U.S. patent application number 16/688963 was filed with the patent office on 2020-03-19 for monoclonal antibodies to human fibroblast growth factor receptor 2 (hfgfr2) and methods of use thereof.
This patent application is currently assigned to DAIICHI SANKYO COMPANY, LIMITED. The applicant listed for this patent is DAIICHI SANKYO COMPANY, LIMITED. Invention is credited to Kokichi HONDA, Reimi KAWAIDA, Chigusa YOSHIMURA.
Application Number | 20200085946 16/688963 |
Document ID | / |
Family ID | 57142992 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200085946 |
Kind Code |
A1 |
YOSHIMURA; Chigusa ; et
al. |
March 19, 2020 |
MONOCLONAL ANTIBODIES TO HUMAN FIBROBLAST GROWTH FACTOR RECEPTOR 2
(HFGFR2) AND METHODS OF USE THEREOF
Abstract
The present invention provides a pharmaceutical composition or a
diagnostic composition targeting human fibroblast growth factor
receptor 2 (hFGFR2).
Inventors: |
YOSHIMURA; Chigusa; (Tokyo,
JP) ; KAWAIDA; Reimi; (Tokyo, JP) ; HONDA;
Kokichi; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DAIICHI SANKYO COMPANY, LIMITED |
TOKYO |
|
JP |
|
|
Assignee: |
DAIICHI SANKYO COMPANY,
LIMITED
TOKYO
JP
|
Family ID: |
57142992 |
Appl. No.: |
16/688963 |
Filed: |
November 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15567936 |
Oct 19, 2017 |
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PCT/JP2016/062297 |
Apr 19, 2016 |
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16688963 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2863 20130101;
G01N 33/577 20130101; C12N 5/10 20130101; A61P 35/00 20180101; G01N
33/6893 20130101; C07K 2317/51 20130101; C07K 2317/565 20130101;
C07K 2317/33 20130101; C07K 2317/76 20130101; G01N 2333/503
20130101; A61K 39/39558 20130101; C07K 2317/24 20130101; C12N 15/09
20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/68 20060101 G01N033/68; C12N 15/09 20060101
C12N015/09; C12N 5/10 20060101 C12N005/10; G01N 33/577 20060101
G01N033/577; A61P 35/00 20060101 A61P035/00; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2015 |
JP |
2015-085942 |
Claims
1-47. (canceled)
48. A monoclonal antibody or an antigen binding fragment thereof,
wherein the monoclonal antibody consists of a heavy chain
comprising a complementarity determining region H1 (CDRH1)
consisting of the amino acid sequence represented by SEQ ID NO: 24,
a CDRH2 consisting of the amino acid sequence represented by SEQ ID
NO: 25, and a CDRH3 consisting of the amino acid sequence
represented by SEQ ID NO: 26; and a light chain comprising a CDRL1
consisting of the amino acid sequence represented by SEQ ID NO: 27,
a CDRL2 consisting of the amino acid sequence represented by SEQ ID
NO: 28, and a CDRL3 consisting of the amino acid sequence
represented by SEQ ID NO: 29.
49. The monoclonal antibody or antigen binding fragment thereof
according to claim 48, wherein the monoclonal antibody or antigen
binding fragment thereof specifically binds to non-denatured human
fibroblast growth factor receptor 2 (hFGFR2) IIIc and denatured
hFGFR2 IIIc in a preparation fixed in formalin.
50. The monoclonal antibody or antigen binding fragment thereof
according to claim 48, wherein the monoclonal antibody comprises a
heavy chain variable region and a light chain variable region
described in any one of the following (i) to (iv): (i) a heavy
chain variable region of SEQ ID NO: 3 and a light chain variable
region of SEQ ID NO: 6; (ii) a heavy chain variable region in which
the framework region of the heavy chain variable region is 95% of
more identical to the framework region of SEQ ID NO: 3 and a light
chain variable region in which the framework region of the light
chain variable region is 95% of more identical to the framework
region of SEQ ID NO: 6; (iii) a heavy chain variable region derived
from SEQ ID NO: 3 by incorporating a substitution, deletion,
insertion, or addition of 1 or more amino acids into the framework
region, and a light chain variable region derived from SEQ ID NO: 6
by incorporating a substitution, deletion, insertion, or addition
of 1 or more amino acids into the framework region; and (iv) a
heavy chain variable region encoded by a polynucleotide that
hybridizes under stringent conditions to a polynucleotide that
encodes SEQ ID NO: 3, and a light chain variable region encoded by
a polynucleotide that hybridizes under stringent conditions to a
polynucleotide that encodes SEQ ID NO: 6.
51. The monoclonal antibody or antigen binding fragment thereof
which has the following properties (i) to (iii): (i) specifically
binds to non-denatured human fibroblast growth factor receptor 2
(hFGFR2) IIIc; (ii) specifically binds to none of the following:
non-denatured human fibroblast growth factor receptor 1 (hFGFR1);
non-denatured human fibroblast growth factor receptor 3 (hFGFR3);
and non-denatured human fibroblast growth factor receptor 4
(hFGFR4); and (iii) specifically binds to denatured hFGFR2 IIIc in
a preparation fixed in formalin, wherein the monoclonal antibody or
antigen binding fragment thereof binds to a site on hFGFR2 Mc which
is recognized by an antibody or an antigen binding fragment thereof
according to claim 3 or competes with an antibody or an antigen
binding fragment thereof according to claim 3 for binding to hFGFR2
IIIc.
