e.g., F(ab').sub.2 bi-specific
antibody).
[0040] The anti-cadherin antibody of the present invention and the
antibody fragment thereof may be produced through any suitable
method such as in vivo, cultured cells, in vitro translation
reaction, and recombinant DNA expression system.
[0041] Techniques of producing monoclonal antibodies and
antibody-producing cells (hybridomas) are generally known in the
art (Campbell, "Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology," Elsevier Science
Publishers, Amsterdam, The Netherlands, 1984; and St. Groth et al.,
J. Immunol. Methods 35: 1-21, 1980). In one specific procedure, a
protein or a fragment thereof encoded by a cadherin gene serving as
an immunogen is subcutaneously or intraperitoneally injected for
immunization to any animal (e.g., mouse or rabbit) which is known
to produce an antibody. In immunization, an adjuvant may be
employed, and such an adjuvant is well known in the art.
[0042] The polyclonal antibody may be produced by isolating an
anti-serum containing antibodies from an immunized animal and
screening for the presence of an antibody having a target
specificity through a technique well known in the art (e.g., ELISA,
Western blotting, or radioimmunoassay).
[0043] The monoclonal antibody may be produced by removing spleen
cells from an immunized animal and fusing the cells with myeloma
cells, to thereby produce hybridomas which can produce monoclonal
antibodies. Hybridoma cells producing an antibody which can
recognize a protein of interest or a fragment thereof may be
selected based on a technique well known in the art (e.g., ELISA,
Western blotting, or radioimmunoassay). Then, the hybridoma
secreting an antibody of interest is cloned, and the obtained cells
are cultured under appropriate conditions. The thus-secreted
antibody is recovered and purified through a method well known in
the art (e.g., ion-exchange column chromatography or affinity
chromatography). In an alternative procedure, a human monoclonal
antibody may be produced by use of a xenomouse strain (see Green,
J. Immunol. Methods 231: 11-23, 1999; and Wells, Eek, Chem. Biol.
2000 August; 7(8): R185-6). Currently, monoclonal antibody
production based on phage display involving no immunization is
carried out. The monoclonal antibody of the present invention is a
single-molecular-species antibody produced by single-species of
antibody-producing cells or a DNA fragment obtained therefrom and
encoding the antibody. The monoclonal antibody may be produced
through any of the aforementioned methods.
[0044] The DNA fragment encoding a monoclonal antibody can be
readily isolated and sequenced through a routine method (e.g., by
use of an oligonucleotide probe which can binds specifically to
genes encoding the heavy chain and light chain of the monoclonal
antibody). A hybridoma cell is a preferred starting material for
producing such a DNA fragment. After isolation, such a DNA fragment
is inserted into an expression vector, and the vector is recombined
to host cells such as E. coli cells, monkey COS cells, Chinese
hamster ovary (CHO) cells or myeloma cells in which no
immunoglobulin is produced unless the cells are transformed. The
monoclonal antibody of interest is produced by the recombinant host
cells. In an alternative mode, an antibody or an antibody fragment
can be isolated from an antibody phage library produced through a
technique of McCafferty et al. (Nature 348: 552-554 (1990)).
[0045] The host cell employed for monoclonal antibody expression is
preferably a mammal-origin host cell. A host cell most suited to a
monoclonal antibody to be expressed may be selected. The host cell
is not limited and typical examples thereof include CHO-originating
cell line (Chinese hamster ovary cell), CV1 (monkey kidney), COS
(CV1 derivative expressing SV40T antigen), SP2/0 (mouse myeloma),
P3x63-Ag3.653 (mouse myeloma), 293 (human kidney), and 293T (293
derivative expressing SV40T antigen). The host cell system may be
obtained from a commercial facility, the American Tissue Culture
Collection (ATCC), or an organization which published a relevant
document.
[0046] The host cell is preferably a dhfr gene expression-defective
CHO-originating cell line (deletion in dhfr gene expression) or
SP2/0 (see Urland, G. et al., Effect of gamma rays at the
dihydrofolate reductase locus: deletions and inversions; Somat.
Cell. Mol. Genet. Vol. 12, 1986, p. 5555-566; and Schulman, M. et
al., A better cell line for making hybridomas secreting specific
antibodies, Nature Vol. 276, 1978, p. 269-270). The host cell is
more preferably a DHFR-deleted CHO. Transfection of a plasmid into
host cells may be performed through any technique. Transfection
technique is not limited and specific examples thereof include
transfection (including calcium phosphate method, DEAE method,
lipofection, and electroporation), DNA incorporation by use of an
envelope (e.g., Sendai virus), micro-injection, and infection by
use of a viral (e.g., retrovirus or adenovirus) vector (see Current
Protocols in Molecular Biology, Chapter 9 Introduction of DNA into
Mammalian Cells, John Wiley and Sons, Inc.). Among them,
incorporation of a plasmid into host cells through electroporation
is particularly preferred.
[0047] The recognition site in cadherin of the anti-cadherin
antibody of the present invention is preferably a region from 1 to
655 of SEQ ID NO: 2.
[0048] The anti-cadherin antibody of the present invention is
preferably produced from a hybridoma PPMX2016, PPMX2025, PPMX2029,
PPAT-052-02, PPAT-052-03, PPAT-052-09, PPAT-052-24, PPAT-052-25,
PPAT-052-26, or PPAT-052-28, or an transgenic CHO cell line
PPAT-052-27c, PPAT-052-02c, PPAT-052-03c, PPAT-052-09c,
PPAT-052-21c, PPAT-052-24c, PPAT-052-25c, PPAT-052-26c,
PPAT-052-28c, or PPAT-052-29c. In the present specification, the
numbers attached to PPMX or PPAT are given to either corresponding
antibody-producing cells or antibodies produced by the
antibody-producing cells.
[0049] A radioactive metal which is bound to the anti-cadherin
antibody is preferably a cytotoxic radioactive metal when the
radioactive metal-labeled anti-cadherin antibody is used as a
cancer therapeutic agent, and a non-cytotoxic radioactive metal
when the radioactive metal-labeled anti-cadherin antibody is used
as a cancer diagnostic agent.
[0050] Examples of the cytotoxic radioactive metal include
yttrium-90 (.sup.90Y), rhenium-186 (.sup.186Re), rhenium-188
(.sup.188Re), copper-67 (.sup.67Cu), iron-59 (.sup.59Fe),
strontium-89 (.sup.89Sr), gold-198 (.sup.198Au),)mercury-203
(.sup.203Hg), lead-212 (212Pb),)dysprosium-165 (.sup.165Dy),
ruthenium-103 (.sup.103Ru), bismuth-212 (212Bi), (.sup.212Bi)
bismuth-213 (.sup.213Bi),)holmium-166 (.sup.166Ho), samarium-153
(153Sm), and lutetium-177 (.sup.177Lu).
[0051] Among these radioactive metals, .sup.90Y, .sup.153Sm, and
.sup.177Lu are preferred, from the viewpoints of half-life,
radiation energy, ease of labeling reaction, percent of labeling,
complex stability, etc.
[0052] A non-cytotoxic radioactive metal suitably employed in a
cancer diagnostic agent is not limited and examples thereof include
technetium-99m (.sup.99mTc), indium-111)indium-113m (.sup.1l3mIn),
gallium-67 (.sup.67Ga), gallium-68 (.sup.68Ga), thallium-201
(.sup.201Tl), chromium-51 (.sup.51Cr), cobalt-57 (.sup.57Co),
cobalt-58 (.sup.58Co), cobalt-60 (.sup.60Co), strontium-85
(.sup.85Sr), mercury-197 (.sup.197Hg), and copper-64
(.sup.64Cu).
[0053] For bonding a radioactive metallic element to the
anti-cadherin antibody, in a preferred mode, a metal-chelating
reagent is reacted with the anti-cadherin antibody, and the product
is further reacted with a radioactive metallic element, to thereby
form a complex. In the thus-produced modified antibody, the
radioactive metallic element is bound to the anti-cadherin antibody
via the metal-chelating reagent.
[0054] Examples of the metal-chelating reagent for forming such a
complex include (1) quinoline derivatives such as
8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine,
oxyquinoline sulfate, O-acetyloxine, O-benzoyloxine,
O-p-nitrobenzoyloxine, and quinolone compounds having a quinoline
skeleton (e.g., norfloxacin, ofloxacin, enoxacin, ciprofloxacin,
lomefloxacin, tosfloxacin, fleroxacin, and sparfloxacin); (2)
compounds such as chloranilic acid, aluminon, thiourea, pyrogallol,
cupferron, Bismuthiol (II), galloyl gallic acid, thiolide,
2-mercaptobenzothiazole, and tetraphenylarsonium chloride; (3)
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), and compounds having a
similar skeleton (dihydroxyethylglycine, diaminopropanolte
traacetic acid, ethylenediamine diacetic acid,
ethylenediaminedipropionic acid hydrochloride,
hydroxyethylethylenediaminetriacetic acid,
ethylenediaminetetrakis(methylenesulfonic acid), glycol ether
diaminetetraacetic acid, hexamethylenediaminetetraacetic acid,
hydroxyethyliminodiacetic acid, iminodiacetic acid,
diaminopropanetetraacetic acid, nitrilotriacetic acid,
nitrilotripropionic acid, nitrilotris(methylenesulfonic acid)
trisodium salt, triethylenetetraminehexaacetic acid, methyl DTPA,
cyclohexyl DTPA, aminobenzyl EDTA, isothiocyanobenzyl EDTA,
isothiocyanobenzyl DTPA, methylisothiocyanobenzyl DTPA,
cyclohexylisothiocyanobenzyl DTPA, maleimidopropylamidobenzyl EDTA,
maleimidopentylamidobenzyl EDTA, maleimidodecylamidobenzyl EDTA,
maleimidopentylamidobenzyl DTPA, and maleimidodecylamidobenzyl
DTPA); and (4)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA),
1,4,7,10-tetraazacyclododecane (Cyclen),
1,4,8,11-tetraazacyclotetradecan (Cyclam), isothiocyanobenzyl DOTA,
and isothiocyanobenzyl NOTA.
[0055] Among these metal-chelating reagents, isothiocyanobenzyl
DOTA, methylisothiocyanobenzyl DTPA, cyclohexylisothiocyanobenzyl
DTPA are preferred, from the viewpoints of ease of incorporation
reaction of metal-chelate to antibody, percent of labeling, complex
stability, etc.
[0056] The radioactive metallic element may be bound to the
anti-cadherin antibody through a routine method. In one procedure,
a metal-chelating reagent is reacted with an anti-cadherin
antibody, to thereby prepare a label precursor, and the precursor
is reacted with a radioactive metallic element.
[0057] In the cancer therapeutic agent and cancer diagnostic agent
of the present invention, the ratio by mole of anti-cadherin
antibody to metal-chelating reagent is important for accumulation
in cancer cells and anti-cancer effect. The mole ratio
(anti-cadherin antibody:chelating reagent) is preferably 1:0.1 to
1:4.5, more preferably 1:0.5 to 1:3. In order to attain such mole
ratios, the anti-cadherin antibody and the chelating reagent are
preferably added to react at a ratio of 1:0.1 to 1:less than 5,
particularly preferably 1:1 to 1:3. The number of chelate
molecule(s) per anti-cadherin antibody may be calculated by
measuring molecular weight through MALDI-TOF mass analysis or a
similar technique, and comparing the molecular weight of an
un-modified antibody to that of a modified antibody (U.S. Pat. No.
7,514,078, Lu et al., J. Pharm. Sci. 94(4), 2005, p. 788-797, and
Tedesco et al., J. Clin. Onco. 23 (16S), 2005, 4765).
Alternatively, the number of chelate molecule(s) per anti-cadherin
antibody may be determined through chelatometric titration. One
known method employs an alkaline earth metal colorimetric reagent
(arsenazo III) (Bradyr et al., Nucl. Med. Biol. 31, 795-802, 2004,
and Dadachova et al., Nucl. Med. Biol. 26, 977-982, 1999).
[0058] The cancer therapeutic agent or cancer diagnostic agent of
the present invention may be provided as a labeled formulation or a
kit formulation containing a label precursor. Either formulation
may be employed in the present invention. In the case of labeled
formulation, a cancer therapeutic agent or a cancer diagnostic
agent containing a labeled anti-cadherin antibody may be
administered as is. In the case of a kit formulation, the agent may
be administered after labeling with a radioactive metallic element
of interest.
[0059] The anti-cadherin antibody containing a radioactive metallic
element bound thereto highly accumulates in cancer tissue and
exhibits high cancer cell-toxic activity. Thus, the antibody is a
useful cancer therapeutic agent which less damages the tissue other
than cancer tissue and which has high safety. Also, the
anti-cadherin antibody containing a radioactive metallic element
bound thereto has an anti-cancer activity remarkably higher than
that of a corresponding anti-cadherin antibody. The anti-cancer
activity is remarkably high particularly when the mole ratio of
antibody to chelating agent is 1:0.1 to 1:4.5.
[0060] The cancer therapeutic agent of the present invention may be
used in combination with another anti-cancer agent. Examples of
such an anti-cancer agent include an alkylating agent, an
antimetabolite, a microtubule inhibitor, an antibiotic anti-cancer
agent, a topoisomerase inhibitor, a platinum agent, a molecular
target drug, a hormone agent, and a biologics. Examples of the
alkylating agent include nitrogen mustard anti-cancer agents (e.g.,
cyclophosphamide), nitrosourea anti-cancer agents (e.g.,
ranimustine), and dacarbazine. Examples of the antimetabolite
include 5-FU, UFT, carmofur, capecitabine, tegafur, TS-1,
gemcitabine, and cytarabine. Examples of the microtubule inhibitor
include alkaloid anti-cancer agents (e.g., vincristine) and taxane
anti-cancer agents (e.g., docetaxel and paclitaxel). Examples of
the antibiotic anti-cancer agent include mitomycin C, doxorubicin,
epirubicin, daunorubicin, and bleomycine. Examples of the
topoisomerase inhibitor include irinotecan and nogitecan having
topoisomerase I inhibiting activity and etoposide having
topoisomerase II inhibiting activity. Examples of the platinum
agent include cisplatin, paraplatin, nedaplatin, and oxaliplatin.
Examples of the molecular target drug include trastuzumab,
rituximab, imatinib, gefitinib, erlotinib, bevacizumab, bortezomib,
sunitinib, and sorafenib. Examples of the hormone agent include
dexamethasone, finasteride, and tamoxifen. Examples of the
biologics include interferons .alpha., .beta., and .gamma. and
interleukin 2.
[0061] The cancer therapeutic agent of the present invention may be
used in combination with a cancer therapy. Examples of the cancer
therapy include surgery, radiation therapies (including gamma knife
therapy, Cyber knife therapy, boron neutron capture therapy, and
proton beam/heavy ion beam therapy), MR-guided focused ultrasound
surgery, cryotherapy, radiofrequency ablation, percutaneous ethanol
injection therapy, and embolotherapy.
[0062] The cancer therapeutic agent of the present invention is
effective on various cancers of a mammal (including a human).
Examples of the target cancer include carcinomas such as pharyngeal
cancer, laryngeal cancer, tongue cancer, lung cancer, breast
cancer, esophageal cancer, stomach cancer, colorectal cancer,
uterine cancer, ovarian cancer, liver cancer, pancreatic cancer,
gallbladder cancer, kidney cancer, prostatic cancer, malignant
melanoma, and thyroid cancer; and sarcomas such as osteosarcoma,
chondrosarcoma, rhabdomyosarcoma, leiomyosarcome, liposarcoma,
angiosarcoma, fibrosarcoma, leukemia, malignant lymphoma, and
myeloma.