52. The monoclonal antibody or antigen binding fragment thereof
according to claim 48, wherein the monoclonal antibody comprises
the amino acid sequence of a heavy chain represented by SEQ ID NO:
15 and the amino acid sequence of a light chain represented by SEQ
ID NO: 19.
53. The monoclonal antibody or antigen binding fragment thereof
according to claim 48, wherein the monoclonal antibody or antigen
binding fragment thereof binds to neither non-denatured human
fibroblast growth factor receptor 2 (hFGFR2) IIIb nor denatured
hFGFR2 IIIb in a preparation fixed in formalin.
54. A monoclonal antibody or an antigen binding fragment thereof
which is obtained by a method comprising the following steps (i)
and (ii): (i) culturing a cell comprising a polynucleotide encoding
a monoclonal antibody according to claim 48 or a vector comprising
a polynucleotide encoding a monoclonal antibody according to claim
48; and (ii) recovering the monoclonal antibody or antigen binding
fragment thereof from the cultures of step (i).
55. A composition comprising a monoclonal antibody or an antigen
binding fragment thereof according to claim 48.
56. A composition comprising a monoclonal antibody or an antigen
binding fragment thereof according to claim 48, wherein the
composition can detect or assay for hFGFR2 IIIc in a tissue
preparation which is paraffin-embedded and then deparaffinized, the
tissue preparation comprising the monoclonal antibody or antigen
binding fragment thereof according to claim 1.
57. The composition according to claim 56, wherein the preparation
is subjected to enzymatic treatment following the deparaffinization
treatment.
58. The composition according to claim 57, wherein the enzymatic
treatment is the reaction of protease at 20 to 38.degree. C.
59. A pharmaceutical composition comprising an antibody or an
antigen binding fragment thereof according to claim 48 that
specifically binds to hFGFR2 Mc.
60. A method for detecting or assaying hFGFR2 IIIc, comprising the
following step (i) or steps (i) and (ii): (i) contacting a
monoclonal antibody or an antigen binding fragment of the antibody
according to claim 48 or a composition comprising a monoclonal
antibody or an antigen binding fragment thereof according to claim
48 with a test preparation; and (ii) determining the test
preparation to be positive when hFGFR2 IIIc is detected or assayed
in the test preparation or when the expression level of hFGFR2 IIIc
in the test preparation is equivalent to or higher than
predetermined references; and determining the test preparation to
be negative when hFGFR2 Mc is not detected or assayed in the test
preparation or when the expression level of hFGFR2 IIIc in the test
preparation is equivalent to or lower than the predetermined
references.
61. The method according to claim 60, wherein the method is used in
a method for testing or diagnosing a hFGFR2 IIIc-positive
patient.
62. The method according to claim 61, wherein the testing or
diagnosing is for cancer.
63. A method for identifying a suitable individual for treating
with a pharmaceutical composition comprising an antibody
specifically binding to hFGFR2 or an antigen binding fragment of
the antibody, the method comprising the following step (i) or steps
(i) and (ii) or steps (i)-(iii): (i) contacting an antibody or an
antigen binding fragment of the antibody according to claim 48 or a
composition comprising a monoclonal antibody or an antigen binding
fragment thereof according to claim 48 with an individual-derived
sample; (ii) determining the individual to be positive when hFGFR2
IIIc is detected or assayed in the individual-derived sample or
when the expression level of hFGFR2 IIIn the individual derived
sample is equivalent to or higher than predetermined references,
and determining the individual to be negative when hFGFR2 IIIc is
not detected or assayed in the individual-derived sample or when
the expression level of hFGFR2 IIIc in the individual-derived
sample is equivalent to or lower than the predetermined references;
and (iii) treating the individual who is determined to be positive
with the pharmaceutical composition.
64. A method for treating a patient that is positive for hFGFR2
IIIc, comprising administering a pharmaceutical composition
comprising an antibody or antigen binding fragment thereof
according to claim 48 to a patient in need thereof.
65. A kit for testing or diagnosing a hFGFR2 IIIc-patient disease,
comprising an antibody or an antigen binding fragment of the
antibody according to claim 48.
66. The kit according to claim 65, wherein the testing or
diagnosing is for cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional Application of U.S.
patent application Ser. No. 15/567,936, filed on Oct. 19, 2017,
which is U.S. National Stage Application of International Patent
Application No. PCT/JP2016/062297, filed on Apr. 19, 2016, which
claims the benefit of priority to Japanese Patent Application No.
2015-085942, filed Apr. 20, 2015, the entireties of which are
incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, is
named 098065-0254 SL.txt and is 48.2 kb in size.
TECHNICAL FIELD
[0003] The present invention relates to a novel antibody, a
functional fragment of the antibody, a modified form of the
antibody, a nucleotide comprising a nucleotide sequence encoding
the amino acid sequence of the antibody, a vector having an insert
of the nucleotide, a cell comprising the nucleotide or the vector,
a method for producing the antibody, comprising the step of
culturing the cell, a pharmaceutical composition, a composition for
diagnosis or testing, a kit, etc.