[0063] The cancer therapeutic agent of the present invention may be
dissolved in an aqueous solution, preferably a physiologically
adaptable buffer such as Hanks' solution, Ringer's solution, or
buffered physiological saline. Also, the therapeutic agent may have
the form of suspension, solution, emulsion, or the like in an oily
or aqueous vehicle.
[0064] The dosage of the cancer therapeutic agent of the present
invention, which varies in accordance with the symptom,
administration route, body weight, age, etc. of a patient in need
thereof, is preferably, for example, 37 to 3,700 MBq for one
treatment of adult.
[0065] The cancer therapeutic agent of the present invention is
generally administered parenterally. For example, the cancer
therapeutic agent is injected (e.g., subcutaneously, intravenous,
intramuscle, intraperitoneally) or administered transdermally,
transmucosally, transnasally, transplumonarily, etc.
[0066] The cancer diagnostic agent of the present invention may be
used in tumor imaging. In the case where a patient has a tumor in
which CDH3 protein is expressed, the cancer diagnostic agent of the
present invention accumulates in the tumor. Thus, the tumor can be
imaged by detecting radiation by means of an apparatus such as a
single photon emission computed tomograph (SPECT), a positron
emission tomograph (PET), or a scintillation camera. For example,
by use of the cancer diagnostic agent of the present invention, the
therapeutic effect of the cancer therapeutic agent of the present
invention can be predicted before administration of the therapeutic
agent. The diagnostic agent is administered to a patient before the
treatment, and the tumor is imaged. When high accumulation is
observed, the superior effect of the therapeutic agent can be
predicted as potent. The diagnostic agent may be used for
determining the therapeutic effect. The diagnostic agent of the
present invention is administered to a patient who has received the
treatment with the therapeutic agent of the present invention or
any other treatment so as to image the tumor. Through monitoring
the time-dependent variation in accumulation of the diagnostic
agent, the expansion or shrinkage of the tumor over time can be
observed.
[0067] The antibody for use as a diagnostic agent preferably
recognizes an epitope competitive to a therapeutic agent. More
preferably, the antibody recognizes the same epitope as that
recognized by the therapeutic agent. Most preferably, the
therapeutic agent and the diagnostic agent are the same
antibody.
[0068] The cancer diagnostic agent of the present invention is
generally administered to a subject intravenously. However, the
cancer diagnostic agent may also be administered arterially. The
dosage thereof, which varies in accordance with the symptom,
administration route, body weight, age, etc. of a patient in need
thereof, is preferably, for example, 37 to 1,120 MBq for one
treatment of adult.
EXAMPLES
[0069] The present invention will next be described in detail by
way of examples, which should not be construed as limiting the
invention thereto.
Example 1
Production of Soluble CDH3 Antigen
[0070] Soluble CDH3 (sCDH3) protein in which the C-terminal
transmembrane region had been deleted was produced to serve as an
immunogen for producing an anti-CDH3 antibody.
(1) Production of Soluble CDH3 Antigen Expression Vector
[0071] PCR was performed by use of a CDH3 full-length cDNA as a
template and a forward primer (SEQ ID NO: 3:
CGCGGTACCATGGGGCTCCCTCGT, (hCDH3FullFW)) and a reverse primer (SEQ
ID NO: 4: CCGTCTAGATAACCTCCCTTCCAGGGTCC, (hCDH3SolbRV)), which had
been designed so as to amplify a segment corresponding to the CDH3
extracellular region (1-654 in SEQ ID NO: 2, hereinafter referred
to as sCDH3cDNA). The reaction was performed by use of KOD-Plus
(product of Toyobo) and under the following conditions: 94.degree.
C.-15 sec, 55.degree. C.-30 sec, and 68.degree. C.-90 sec (30
cycles)).
[0072] After completion of the PCR reaction, the reaction mixture
was subjected to agarose gel electrophoresis, and a gel piece
containing a band of a target size (about 2.0 kbp) was cut out. The
target sCDH3cDNA was recovered from the gel piece by use of a QIA
quick gel extraction kit (product of Quiagen).
[0073] In order to insert sCDH3cDNA into an expression vector
pEF4/myc-HisB, sCDH3cDNA was treated with two restriction enzymes
KpnI and XbaI. The thus-obtained fragment was inserted into
pEF4/myc-HIsB which had been treated with the same restriction
enzymes KpnI and XbaI, by use of T4 DNA ligase through a routine
technique, whereby an expression vector pEF4-sCDH3-myc-His was
yielded.
(2) Expression of Soluble CDH3 Protein
[0074] According to a protocol of an FuGENE6 transfection reagent,
8.times.10.sup.5 CHO cells were inoculated to a 10-cm-diameter dish
on the day before transfection, and the cells were cultured
overnight. Thereafter, an expression vector pEF4-sCDH3-myc-His (8
.mu.g) and an FuGENE6 regent (16 were mixed with serum-free RPMI
1640 medium (400 .mu.L), and the mixture was allowed to stand at
room temperature for 15 minutes. The resultant mixture was added to
the cell culture liquid for transfection. Two days after
transfection, cloning was performed through limiting dilution by
use of a selection reagent (Zeocin).
[0075] Soluble CDH3-expressing CHO cells were selected through
Western blotting by use of an anti-c-Myc monoclonal antibody
(product of SANTA CRUZ BIOTECHNOLOGY). Cell lines which exhibited
high level of secretion into the culture supernatant and high
proliferation were selected to obtain a soluble CDH3-expressing CHO
cell line (EXZ1702). The thus-selected soluble CDH3-expressing CHO
cells (EXZ1702) were cultured for 72 hours in three roller bottles
(each culture area: 1,500 cm.sup.2) with serum-free medium
CHO-S-SFM-II (333 mL/bottle) (product of Invitrogen), and the
culture supernatants were recovered. The thus-obtained culture
supernatant was subjected to affinity chromatography by means of
HisTrap (registered trademark) HP column (product of GE Healthcare
Bio-science) and gel filtration chromatography by means of Superdex
(registered trademark) 200 pg column (product of GE Healthcare
Bio-science), to thereby acquire soluble CDH3 protein.
Example 2
Establishment of CDH3-Expressing CHO Cell Line
[0076] For obtaining a cell line for anti-CDH3 antibody screening,
a CHO cell line expressing full length CDH3 was established.
(1) Production of CDH3 Gene Expression Vector
[0077] In order to insert full-length human CDH3 DNA represented by
SEQ ID NO: 1 into a mammal expression vector pEF4/myc-HisB (product
of Invitrogen), the full-length human CDH3 DNA was treated with two
restriction enzymes KpnI (product of Takara Bio Inc.) and XbaI
(product of Takara Bio Inc.) at 37.degree. C. for one hour. The
thus-obtained fragment was inserted into pEF4/myc-HisB which had
been treated with the same restriction enzymes KpnI and XbaI, by
use of T4 DNA ligase (product of Promega) through a routine
technique, whereby an expression vector pEF4-CDH3-myc-His was
produced.
(2) Acquisition of Stable CDH3-Expressing Line
[0078] According to a protocol of an FuGENE (registered trademark)
6 transfection reagent (product of Roche Diagnostics K.K.),
8.times.10.sup.5 CHO cells were inoculated to a 10-cm-diameter dish
on the day before transfection, and the cells were cultured
overnight. Thereafter, an expression vector pEF4-CDH3-myc-His (8
.mu.g) and an FuGENE6 regent (16 .mu.L) were mixed with serum-free
RPMI 1640 medium (product of SIGMA-ALDRICH) (400 .mu.L), and the
mixture was allowed to stand at room temperature for 15 minutes.
The resultant mixture was added to the cell culture liquid for
transfection. Two days after transfection, cloning was performed
through limiting dilution by use of a selection reagent
(Zeocin).
[0079] Clones of CDH3 full-length expressing CHO were selected
through Western blotting by use of an anti-c-Myc monoclonal
antibody (product of SANTA CRUZ BIOTECHNOLOGY). As a result, a CDH3
full-length expressing CHO cell line (EXZ1501) was selected as a
cell line which exhibited high level of expression and high
proliferation. The reaction between EXZ1501 and a commercial
anti-CDH3 antibody (product of R&D SYSTEMS) was confirmed
through flow cytometry. That is, CDH3 protein expression on the
cell membrane of EXZ1501 was confirmed.
Example 3
Production of Anti-CDH3 Monoclonal Antibody
(1) Production of Monoclonal Antibody by Use of Soluble CDH3
Protein as an Immunogen
[0080] Soluble CDH3 protein (50 .mu.g) dissolved in physiological
saline was mixed with an equal amount of Titer-MAX Gold (registered
trademark) (product of Titer Max), and the mixture was
intraperitoneally and subcutaneously injected to MRL/lpr mice
(Japan SLC inc.) for initial immunization. Subsequent immunization
procedures were performed by intraperitoneally and subcutaneously
injecting, to the mice, a mixture of soluble CDH3 protein (25
.mu.g) and Titer-MAX Gold prepared in the same manner. Three days
after final immunization, spleen cells were prepared from the mice
under aseptic conditions, and the cells were fused with mouse
myeloma cells SP2/O--Ag14 or P3-X63-Ag8.653 through a generally
employed polyethylene glycol method.
(2) Selection of Anti-CDH3 Antibody-Producing Hybridomas
[0081] Selection of anti-CDH3 antibodies were performed through
flow cytometry by use of a full-length CDH3-expressing CHO cell
line (EXZ1501).
[0082] Specifically, full-length CDH3-expressing CHO cells
(EXZ1501) were removed from a culture plate by treating with 2 mM
EDTA-PBS and suspended in FACS solution to a cell concentration of
1.times.10.sup.6 cells/mL. The cell suspension was inoculated to a
96-well plate to a concentration of 50 .mu.L/well, and a hybridoma
culture supernatant was added thereto, followed by reaction at
4.degree. C. for 60 minutes. The plate was washed twice with FACS
solution (200 .mu.L/well), and Alexa Fluor 488-labeled anti-mouse
IgG.cndot.goat F(ab')2 (product of Invitrogen) was added thereto,
followed by reaction at 4.degree. C. for 30 minutes. Subsequently,
the plate was washed twice with FACS solution, and flow cytometry
was performed, to thereby select hybridomas producing an antibody
which binds to CDH3-expressing CHO cells. As a result, 40 clones
PPMX2016 to PPAT-052-28 were obtained. Through flow cytometry, it
was confirmed that all the hybridomas reacted with CDH3-expressing
CHO cells (EXZ1501) and NCI-H358 but do not react with CHO cells.
Antibodies were purified from the hybridoma culture supernatant by
means of Protein G column and employed in the subsequent
experiments. Among the selected hybridomas, PPMX2016 (NITE BP-897),
PPMX2025 (NITE BP-898), PPMX2029 (NITE BP-899), PPAT-052-02 (NITE
BP-1034), PPAT-052-03 (NITE BP-1035), PPAT-052-09 (NITE BP-1036),
PPAT-052-24 (NITE BP-1037), PPAT-052-25 (NITE BP-1038), PPAT-052-26
(NITE BP-1039), and PPAT-052-28(NITE BP-1040) were deposited with
Incorporated Administrative Agency, the National Institute of
Technology and Evaluation, Patent Microorganisms Depositary (2-5-8,
Kazusakamatari, Kisarazu-shi, Chiba, Japan) on Feb. 10, 2010 and
Jan. 18, 2011.
Example 4
Cloning of Antibody Genes
[0083] (1) A DNA fragment encoding the V-region of a mouse
monoclonal antibody to human CDH3 was cloned through the following
procedure. Cytoplasmic RNA was isolated from the mouse hybridoma
cells through a method disclosed in a document (Gough, "Rapid and
quantitative preparation of cytoplasmic RNA from small numbers of
cells," Analytical Biochemistry, 173, p. 93-95 (1988)), with the
proviso that instead of the dissolution buffer disclosed in the
document, a TNE buffer (i.e., 25 mM Tris-HCl, pH: 7.5; 1% NP-40;
150 mM NaCl; 1 mM EDTA, pH: 8.0)was employed. More specifically,
5.times.10.sup.6 hybridoma cells were suspended in the TNE buffer
(200 .mu.L), to thereby dissolve cell membranes, and cell nuclei
were removed through centrifugation. To the thus-obtained
cytoplasma supernatant (about 200 .mu.L), an extraction buffer (10
mM Tris-HCl, pH: 7.5; 0.35M NaCl; 1% (w/v) SDS; 10 mM EDTA, pH:
8.0; 7M urea) (200 .mu.L) was added. The mixture was subjected to
extraction with phenol and chloroform. To the thus-obtained RNA
solution, glycogen (product of Roche, Cat No. 901393) serving as a
carrier was added. Then, ethanol was added to precipitate the
product. The RNA precipitate was dissolved in sterilized distilled
water (10 to 50 .mu.L) to a cytoplasmic RNA concentration of 0.5 to
2 .mu.g/.mu.L. (2) Production of cDNA Library from RNA Prepared
from Hybridomas
[0084] For synthesizing a single-strand cDNA, there was prepared a
reaction mixture (20 .mu.L) containing the above-prepared
cytoplasmic RNA (0.5 to 3 .mu.g), 50 mM Tris-HCl (pH: 8.3, room
temperature), 75 mM KCl, 3 mM MgCl.sub.2, and 10 mM DTT), a random
primer (100 ng), 0.5 mM dNTP, and Superscript II (reverse
transcriptase, product of Invitrogen) (200 units). The mixture was
incubated at 42.degree. C. for 50 minutes. The thus-synthesized
cDNA library was employed as a template of polymerase chain
reaction (PCR) without performing further treatment.
(3) Amplification of a Gene Encoding a Variable Region of Anti-CDH3
Antibody Through PCR
[0085] All the primers employed in the experiments were synthesized
by Hokkaido System Science Co., Ltd.
a. Primers for Use in PCR Amplification of a Gene Encoding Mouse
L-Chain V-Region
[0086] The following two primer sets were employed: (i) a DNA
primer having, at the 5' end, a homology to the FR1 part and 4-set
primers having, at the 3' end, a homology to a J-chain gene in the
mouse L-chain, and (ii) 7-set primers having, at the 5' end, a
homology to the L-chain signal part and an antisense primer having,
at the 3' end, a homology to the KC part (KVL antisense primer).
Polymerase chain reaction was performed by use of the two primer
sets, whereby a mouse immunoglobulin L-chain variable region DNA
fragment was obtained from the cDNA. The primer sequences are as
follows.
(i) 4-Set Sense Primers for Mouse L-Chain Variable Region
Cloning
[0087] According to "Phage Display--A Laboratory Manual-, Barbas
Burton Scott Silverman," PROTOCOL 9.5, 17 sense primers and 3
reverse primers were synthesized by Hokkaido System Science Co.,
Ltd.
VK Sense (FR1 Part)
[0088] A mixture of the following 17 primers was employed as a VK
sense primer.