BACKGROUND ART
[0004] Fibroblast growth factors (FGFs) are known to play an
important role in embryogenesis, tissue homeostasis, and metabolism
via FGF receptor (FGFR) signals (Non Patent Literature 1). In
humans, there are 22 FGFs (FGF1 to FGF14 and FGF16 to FGF23) and 4
FGF receptors (FGFR1 to FGFR4; hereinafter, collectively referred
to as "FGFRs") having a tyrosine kinase domain. These FGFRs are
each composed of an extracellular region comprising a ligand
binding site composed of 2 or 3 immunoglobulin-like domains (IgD1
to IgD3), a single-pass transmembrane region, and an intracellular
region comprising the tyrosine kinase domain. FGFR1, FGFR2, and
FGFR3 each have two splicing variants called IIIb and IIIc. These
isoforms differ in the sequence of approximately 50 amino acids in
the latter half of IgD3 and exhibit distinctive tissue distribution
and ligand specificity. It is generally known that the IIIb isoform
is expressed in epithelial cells, while the IIIc isoform is
expressed in mesenchymal cells. The binding of FGFs to FGFRs
induces the activation of many signaling pathways (Non Patent
Literature 1). As a result, FGFs and their corresponding receptors
control a wide range of cell functions including growth,
differentiation, migration, and survival.
[0005] The abnormal activation of FGFRs is known to participate in
particular types of malignant tumor development in humans (Non
Patent Literature 1 and 2). Particularly, FGFR2 signal
abnormalities such as the overexpression of FGFR2 and its ligand,
receptor mutations or gene amplification, and isoform switching,
have been found to be associated with cancer (Non Patent Literature
2, 3, 4, 5, 6 and 7).
[0006] As mentioned above, the possibility of FGFR2 as an excellent
therapeutic target for cancer has been suggested. In fact,
monoclonal antibodies against FGFR2 have been obtained and are
under clinical trial (Non Patent Literatures 8, 9, 10, and 11).
[0007] For these reasons, the provision of methods capable of
detecting expression of FGFR2 and its splicing variants is useful
in the testing or diagnosis of FGFR2-related diseases such as
cancer or of FGFR2 expression.
[0008] Many monoclonal antibodies which recognize human FGFR2 are
known. However, very few of these known antibodies are capable of
being used for immunohistological staining. For instance, only one
clone known as 1G3 (Non Patent Literature 12) recognises denatured
FGFR2 when fixed in formalin, which means it is capable of
immunohistological staining. Neither antibody cross-reactivity to
the denatured form of other FGFR families when fixed in formalin,
nor selective recognition of the denatured human FGFR2 splicing
variants IIIb and IIIc when fixed in formalin, have been
reported.
[0009] A monoclonal antibody which selectively recognizes a
denatured splicing variant IIIb of human FGFR2 fixed in formalin
has been reported (Patent Literature 1). However, no monoclonal
antibody which selectively recognizes a denatured human FGFR2 IIIc
has been identified.
CITATION LIST
Patent Literature
[0010] Patent Literature 1: WO2013/154206
Non Patent Literature
[0010] [0011] Non Patent Literature 1: Eswarakumar, V. P., et al.,
J. Cytokine Growth Factor Rev., Apr. 2005, Vol. 16 (No. 2), p.
139-149, published online on Feb 1, 2005, Review [0012] Non Patent
Literature 2: Turner, N. and Grose, R., Nat. Rev. Cancer, Feb.
2010, Vol. 10 (No. 2), p. 116-129, Review [0013] Non Patent
Literature 3: Easton, D. F., et al., Nature, Jun. 28, 2007, Vol.
447 (No. 7148), p. 1087-1093 [0014] Non Patent Literature 4: Hunter
D J, et al., Nat. Genet., Jul. 2007, Vol. 39 (No. 7), p. 870-874,
published online on May 27, 2007 [0015] Non Patent Literature 5:
Katoh, Y. and Katoh, M., Int. J. Mol. Med., Mar. 2009, Vol. 23 (No.
3), p. 307-311, Review [0016] Non Patent Literature 6: Chaffer, C.
L., et al., Differentiation, Nov. 2007, Vol. 75 (No. 9), p.
831-842, published online on Aug. 14, 2007, Review [0017] Non
Patent Literature 7: Carstens, R. P., et al., Oncogene, Dec. 18,
1997, Vol. 15 (No. 25), p. 3059-3065 [0018] Non Patent Literature
8: Zhao, W. M., et al., Clin. Cancer Res., Dec. 1, 2010, Vol. 16
(No. 23), p. 5750-5758, published online on Jul. 29, 2010 [0019]
Non Patent Literature 9: Bai, A., et al., Cancer Res., Oct. 1,
2010, Vol. 70 (No. 19), p. 7630-7639, published online on Aug. 13,
2010 [0020] Non Patent Literature 10: Clinical Trials. gov,
Clinical Trials. gov Identifier: NCT01881217, published online on
Jun. 13, 2013 [0021] Non Patent Literature 11: Clinical Trials.
gov, Clinical Trials. gov Identifier: NCT02368951, published online
on Feb. 16, 2015 [0022] Non Patent Literature 12: Vermeulen, J. F.,
et al., PloS One, published in 2013, Vol. 8 (No. 1), e53353
SUMMARY OF THE INVENTION
Technical Problem
[0023] An object of the present invention is to provide an antibody
against FGFR2.
[0024] Another object of the present invention is to provide a
composition for diagnosis or testing, etc., comprising an
anti-FGFR2 antibody.
[0025] An alternative object of the present invention includes the
provision of a nucleotide encoding the amino acid sequence of the
antibody, a vector having an insert of the nucleotide, a cell
comprising the nucleotide or the vector, a method for producing the
antibody, comprising the step of culturing the cell, etc.