TABLE-US-00001 (degeneracy 2): SEQ ID NO: 5 5'-GAY ATC CAG CTG ACT
CAG CC-3' (degeneracy 4): SEQ ID NO: 6 5'-GAY ATT GTT CTC WCC CAG
TC-3' (degeneracy 8): SEQ ID NO: 7 5'-GAY ATT GTG MTM ACT CAG TC-3'
(degeneracy 8): SEQ ID NO: 8 5' GAY ATT GTG YTR ACA CAG TC-3'
(degeneracy 8): SEQ ID NO: 9 5' GAY ATT GTR ATG ACM CAG TC-3'
(degeneracy 16): SEQ ID NO: 10 5' GAY ATT MAG ATR AMC CAG TC-3'
(degeneracy 12): SEQ ID NO: 11 5' GAY ATT CAG ATG AYD CAG TC-3'
(degeneracy 4): SEQ ID NO: 12 5' GAY ATY CAG ATG ACA CAG AC-3'
(degeneracy 4): SEQ ID NO: 13 5' GAY ATT GTT CTC AWC CAG TC-3'
(degeneracy 8): SEQ ID NO: 14 5' GAY ATT GWG CTS ACC CAA TC-3'
(degeneracy 16): SEQ ID NO: 15 5' GAY ATT STR ATG ACC CAR TC-3'
(degeneracy 16): SEQ ID NO: 16 5' GAY RTT KTG ATG ACC CAR AC-3'
(degeneracy 12): SEQ ID NO: 17 5' GAY ATT GTG ATG ACB CAG KC-3'
(degeneracy 4): SEQ ID NO: 18 5' GAY ATT GTG ATA ACY CAG GA-3'
(degeneracy 4): SEQ ID NO: 19 5' GAY ATT GTG ATG ACC CAG WT-3'
(degeneracy 2): SEQ ID NO: 20 5' GAY ATT GTG ATG ACA CAA CC-3'
(degeneracy 2): SEQ ID NO: 21 5' GAY ATT TTG CTG ACT CAG TC-3'
J Antisense (4-Set Primers)
J1/J2 Antisense Primer (1)
TABLE-US-00002 [0089] (degeneracy 8): SEQ ID NO: 22 5'-GGS ACC AAR
CTG GAA ATM AAA-3'
J4 Antisense Primer (2)
TABLE-US-00003 [0090] SEQ ID NO: 23 5'-GGG ACA AAG TTG GAA ATA
AAA-3':
J5 Antisense Primer (3)
TABLE-US-00004 [0091] SEQ ID NO: 24 5'-GGG ACC AAG CTG GAG CTG
AAA-3':
J1/J2, J4, J5 Antisense Primer Mixture (4)
(ii) 7-Set Primers for Mouse L-Chain Variable Region Cloning VK
Sense (Signal Peptide Part)
[0092] The primers were obtained through nucleotide sequence
modification of a mouse Ig-primer set (Novagen; Merck, Cat. No.
69831-3) such that restriction enzyme sites were removed. A-set
sense primer
TABLE-US-00005 SEQ ID NO: 25 5'-ATGRAGWCACAKWCYCAGGTCTTT-3':
B-Set Sense Primer
TABLE-US-00006 [0093] SEQ ID NO: 26
5'-ATGGAGACAGACACACTCCTGCTAT-3':
C-Set Sense Primer
TABLE-US-00007 [0094] SEQ ID NO: 27
5'-ATGGAGWCAGACACACTSCTGYTATGGGT-3':
D-Set Sense Primer (Mixture of the Following 2 Primers)
TABLE-US-00008 [0095] SEQ ID NO: 28
5'-ATGAGGRCCCCTGCTCAGWTTYTTGGIWTCTT-3': SEQ ID NO: 29
5'-ATGGGCWTCAAGATGRAGTCACAKWYYCWGG-3':
E-Set Sense Primer (Mixture of the Following 3 Primers)
TABLE-US-00009 [0096] SEQ ID NO: 30
5'-ATGAGTGTGCYCACTCAGGTCCTGGSGTT-3': SEQ ID NO: 31
5'-ATGTGGGGAYCGKTTTYAMMCTTTTCAATTG-3': SEQ ID NO: 32
5'-ATGGAAGCCCCAGCTCAGCTTCTCTTCC-3':
F-Set Sense Primer (Mixture of the Following 4 Primers)
TABLE-US-00010 [0097] SEQ ID NO: 33
5'-ATGAGIMMKTCIMTTCAITTCYTGGG-3': SEQ ID NO: 34
5'-ATGAKGTHCYCIGCTCAGYTYCTIRG-3': SEQ ID NO: 35
5'-ATGGTRTCCWCASCTCAGTTCCTTG-3': SEQ ID NO: 36
5'-ATGTATATATGTTTGTTGTCTATTTCT-3':
G-Set Sense Primer (Mixture of the Following 4 Primers)
TABLE-US-00011 [0098] SEQ ID NO: 37
5'-ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3': SEQ ID NO: 38
5'-ATGGATTTWCARGTGCAGATTWTCAGCTT-3': SEQ ID NO: 39
5'-ATGGTYCTYATVTCCTTGCTGTTCTGG-3': SEQ ID NO: 40
5'-ATGGTYCTYATVTTRCTGCTGCTATGG-3':
KVL Antisense Primer
TABLE-US-00012 [0099] SEQ ID NO: 41 ACTGGATGGTGGGAAGATGGA:
B. Primers for Use in PCR Amplification of a Gene Encoding Mouse
H-Chain V-Region
[0100] The following two primer sets were employed 4-set primers
having, at the 5' end, a homology to the mouse H-chain signal part
and a primer having, at the 3' end, a homology to the KC part; and
1-set primers having, at the 5' end, a homology to the FR1 part and
2-set primers having, at the 3' end, a homology to the mouse
H-chain constant region (IGHC). Polymerase chain reaction was
performed by use of the two primer sets, whereby a mouse
immunoglobulin H-chain variable region DNA fragment was isolated
from the cDNA. The primer sequences are as follows.
(i) Primers for Mouse H-Chain Variable Region Cloning VH Sense
(Signal Part: 4-Set Primers)
[0101] These primers were synthesized according to Current
Protocols in Immunology (John Wiley and Sons, Inc.), Unit 2.12
Cloning, Expression, and Modification of Antibody V Regions (Table
2.12.2).
TABLE-US-00013 (degeneracy: 32): SEQ ID NO: 42 5'-ATG GRA TGS AGC
TGK GTM ATS CTC TT-3' (degeneracy: 8): SEQ ID NO: 43 5'-ATG RAC TTC
GGG YTG AGC TKG GTT TT-3' SEQ ID NO: 44 5'-ATG GCT GTC TTG GGG CTG
CTC TTC T-3': (degeneracy: 32): SEQ ID NO: 45 5'-ATG GRC AGR CTT
ACW TYY-3'
(ii) Primers for Mouse H-Chain Variable Region Cloning VH Sense
(FR1 Part)
[0102] These primers were designed by nucleotide sequence
modification of sense primers disclosed in a document (Tan et al,
"Superhumanized" Antibodies: Reduction of Immunoogenic Potential by
Complementarity-Determining Region Grafting with Human Germline
Sequences: Application to an Anti-CD281, Journal of Immunology 169
(2002), p. 1119-1125).
TABLE-US-00014 (degeneracy: 256): SEQ ID NO: 46 5'-SAG GTS MAR CTK
SAG SAG TCW GG-3'
VH Antisense (Antisense Primer Common to 3 and 4)
[0103] The primer was designed through degeneration of the
nucleotide sequence so that the primer can be annealed with all the
isoforms of mouse IgG.
TABLE-US-00015 (degeneracy: 6): SEQ ID NO: 47 5'-CAS CCC CAT CDG
TCT ATC C-3'
Example 5
Production of Chimera Anti-CDH3 Immunoglobulin Expression
Vector
Production of Expression Plasmid
[0104] Through PCR employing DNA Engine (Peltier Thermal Cycler, MJ
Research, Bio-Rad), each variable region of the L-chain and the
H-chain of an anti-CDH3 mouse monoclonal antibody was amplified by
use of the primers described in Example 4. Each of the
thus-amplified DNA fragments was incorporated into a sub-cloning
vector pGEM (product of Promega). The nucleotide sequence of the
DNA fragment was determined by use of a universal primer which
binds to T7 an 6. SP6 promoter of the vector. The thus-obtained
nucleotide sequences of the L-chain and H-chain variable regions of
the anti-CDH3 antibody were searched by IMGT/V-QUEST Search page
(http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=mouseIg),
whereby completion of cloning of the antibody genes was
confirmed.
[0105] Next, a gene encoding the human C.kappa. region was linked
to the cloned gene encoding the V region of the L-chain of the
anti-CDH3 antibody, and a gene encoding the human C.kappa.1 region
was linked to the gene encoding the V region of the H-chain. The
thus-designed L-chain and H-chain chimeric antibody genes were
synthesized in full length by GenScript. At the time, frequency of
codon usage was optimized so as to obtain efficient gene expression
in producing cells (according to a method disclosed in Kim et al.,
Codon optimization for high-level expression of human
erythropoietin (EPO) in mammalian cells, Gene, 199, 1997, p.
293-301). Specifically, in the case of L-chain, for the purpose of
effective translation, an essential DNA sequence (Kozak, M., J., At
least six nucleotides preceding the AUG initiator codon enhance
translation in mammalian cells. J. Mol. Biol. 196, p. 947-950,
1987), signal peptide of mouse IGKV, the V region of the L-chain of
the anti-CDH3 antibody, and the human C.kappa. region were
juxtaposed in this order, and restriction enzyme sites were added
to both ends (NheI on the 5' side and EcoRI on the 3' side). The
chimera H-chain was prepared in the same manner. Each of the
synthesized genes was cut with NheI and EcoRI, and the cut fragment
was incorporated into an expression vector pCAGGS between the NheI
site and the EcoRI site, to thereby produce an anti-CDH3 chimeric
antibody L-chain expression vector pCAGGS-IGK and H-chain
expression vector pCAGGS-IGH.
Example 6
Production of Chimera Anti-CDH3 Immunoglobulin Stable Expression
Vector
[0106] For realizing high-level expression of a genetically
modified antibody gene in CHO cells, there was produced an
expression vector into which a dihydrofolate reductase (dhfr) gene
linked to a CMV promoter sequence and having poly A signal had been
incorporated.
[0107] For producing a chimeric antibody-stably
expressing/producing cell line, there was produced a pCAGGS
expression vector into which a dhfr gene had been incorporated.
Specifically, into pCAGGS-IGH and pCAGGS-IGK, which are transient
expression vectors, a dhfr gene having a CMV promoter and poly A
signal was incorporated. Each of a mouse dhfr gene having a CMV
promoter and a Kozak sequence and SV40 poly A signal was amplified
through PCR. These genes in mixture form were linked together
through PCR, and an HindIII site was added to both ends of the
linked product, to thereby acquire a gene fragment of HindIII-CMV
promoter-Kozak-dhfr-poly A-HindIII. The fragment was inserted into
pCAGGS-IGH or pCAGGS-IGK at the HindIII sites, to thereby obtain
pCAGGS-IGH-CMVp-dhfr-A and pCAGGS-IGK-CMVp-dhfr-A. These expression
vectors enable chimeric antibody expression with a CAG promoter,
and expression of a dhfr gene with a CMV promoter, whereby a
chimeric antibody can be effectively produced through gene
amplification.
Example 7
Establishing a Chimera Anti-CDH3-Producing CHO Cell Line
[0108] CHO dhfr cells (G. Urlaub et al., Isolation of Chinese
hamster cell mutants deficient in dihydrofolate reductase activity,
Proc. Natl. Acad. Sci. USA 77, p. 4216-4220, 1980) were
simultaneously transformed by use of two plasmids (linear plasmids
obtained by cutting circular plasmids with PvuI in an
ampicillin-resistant gene); i.e., a pCAGGS-IGK-CMV-dhfr-A vector
for chimera anti-CDH3 L chain expression and a
pCAGGS-IGH-CMV-dhfr-A vector for chimera anti-CDH3 H chain
expression. Electroporation was performed by means of Amaxa
(product of Lonza). DNA fragment (2 .mu.g/sample; in the case of
L-chain plasmid or H-chain plasmid) was added to a 0.1 mL Amaxa
electroporation CHO buffer containing 3.times.10.sup.3 cells and a
pulse was applied.
[0109] The cells which had undergone electroporation were added to
an Iscove's Modified Dulbecco medium (IMDM: free of HT) containing
10% dialyzed FBS that is free of HT (H: hypoxanthine, T:
thymidine). Three days after transfection, the medium was changed
to an IMDM medium free of 10% dialyzed FBS, 2 mM L-glutamine, and
HT, and neo+ transformed cells were selected by use of 1 mg/mL
G418, to thereby acquire clones of a chimeric antibody-producing
positive cell line. Subsequently, gene amplification was performed
by use of the clones selected by using G418. 2-Round amplification
was performed in 250 nM and 1,000 nM methotrexate (MTX), and cell
lines which can produce a chimera CDH3 antibody (about 50 to 100
mg/L-culture supernatant) were established. The thus established
chimera anti-CDH3 antibody-stably expressing CHO cell lines were
deposited with Incorporated Administrative Agency, the National
Institute of Technology and Evaluation, Patent Microorganisms
Depositary.
TABLE-US-00016 TABLE 1 Cell line Accession No. PPAT-052-02c NITE
BP-1041 PPAT-052-03c NITE BP-1042 PPAT-052-09c NITE BP-1043
PPAT-052-21c NITE BP-1044 PPAT-052-24c NITE BP-1045 PPAT-052-25c
NITE BP-1046 PPAT-052-26c NITE BP-1047 PPAT-052-27c NITE BP-1048
PPAT-052-28c NITE BP-1049 PPAT-052-29c NITE BP-1050
Example 8
Acquisition of Purified Antibodies
[0110] The antibodies were purified from the culture supernatant by
use of protein A.
Example 9
Confirmation of Affinity
[0111] Through a competitive method, the affinity of the mouse
anti-CDH3 antibody was compared with that of the chimera anti-CDH3
antibody. In the competitive method, the affinity of the anti-CDH3
antibody was determined through flow cytometry (BD, FACS Calibur)
by use of cancer cells NCI-H358, which are known to be CDH3 high
expression cells.
[0112] Specifically, an antibody serially diluted sample (400
.mu.g/mL to 24 ng/mL) (50 .mu.L) and an Alexa488-labeled antibody
(4 .mu.g/mL) (50 .mu.L) were added to and mixed on a 96-well plate.
NCI-H358 cells were removed from a culture plate through treatment
with 2 mM EDTA-PBS, and the cells were suspended in an FACS
solution (1% BSA PBS) to a concentration of 1.5.times.10.sup.6/mL.
An aliquot (100 .mu.L) of the suspension was added to the wells
containing the antibody mixture. After addition, reaction was
performed at room temperature for 60 minutes, and the plate was
washed twice with an FACS solution (200 .mu.L/well). Subsequently,
the fluorescence intensity (GEO mean) of each well was determined
through flow cytometry.
[0113] The percent of binding inhibition of the Alexa488-labeled
antibody was calculated from a GEO mean value, as compared with
that obtained by the reaction only with the Alexa488-labeled
antibody (1 pg/mL). The antibody concentration showing 50%
inhibition was calculated, and the data were compared.
[0114] FIG. 1 shows the affinity evaluation of PPMX2016 (mouse
antibody) and PPAT-052-27c (chimeric antibody thereof). Virtually
no difference in affinity was observed between the two
antibodies.
Example 10
Production of Labeled Antibodies
(1) Bonding DOTA to Antibody
[0115] An antibody was dissolved in a buffer (50 mM Bicine-NaOH,
150 mM NaCl, pH: 8.5) to an antibody concentration of 10 mg/mL.
Separately, isothiocyanobenzyl DOTA (B-205, product of
Macrocyclics) was dissolved in DMSO to a concentration of 10 mg/mL.