[0026] A further alternative object of the present invention is to
provide a pharmaceutical composition and a method of treatment
and/or a composition for use in a method of treatment.
Solution to the Problem
[0027] The present inventors have conducted diligent studies to
attain the objects and consequently completed the present invention
by developing a novel anti-FGFR2 antibody and have found that FGFR2
can be detected using the antibody.
[0028] The present invention relates to: [0029] (1) A monoclonal
antibody or an antigen binding fragment thereof which has the
following properties (i) to (iii): [0030] (i) specifically binds to
non-denatured human fibroblast growth factor receptor 2 (hFGFR2)
IIIc; [0031] (ii) specifically binds to none of the following:
non-denatured human fibroblast growth factor receptor 1 (hFGFR1);
non-denatured human fibroblast growth factor receptor 3 (hFGFR3);
and non-denatured human fibroblast growth factor receptor 4
(hFGFR4); and [0032] (iii) specifically binds to denatured hFGFR2
IIIc in a preparation fixed in formalin; [0033] (2) The monoclonal
antibody or antigen binding fragment thereof according to (1),
wherein the monoclonal antibody or antigen binding fragment thereof
specifically binds to non-denatured human fibroblast growth factor
receptor 2 (hFGFR2) IIIb and denatured hFGFR2 IIIb in a preparation
fixed in formalin; [0034] (3) The monoclonal antibody or antigen
binding fragment thereof according to (1) or (2), wherein the
monoclonal antibody consists of a heavy chain comprising a CDRH1
consisting of the amino acid sequence represented by SEQ ID NO: 30
(FIG. 20) or an amino acid sequence derived from the amino acid
sequence by the substitution of one or two amino acids, a CDRH2
consisting of the amino acid sequence represented by SEQ ID NO: 31
(FIG. 20) or an amino acid sequence derived from the amino acid
sequence by the substitution of one or two amino acids, and a CDRH3
consisting of the amino acid sequence represented by SEQ ID NO: 32
(FIG. 20) or an amino acid sequence derived from the amino acid
sequence by the substitution of one or two amino acids; and a light
chain comprising a CDRL1 consisting of the amino acid sequence
represented by SEQ ID NO: 33 (FIG. 20) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids, a CDRL2 consisting of the amino acid sequence
represented by SEQ ID NO: 34 (FIG. 20) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids, and a CDRL3 consisting of the amino acid sequence
represented by SEQ ID NO: 35 (FIG. 20) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids; [0035] (4) The monoclonal antibody or antigen
binding fragment thereof according to (3), wherein the monoclonal
antibody consists of a heavy chain comprising a CDRH1 consisting of
the amino acid sequence represented by SEQ ID NO: 30 (FIG. 20), a
CDRH2 consisting of the amino acid sequence represented by SEQ ID
NO: (FIG. 20), and a CDRH3 consisting of the amino acid sequence
represented by SEQ ID NO: 32 (FIG. 20); and a light chain
comprising a CDRL1 consisting of the amino acid sequence
represented by SEQ ID NO: 33 (FIG. 20), a CDRL2 consisting of the
amino acid sequence represented by SEQ ID NO: 34 (FIG. 20), and a
CDRL3 consisting of the amino acid sequence represented by SEQ ID
NO: 35 (FIG. 20); [0036] (5) The monoclonal antibody or antigen
binding fragment thereof according to (1) or (2), wherein the
monoclonal antibody comprises the amino acid sequences of a heavy
chain variable region and a light chain variable region described
in any one of the following (i) to (iv): [0037] (i) the amino acid
sequence of a heavy chain variable region represented by SEQ ID NO:
8 (FIG. 15B) and the amino acid sequence of a light chain variable
region represented by SEQ ID NO: 10 (FIG. 15D); [0038] (ii) an
amino acid sequence 95% or more identical to the amino acid
sequence of a heavy chain variable region represented by SEQ ID NO:
8 (FIG. 15B) and an amino acid sequence 95% or more identical to
the amino acid sequence of a light chain variable region
represented by SEQ ID NO: 10 (FIG. 15D); [0039] (iii) an amino acid
sequence derived from the amino acid sequence of a heavy chain
variable region represented by SEQ ID NO: 8 (FIG. 15B) by the
substitution, deletion, insertion, or addition of 1 to several
amino acids and an amino acid sequence derived from the amino acid
sequence of a light chain variable region represented by SEQ ID NO:
10 (FIG. 15D) by the substitution, deletion, insertion, or addition
of 1 to several amino acids; and [0040] (iv) an amino acid sequence
encoded by the nucleotide sequence of a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
comprising a nucleotide sequence encoding the amino acid sequence
of a heavy chain variable region represented by SEQ ID NO: 8 (FIG.