The two solutions were mixed together so as to adjust the ratio by
mole of antibody to DOTA 1:1 (adding ratio 1:1), 1:3 (adding ratio
1:3), or 1:10 (adding ratio 1:10), and the mixture was stirred and
allowed to stand at 25.degree. C. for 17 hours. After termination
of reaction, the reaction mixture was purified by means of a
desalting column (PD-10, product of GE Healthcare, 17-0435-01) with
PBS. The following antibodies were used: PPMX2016, PPMX2025,
PPMX2029, PPAT-052-27c, and PPAT-052-28c.
(2) Determination of Percent of Chelate Incorporation
[0116] The percent of chelate incorporated into antibody was
determined through chelatometric titration. The modification
antibody protein concentration was determined through a customary
method in advance, and the number of moles of modification antibody
was calculated from the molecular weight of IgG. To 1-mg/mL
standard copper solution (100 whose concentration had been
determined through atomic absorption spectrometry, an arsenazo III
reagent (0.776 mg) and metal-free 5M ammonium acetate (product of
Sigma Aldrich) solution (3 mL) were added, and ultrapure water was
added to the solution to adjust the final volume to 10 mL. The
resultant solution was stored at room temperature in the dark to
prepare the arsenazo III solution. DOTA was dissolved in ultrapure
water, to thereby prepare a DOTA standard solution. A modification
antibody was dissolved in ultrapure water, to thereby prepare a
modification antibody solution. The DOTA standard solution or the
modification antibody solution (10 .mu.L) was admixed with the
arsenazo III solution (190 .mu.L), and the mixture was incubated at
37.degree. C. for 30 minutes. Subsequently, the absorbance of the
mixture was measured at a wavelength of 630 nm. A standard curve
was drawn from the absorbance measurements of DOTA standard
solutions. By the standard curve, the number of DOTA molecule(s)
bound to the modification antibody was calculated (average number
of DOTA modification).
[0117] Table 2 shows the results (DOTA-adding ratio and actual
average number of modifying DOTA). As is clear from Table 2, the
number of DOTA molecules bound to the antibody was found to be
determined by the adding ratio of DOTA.
TABLE-US-00017 TABLE 2 Av. no. of Antibody to DOTA-adding ratio
modifying DOTA PPMX2025 (adding ratio 1:1) 0.9 PPMX2025 (adding
ratio 1:3) 2.0 PPMX2025 (adding ratio 1:10) 5.3 PPMX2016 (adding
ratio 1:1) 0.7 PPMX2016 (adding ratio 1:3) 2.1 PPMX2016 (adding
ratio 1:10) 5.2 PPAT-052-27c (addding ratio 1:3) 1.8 (Note)
PPAT-052-27c was tested only at an adding ratio of 1:3.
(3) Preparation of .sup.67Ga-, or .sup.90Y-Labeled Antibodies
[0118] (i) Labeling with .sup.67Ga or .sup.111In
[0119] Each of the purified PPMX2016, PPMX2025, PPMX2029, and
PPAT-052-27c antibodies and PPAT-052-28c antibody was dissolved in
a buffer (0.25M ammonium acetate-HCl, pH: 5.5) to a concentration
of 6 mg/mL. A .sup.67GaCl.sub.3 solution (product of Fuji Film RI
Pharma) or a .sup.111InCl.sub.3 solution (product of MDS Nordion
Inc.) was added to the antibody solution, and the mixture was
incubated at 45.degree. C. for one hour.
(ii) Labeling with .sup.90Y
[0120] Each of the purified PPMX2029 and PPAT-052-27c antibodies
was dissolved in a buffer (0.25M ammonium acetate-HCl, pH: 5.5) to
a concentration of 6 mg/mL. A .sup.90YCl.sub.3 solution (product of
Nuclitec) was added to the antibody solution, and the mixture was
incubated at 45.degree. C. for one hour.
(iii) Determination of Percent of Labeling
[0121] An aliquot of the labeling reaction mixture was sampled and
subjected to thin-layer chromatography (61885, product of PALL), to
thereby determine the percent of labeling. Physiological saline was
employed as an eluent, and the radioactivity was measured at the
top and bottom ends of a strip by means of a .gamma.-counter. The
percent of labeling was calculated by the following equation.
Percent of labeling=(bottom end count/(top end count+bottom end
count)).times.100(%)
[0122] When the percent of labeling reached 90% or higher, the
labeled antibody was used in the subsequent experiment. The labeled
antibodies were purified a desalting column (PD-10, product of GE
Healthcare, 17-0435-01) with PBS.
Example 11
Investigation of Relationship Between Percent of
Chelate-Incorporation and Bio-Distribution (i.e., Distribution in
the Body)
[0123] PPMX2016, PPMX2025, and PPMX2029 were investigated in terms
of bio-distribution by virtue of difference of percent of chelate
incorporation values (DOTA-adding ratio 1:1, 1:3, and 1:10).
[0124] Firstly, NCI-H358 was cultured in a 10% FBS-containing
RPMI1640 medium, and the cultured cells were subcutaneously
transplanted to the right ventral region of each of the nude mice
(female, 7-week old, CLEA Japan Inc.) at a cell concentration of
1.times.10.sup.7 cells/mouse. The mice were bred until the average
tumor volume reached 100 to 150 mm.sup.3.
[0125] Then, to the NCI-H358-transplanted mice,
.sup.67Ga-DOTA-PPMX2016 antibody (adding ratio 1:3 and 1:10),
.sup.67Ga-DOTA-PPMX2025 antibody (adding ratio 1:3 and 1:10), or
.sup.67(Ga-DOTA-PPMX2029 antibody(adding ratio 1:3 and 1:10) was
administered at a dose of 370 kBq/mouse.
[0126] Ninety-six hours after administration, the mice were
sacrificed to anatomy, and tissues and the tumor were removed. The
weight of each tissue and the tumor weight were measured, the
radioactivity was measured by means of a .gamma.-counter and % ID/g
was calculated by the following equation.
% ID/g=(accumulated RI amount/total administered RI
amount.times.100(%))/weight (g) [E2]
[0127] FIGS. 2 to 7 show the results. All the tested antibodies
exhibited enhanced accumulation in the tumor, at a DOTA-adding
ratio of 1:3, as compared with the case at a ratio of 1:10. Such
enhanced accumulation results in enhancement of therapeutic effect.
In addition, adverse side effects, which would otherwise be caused
by retention of a radioactive substance in non-targeted organs, can
be avoided.
[0128] FIG. 8 shows the result of administration of
.sup.67Ga-DOTA-PPMX2025 antibody (use ratio 1:1, 1:3, and 1:10) to
non-cancer-bearing nude mice (female, 7-week old, CLEA Japan Inc.)
at 370 kBq/mouse. The antibody was accumulated highly in the liver
when the adding ratio was 1:10, whereas accumulation in the liver
was low when the ratio was 1:3 or 1:1, which indicates that adverse
side effects such as radioactive damage are prevented on the
liver.
Example 12
Investigation of Behavior of Anti-Caldina Chimeric Antibody in the
Body
[0129] The behaviors of chimera antibodies PPAT-052-27c and
PPAT-052-28c in the body were investigated.
[0130] Firstly, NCI-H1373 was cultured in a 10% FBS-containing
RPMI1640 medium, and the cultured cells were subcutaneously
transplanted into to the right ventral region of each of the nude
mice (female, 9-week old, CLEA Japan Inc.) at a cell concentration
of 4.times.10.sup.6 cells/mouse. The mice were bred until the
average tumor volume reached 100 to 150 mm.sup.3.
[0131] Then, to the NCI-H1373-transplaned mice,
.sup.111In-DOTA-PPAT-052-27c (adding ratio 1:3) or
.sup.111In-DOTA-PPAT-052-28c (adding ratio 1:3) at a dose of 370
kBq/mouse.
[0132] Forty-eight or ninety-six hours after administration, the
mice were sacrificed to anatomy, and tissues and the tumor were
removed. The weight of each tissue and the tumor weight were
measured, the radioactivity was measured by means of a
.gamma.-counter and % ID/g was calculated.
[0133] FIGS. 9 to 11 show the results. PPAT-052-27c exhibited a
percent accumulation in the tumor as high as 46% ID/g 48 hours
after administration. PPAT-052-28c exhibited a percent accumulation
in the tumor as high as 41% ID/g 48 hours after administration and
52% ID/g 96 hours after administration.
Example 13
Xenograft Test
[0134] NCI-H358 was cultured in a 10% FBS-containing RPMI1640
medium, and the cultured cells were subcutaneously transplanted
into to the right ventral region of each of the nude mice (female,
7-week old, CLEA Japan Inc.) at a cell concentration of
1.times.10.sup.7 cells/mouse.
[0135] The NCI-H358-transplaned mice were divided into six groups
(n=8). .sup.90Y-DOTA-PPMX2029 antibody (adding ratio 1:3) was
administered at a dose of 7.4 MBq/mouse, 5.6 MBq/mouse, 3.7
MBq/mouse, and 1.9 MBq/mouse. As control groups, unlabeled PPMX2029
was administered at a dose of 80 .mu.g/mouse, and physiological
saline was administered at a dose of 100 .mu.L/mouse. In all the
groups, administration was performed when the average tumor volume
reached 100 to 150 mm.sup.3.
[0136] After administration, the body weight and the tumor volume
were measured twice a week (every 3 or 4 days). This observation
wad continued to day 51 after administration.
[0137] FIG. 12 shows the test results. .sup.90Y-DOTA-PPMX2029
antibody (adding ratio 1:3) exhibited anti-tumor effect
proportional to the radioactivity.
[0138] Separately, NCI-H1373 was cultured in a 10% FBS-containing
RPMI1640 medium, and the cultured cells were subcutaneously
transplanted into to the right ventral region of each of the nude
mice (female, 7-week old, CLEA Japan Inc.) at a cell concentration
of 5.times.10.sup.6 cells/mouse.
[0139] The NCI-H1373-transplaned mice were divided into four groups
(n=8). .sup.90Y-DOTA-PPAT-052-27c antibody (adding ratio 1:3) was
administered at a dose of 5.6 MBq/mouse and 3.7 MBq/mouse. As
control groups, unlabeled PPMX2029 was administered at a dose of 60
.mu.g/mouse, and physiological saline was administered at a dose of
100 .mu.L/mouse. In all the groups, administration was performed
when the average tumor volume reached 100 to 150 mm.sup.3.
[0140] After administration, the body weight and the tumor volume
were measured twice a week (every 3 or 4 days). This observation
wad continued to day 26 after administration.
[0141] FIG. 13 shows the test results. .sup.90Y-DOTA-PPAT-052-27c
(adding ratio 1:3) exhibited anti-tumor effect proportional to the
radioactivity.
Example 14
Immunohistochemical Staining
[0142] CDH3 protein expression in a clinical cancer specimen was
confirmed by immunohistochemical staining of a cancer specimen
tissue array.
[0143] As cancer specimen tissue arrays, employed were tissues of
pancreatic cancer (adenocarcinoma), lung cancer (adenocarcinoma),
lung cancer (squamous cell carcinoma), and colorectal cancer
(adenocarcinoma), which are the products of Shanghai Outdo Biotech
Co., Ltd.
[0144] Each tissue array slide was dewaxed and activated with 10 mM
Tris 1 mM EDTA (pH: 9.0) at 95.degree. C. for 40 minutes.
Endogenous peroxidase in the array slide was inactivated with a
blocking agent, which is included in the ENVISION+Kit (product of
Dako). Subsequently, the tissue array slide was reacted with 5
.mu.g/mL anti-CDH3 antibody 610227 (product of BD BIOSCIENCE) or
with 5 .mu.g/mL anti-HBs antibody Hyb-3423 (negative control) at
4.degree. C. overnight. The antibody solution was washed out, and
the tissue array slide was further reacted with a polymer secondary
antibody reagent which is included in the ENVISION+Kit at room
temperature for 30 minutes. The slide was then color-developed by a
coloring reagent which is included in the ENVISION+Kit, and nuclear
staining was performed by use of a hematoxylin/eosin solution.
[0145] FIG. 14 shows the results. Cancer cells were stained by
anti-CDH3 antibody, but normal cells were not stained.
Sequence CWU 1
1
4712490DNAHomo sapiensCDS(1)..(2490)Homo sapiens 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 2829PRTHomo sapiens 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
324DNAArtificial sequenceDesigned DNA based on CDH3 gene
3cgcggtacca tggggctccc tcgt 24429DNAArtificial sequenceDesigned DNA
based on CDH3 gene 4ccgtctagat aacctccctt ccagggtcc
29520DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody
gene 5gayatccagc tgactcagcc 20620DNAArtificial sequenceDesigned
primer based on anti-CDH3 antibody gene 6gayattgttc tcwcccagtc
20720DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 7gayattgtgm tmactcagtc 20820DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
8gayattgtgy tracacagtc 20920DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 9gayattgtra tgacmcagtc
201020DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 10gayattmaga tramccagtc 201120DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
11gayattcaga tgaydcagtc 201220DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 12gayatycaga tgacacagac
201320DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 13gayattgttc tcawccagtc 201420DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
14gayattgwgc tsacccaatc 201520DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 15gayattstra tgacccartc
201620DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 16gayrttktga tgacccarac 201720DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
17gayattgtga tgacbcagkc 201820DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 18gayattgtga taacycagga
201920DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 19gayattgtga tgacccagwt 202020DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
20gayattgtga tgacacaacc 202120DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 21gayattttgc tgactcagtc
202221DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 22ggsaccaarc tggaaatmaa a 212321DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
23gggacaaagt tggaaataaa a 212421DNAArtificial sequenceDesigned
primer based on anti-CDH3 antibody gene 24gggaccaagc tggagctgaa a
212524DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 25atgragwcac akwcycaggt cttt 242625DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
26atggagacag acacactcct gctat 252729DNAArtificial sequenceDesigned
primer based on anti-CDH3 antibody gene 27atggagwcag acacactsct
gytatgggt 292832DNAArtificial sequenceDesigned primer based on
anti-CDH3 antibody gene 28atgaggrccc ctgctcagwt tyttggnwtc tt
322931DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 29atgggcwtca agatgragtc acakwyycwg g
313029DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 30atgagtgtgc ycactcaggt cctggsgtt 293131DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
31atgtggggay cgktttyamm cttttcaatt g 313228DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
32atggaagccc cagctcagct tctcttcc 283326DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
33atgagnmmkt cnmttcantt cytggg 263426DNAArtificial sequenceDesigned
primer based on anti-CDH3 antibody gene 34atgakgthcy cngctcagyt
yctnrg 263525DNAArtificial sequenceDesigned primer based on
anti-CDH3 antibody gene 35atggtrtccw casctcagtt ccttg
253627DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 36atgtatatat gtttgttgtc tatttct 273729DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
37atgaagttgc ctgttaggct gttggtgct 293829DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
38atggatttwc argtgcagat twtcagctt 293927DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
39atggtyctya tvtccttgct gttctgg 274027DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
40atggtyctya tvttrctgct gctatgg 274121DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
41actggatggt gggaagatgg a 214226DNAArtificial sequenceDesigned
primer based on anti-CDH3 antibody gene 42atggratgsa gctgkgtmat
sctctt 264326DNAArtificial sequenceDesigned primer based on
anti-CDH3 antibody gene 43atgracttcg ggytgagctk ggtttt
264425DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 44atggctgtct tggggctgct cttct 254518DNAArtificial
sequenceDesigned primer based on anti-CDH3 antibody gene
45atggrcagrc ttacwtyy 184623DNAArtificial sequenceDesigned primer
based on anti-CDH3 antibody gene 46saggtsmarc tksagsagtc wgg
234719DNAArtificial sequenceDesigned primer based on anti-CDH3
antibody gene 47casccccatc dgtctatcc 19
* * * * *
References