15B), and an amino acid sequence encoded by the nucleotide sequence
of a polynucleotide that hybridizes under stringent conditions to a
polynucleotide comprising a nucleotide sequence encoding the amino
acid sequence of a light chain variable region represented by SEQ
ID NO: 10 (FIG. 15D); [0041] (6) The monoclonal antibody or antigen
binding fragment thereof according to any one of (1) to (5),
wherein the monoclonal antibody comprises the amino acid sequence
of a heavy chain represented by SEQ ID NO: 21 (FIG. 18B) and the
amino acid sequence of a light chain represented by SEQ ID NO: 23
(FIG. 18D); [0042] (7) The monoclonal antibody or antigen binding
fragment thereof according to (1) or (2), wherein the monoclonal
antibody or antigen binding fragment thereof binds to a site on
hFGFR2 IIIc and/or hFGFR2 IIIb which is recognized by an antibody
or an antigen binding fragment thereof according to any one of (3)
to (6), or competes with an antibody or an antigen binding fragment
thereof according to any one of (3) to (6) for binding to hFGFR2
IIIc and/or hFGFR2 IIIb; [0043] (8) The monoclonal antibody or
antigen binding fragment thereof according to (1), wherein the
monoclonal antibody or antigen binding fragment thereof binds to
neither non-denatured human fibroblast growth factor receptor 2
(hFGFR2) IIIb nor denatured hFGFR2 IIIb in a preparation fixed in
formalin; [0044] (9) The monoclonal antibody or antigen binding
fragment thereof according to (1) or (8), wherein the monoclonal
antibody consists of a heavy chain comprising a CDRH1 consisting of
the amino acid sequence represented by SEQ ID NO: 24 (FIG. 19) or
an amino acid sequence derived from the amino acid sequence by the
substitution of one or two amino acids, a CDRH2 consisting of the
amino acid sequence represented by SEQ ID NO: 25 (FIG. 19) or an
amino acid sequence derived from the amino acid sequence by the
substitution of one or two amino acids, and a CDRH3 consisting of
the amino acid sequence represented by SEQ ID NO: 26 (FIG. 19) or
an amino acid sequence derived from the amino acid sequence by the
substitution of one or two amino acids; and a light chain
comprising a CDRL1 consisting of the amino acid sequence
represented by SEQ ID NO: 27 (FIG. 19) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids, a CDRL2 consisting of the amino acid sequence
represented by SEQ ID NO: 28 (FIG. 19) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids, and a CDRL3 consisting of the amino acid sequence
represented by SEQ ID NO: 29 (FIG. 19) or an amino acid sequence
derived from the amino acid sequence by the substitution of one or
two amino acids; [0045] (10) The monoclonal antibody or antigen
binding fragment thereof according to (9), wherein the monoclonal
antibody consists of a heavy chain comprising a CDRH1 consisting of
the amino acid sequence represented by SEQ ID NO: 24 (FIG. 19), a
CDRH2 consisting of the amino acid sequence represented by SEQ ID
NO: 25 (FIG. 19), and a CDRH3 consisting of the amino acid sequence
represented by SEQ ID NO: 26 (FIG. 19); and a light chain
comprising a CDRL1 consisting of the amino acid sequence
represented by SEQ ID NO: 27 (FIG. 19), a CDRL2 consisting of the
amino acid sequence represented by SEQ ID NO: 28 (FIG. 19), and a
CDRL3 consisting of the amino acid sequence represented by SEQ ID
NO: 29 (FIG. 19); [0046] (11) The monoclonal antibody or antigen
binding fragment thereof according to (1) or (8), wherein the
monoclonal antibody comprises the amino acid sequences of a heavy
chain variable region and a light chain variable region described
in any one of the following (i) to (iv): [0047] (i) the amino acid
sequence of a heavy chain variable region represented by SEQ ID NO:
3 (FIG. 14B) and the amino acid sequence of a light chain variable
region represented by SEQ ID NO: 6 (FIG. 14D); [0048] (ii) an amino
acid sequence 95% or more identical to the amino acid sequence of a
heavy chain variable region represented by SEQ ID NO: 3 (FIG. 14B)
and an amino acid sequence 95% or more identical to the amino acid
sequence of a light chain variable region represented by SEQ ID NO:
6 (FIG. 14D); [0049] (iii) an amino acid sequence derived from the
amino acid sequence of a heavy chain variable region represented by
SEQ ID NO: 3 (FIG. 14B) by the substitution, deletion, insertion,
or addition of 1 to several amino acids and an amino acid sequence
derived from the amino acid sequence of a light chain variable
region represented by SEQ ID NO: 6 (FIG. 14D) by the substitution,
deletion, insertion, or addition of 1 to several amino acids; and
[0050] (iv) an amino acid sequence encoded by the nucleotide
sequence of a polynucleotide that hybridizes under stringent
conditions to a polynucleotide comprising a nucleotide sequence
encoding the amino acid sequence of a heavy chain variable region
represented by SEQ ID NO: 3 (FIG. 14B), and an amino acid sequence
encoded by the nucleotide sequence of a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
comprising a nucleotide sequence encoding the amino acid sequence
of a light chain variable region represented by SEQ ID NO: 6 (FIG.
14D); [0051] (12) The monoclonal antibody or antigen binding
fragment thereof according to any one of (1) and (8) to (11),
wherein the monoclonal antibody comprises the amino acid sequence
of a heavy chain represented by SEQ ID NO: 15 (FIG. 17B) and the
amino acid sequence of a light chain represented by SEQ ID NO: 19
(FIG. 17D); [0052] (13) The monoclonal antibody or antigen binding
fragment thereof according to (1) or (8), wherein the monoclonal
antibody or antigen binding fragment thereof binds to a site on
hFGFR2 IIIc which is recognized by an antibody or an antigen
binding fragment thereof according to any one of (9) to (12), or
competes with an antibody or an antigen binding fragment thereof
according to any one of (9) to (12) for binding to hFGFR2 IIIc;
[0053] (14) A polynucleotide encoding a monoclonal antibody
according to any one of (1) to (13) ; [0054] (15) A vector
comprising a polynucleotide according to (14); [0055] (16) A cell
comprising a polynucleotide according to (14) or a vector according
to (15); [0056] (17) A method for producing a monoclonal antibody
or an antigen binding fragment thereof according to (1), (2), or
(8), comprising the following steps (i) and (ii): [0057] (i)
culturing a cell according to (16); and [0058] (ii) recovering the
monoclonal antibody or antigen binding fragment thereof from the
cultures of step (i); [0059] (18) A monoclonal antibody or an
antigen binding fragment thereof which is obtained by a method
according to (17); [0060] (19) A composition comprising a
monoclonal antibody or an antigen binding fragment thereof
according to any one of (1) to (7) and (18) ; [0061] (20) The
composition according to (19), wherein the composition is used in a
method for detecting or assaying hFGFR2 IIIc and hFGFR2 IIIb in a
tissue preparation which is paraffin-embedded and then
deparaffinized, the tissue preparation comprising the monoclonal
antibody or antigen binding fragment thereof according to any one
of (1) to (7) and (18) (hereinafter, this tissue preparation is
simply referred to as a "preparation"); [0062] (21) The composition
according to (20), wherein the preparation is subjected to heat
treatment following the deparaffinization treatment; [0063] (22)
The composition according to (21), wherein the heat treatment is
performed at 90 to 100.degree. C. and at pH 8 to 10; [0064] (23)
The composition according to any one of (19) to (22), wherein the
composition is used in a method for detecting or assaying hFGFR2
IIIc and hFGFR2 IIIb in a preparation, the method comprising the
step of contacting the monoclonal antibody or antigen binding
fragment thereof according to any one of (1) to (7) and (18) or the
composition according to (19) with the test preparation; [0065]
(24) The composition according to (23), wherein the method for
detecting or assaying hFGFR2 IIIc and hFGFR2 IIIb further comprises
the step of determining the test preparation to be positive when
hFGFR2 IIIc and hFGFR2 IIIb are detected or assayed in the test
preparation or when the expression levels of hFGFR2 IIIc and hFGFR2
IIIb in the test preparation are equivalent to or higher than
predetermined references; and determining the test preparation to
be negative when neither hFGFR2 IIIc nor hFGFR2 IIIb is detected or
assayed in the test preparation or when the expression levels of
hFGFR2 IIIc and hFGFR2 IIIb in the test preparation are equivalent
to or lower than the predetermined references; [0066] (25) The
composition according to any one of (19) to (24), wherein the
composition is used in a method for testing or diagnosing a hFGFR2
IIIc- and hFGFR2 IIIb-positive disease; [0067] (26) The composition
according to (25), wherein the method for testing or diagnosing a
hFGFR2 IIIc- and hFGFR2 IIIb-positive disease comprises determining
a test subject, from which a test preparation determined to be
positive in the detection or assay of hFGFR2 IIIc and hFGFR2 IIIb
is derived, to be suitable for a method for treating or preventing
the hFGFR2 IIIc- and hFGFR2 IIIb-positive disease, comprising the
step of administering an antibody specifically binding to hFGFR2
IIIc and hFGFR2 IIIb or an antigen binding fragment thereof, and
determining a test subject from which a test preparation determined
to be negative therein is derived, to be not suitable for the
method for treating or preventing the hFGFR2 IIIc- and hFGFR2
IIIb-positive disease, comprising the step of administering an
antibody specifically binding to hFGFR2 IIIc and hFGFR2 IIIb or an
antigen binding fragment thereof; [0068] (27) The composition
according to (25) or (26), wherein the hFGFR2 IIIc- and hFGFR2
IIIb-positive disease is hFGFR2 IIIc- and hFGFR2 IIIb-positive
cancer; [0069] (28) A pharmaceutical composition which is
administered to a test subject described in any one of the
following (i) to (iii), the pharmaceutical composition comprising
an antibody specifically binding to hFGFR2 IIIc and hFGFR2 IIIb or
an antigen binding fragment thereof: [0070] (i) a test subject from
which a test preparation is derived, wherein hFGFR2 IIIc and hFGFR2
IIIb are detected or assayed in the test preparation using a
composition according to any one of (19) to (23) and (25);
[0071] (ii) a test subject from which a test preparation determined
to be positive in the detection or assay of hFGFR2 IIIc and hFGFR2
IIIb using a composition according to (24) is derived; and [0072]
(iii) a test subject determined, using a composition according to
(26) or (27), to be suitable for the treatment or prevention of a
hFGFR2 IIIc- and hFGFR2 IIIb-positive disease, comprising the step
of administering an antibody specifically binding to hFGFR2 IIIc
and hFGFR2 IIIb or an antigen binding fragment thereof; [0073] (29)
A method for detecting or assaying hFGFR2 IIIc and hFGFR2 IIIb,
comprising the following step (i) or steps (i) and (ii): [0074] (i)
contacting a monoclonal antibody or an antigen binding fragment of
the antibody according to any one of (1) to (7) and [0075] (18) or
a composition according to (19) to (22) with a test preparation;
and [0076] (ii) determining the test preparation to be positive
when hFGFR2 IIIc and hFGFR2 IIIb are detected or assayed in the
test preparation or when the expression levels of hFGFR2 IIIc and
hFGFR2 IIIb in the test preparation are equivalent to or higher
than predetermined references; and determining the test preparation
to be negative when neither hFGFR2 IIIc nor hFGFR2 IIIb is detected
or assayed in the test preparation or when the expression levels of
hFGFR2 IIIc and hFGFR2 IIIb in the test preparation are equivalent
to or lower than the predetermined references; [0077] (30) A method
for identifying a suitable individual for treating with a
pharmaceutical composition comprising an antibody specifically
binding to hFGFR2 or an antigen binding fragment of the antibody,
the method comprising the following step (i) or steps (i) and (ii):
[0078] (i) contacting an antibody or an antigen binding fragment of
the antibody according to any one of (1) to (7) and (18) or a
composition according to (19) to (22) with an individual-derived
sample; and [0079] (ii) determining the individual to be positive
when hFGFR2 IIIc and hFGFR2 IIIb are detected or assayed in the
individual-derived sample or when the expression levels of hFGFR2
IIIc and hFGFR2 IIIb in the individual-derived sample are
equivalent to or higher than predetermined references, and
determining the individual to be negative when neither hFGFR2 IIIc
nor hFGFR2 IIIb is detected or assayed in the individual-derived
sample or when the expression levels of hFGFR2 IIIc and hFGFR2 IIIb
in the individual-derived sample are equivalent to or lower than
the predetermined references; [0080] (31) A method for detecting or
assaying hFGFR2 IIIc, comprising the following steps (i) to (iii):
[0081] (i) contacting a composition comprising an antibody or an
antigen binding fragment of the antibody according to any one of
(1) to (7) and (18) with a test preparation to detect or assay
hFGFR2 IIIb and hFGFR2 IIIc in the test preparation; [0082] (ii)
contacting a composition comprising an antibody specifically
binding to hFGFR2 IIIb or an antigen binding fragment of the
antibody with the test preparation to detect or assay hFGFR2 IIIb
in the test preparation; and [0083] (iii) comparing the results of
the detection or assay in step (i) with the results of detection or
assay in step (ii) or subtracting the results of detection or assay
in step (ii) from the results of detection or assay in step (i) to
obtain detection or assay results or a value of hFGFR2 IIIc in the
sample; [0084] (32) The method according to any one of (29) to
(31), wherein the method is used in a method for testing or
diagnosing a hFGFR2 IIIc- and hFGFR2 IIIb-positive disease; [0085]
(33) The method according to (30), wherein the method is used in a
method for identifying an individual having a hFGFR2 IIIc- and
hFGFR2 IIIb-positive disease or being at risk thereof; [0086] (34)
A method for treating a hFGFR2 IIIc- and hFGFR2 IIIb-positive
disease, comprising administering a pharmaceutical composition
comprising an antibody specifically binding to hFGFR2 or an antigen
binding fragment of the antibody to a test subject described in any
one of (i) to (iii) of (28); [0087] (35) A kit for testing or
diagnosing a hFGFR2 IIIc- and hFGFR2 IIIb-positive disease,
comprising an antibody or an antigen binding fragment of the
antibody according to any one of (1) to (7) and (18) ; [0088] (36)
The method according to any one of (32) to (34) or the kit
according to (35), wherein the hFGFR2 IIIc- and hFGFR2
IIIb-positive disease is hFGFR2 IIIc- and hFGFR2 IIIb-positive
cancer; [0089] (37) A composition comprising a monoclonal antibody
or an antigen binding fragment thereof according to any one of (1),
(8) to (13), and (18); [0090] (38) The composition according to
(37), wherein the composition is used in a method for detecting or
assaying hFGFR2 IIIc in a tissue preparation paraffin-embedded and
then deparaffinized, the tissue preparation comprising the
monoclonal antibody or antigen binding fragment thereof according
to any one of (1), (8) to (13), and (18) (hereinafter, this tissue
preparation is simply referred to as a "preparation"); [0091] (39)
The composition according to (38), wherein the preparation is
subjected to enzymatic treatment following the deparaffinization
treatment; [0092] (40) The composition according to (39), wherein
the enzymatic treatment is the reaction of protease at 20 to
38.degree. C.; [0093] (41) The composition according to any one of
(37) to (40), wherein the composition is used in a method for
detecting or assaying hFGFR2 IIIc in a preparation, the method
comprising the step of contacting the monoclonal antibody or
antigen binding fragment thereof according to any one of (1), (8)
to (13), and (18) or the composition according to (37) with the
test preparation; [0094] (42) The composition according to (41),
wherein the method for detecting or assaying hFGFR2 IIIc further
comprises the step of determining the test preparation to be
positive when hFGFR2 IIIc is detected or assayed in the test
preparation or when the expression level of hFGFR2 IIIc in the test
preparation is equivalent to or higher than a predetermined
reference; and determining the test preparation to be negative when
no hFGFR2 IIIc is detected or assayed in the test preparation or
when the expression level of hFGFR2 IIIc in the test preparation is
equivalent to or lower than the predetermined reference; [0095]
(43) The composition according to any one of (37) to (42), wherein
the composition is used in a method for testing or diagnosing a
hFGFR2 IIIc-positive disease; [0096] (44) The composition according
to (43), wherein the method for testing or diagnosing a hFGFR2
IIIc-positive disease comprises determining a test subject from
which a test preparation determined to be positive in the detection
or assay of hFGFR2 IIIc is derived, to be suitable for a method for
treating or preventing the hFGFR2 IIIc-positive disease, comprising
the step of administering an antibody specifically binding to
hFGFR2 IIIc or an antigen binding fragment thereof, and determining
a test subject from which a test preparation determined to be
negative therein is derived, to be not suitable for the method for
treating or preventing the hFGFR2 IIIc-positive disease, comprising
the step of administering an antibody specifically binding to
hFGFR2 IIIc or an antigen binding fragment thereof; [0097] (45) The
composition according to (43) or (44), wherein the hFGFR2
IIIc-positive disease is hFGFR2 IIIc-positive cancer; [0098] (46) A
pharmaceutical composition which is administered to a test subject
described in any one of the following (i) to (iii), the
pharmaceutical composition comprising an antibody specifically
binding to hFGFR2 IIIc or an antigen binding fragment thereof:
[0099] (i) a test subject from which a test preparation is derived,
wherein hFGFR2 IIIc is detected or assayed in the test preparation
using a composition according to any one of (37) to (40) and (43) ;
[0100] (ii) a test subject from which a test preparation determined
to be positive in the detection or assay of hFGFR2 IIIc using a
composition according to (42) is derived; and [0101] (iii) a test
subject determined, using a composition according to (44) or (45),
to be suitable for the treatment or prevention of a hFGFR2
IIIc-positive disease, comprising the step of administering an
antibody specifically binding to hFGFR2 IIIc or an antigen binding
fragment thereof; and [0102] (47) The pharmaceutical composition
according to (46), wherein the hFGFR2 IIIc-positive disease is
hFGFR2 IIIc-positive cancer.
BRIEF DESCRIPTION OF DRAWINGS
[0103] FIG. 1 is a diagram showing the results of testing binding
activity of mouse chimeric anti-FGFR2 antibodies FR2-2nd_#028 and
FR2-2nd_#023 against each non-denatured molecule of the human FGFR
family. The vertical axis represents a relative value of the
average fluorescence intensity assayed by flow cytometry.
[0104] FIG. 2A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a commercially available anti-FGFR2 antibody
(18601).
[0105] FIG. 2B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a commercially available anti-FGFR2 antibody
(18601).
[0106] FIG. 2C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a commercially available anti-FGFR2 antibody
(18601).
[0107] FIG. 2D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a commercially available anti-FGFR2 antibody (18601).
[0108] FIG. 3 is a diagram showing the results of immunostaining
blocks of SNU-16 (A), NCI-H716 (B), and KATO III (C) cells using a
commercially available anti-FGFR2 antibody (18601).
[0109] FIG. 4A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#028.
[0110] FIG. 4B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#028.
[0111] FIG. 4C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#028.
[0112] FIG. 4D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a rat anti-FGFR2 antibody FR2-2nd_#028.
[0113] FIG. 5A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028.
[0114] FIG. 5B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028.
[0115] FIG. 5C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028.
[0116] FIG. 5D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a mouse chimeric anti-FGFR2 antibody FR2-2nd_#028.
[0117] FIG. 6 is a diagram showing the results of immunostaining
blocks of SNU-16 (A), NCI-H716 (B), and KATO III (C) cells using a
rat anti-FGFR2 antibody FR2-2nd_#028.
[0118] FIG. 7 is a diagram showing the results of immunostaining
blocks of SNU-16 (A), NCI-H716 (B), and KATO III (C) cells using a
mouse chimeric anti-FGFR2 antibody FR2-2nd_#028.
[0119] FIG. 8 is a diagram showing the results of immunostaining a
xenograft tumor sample of NCI-H716 cells using a rat anti-FGFR2
antibody FR2-2nd_#028 (upper: low magnification, lower: high
magnification of the upper boxed site).
[0120] FIG. 9A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#023.
[0121] FIG. 9B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#023.
[0122] FIG. 9C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a rat anti-FGFR2 antibody FR2-2nd_#023.
[0123] FIG. 9D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a rat anti-FGFR2 antibody FR2-2nd_#023.
[0124] FIG. 10A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023.
[0125] FIG. 10B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023.
[0126] FIG. 10C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023.
[0127] FIG. 10D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a mouse chimeric anti-FGFR2 antibody FR2-2nd_#023.
[0128] FIG. 11 is a diagram showing the results of immunostaining
blocks of SNU-16 (A), NCI-H716 (B), and KATO III (C) cells using a
rat anti-FGFR2 antibody FR2-2nd_#023.
[0129] FIG. 12 is a diagram showing the results of immunostaining
blocks of SNU-16 (A), NCI-H716 (B), and KATO III (C) cells using a
mouse chimeric anti-FGFR2 antibody FR2-2nd_#023.
[0130] FIG. 13A is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR1 IIIb or FGFR1
IIIc molecule, using a commercially available anti-FGFR2 antibody
(ab58201).
[0131] FIG. 13B is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR2 IIIb or FGFR2
IIIc molecule, using a commercially available anti-FGFR2 antibody
(ab58201).
[0132] FIG. 13C is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR3 IIIb or FGFR3
IIIc molecule, using a commercially available anti-FGFR2 antibody
(ab58201).
[0133] FIG. 13D is a diagram showing the results of immunostaining
blocks of 293.alpha. cells forced to express a FGFR4 molecule and
blocks of 293.alpha. cells transfected with an empty vector, using
a commercially available anti-FGFR2 antibody (ab58201).
[0134] FIG. 14A shows a nucleotide sequence encoding the heavy
chain variable region of the rat anti-FGFR2 antibody FR2-2nd_#028
(SEQ ID NO: 2).
[0135] FIG. 14B shows the amino acid sequence of the heavy chain
variable region of the rat anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID
NO: 3).
[0136] FIG. 14C shows a nucleotide sequence encoding the light
chain variable region of the rat anti-FGFR2 antibody FR2-2nd_#028
(SEQ ID NO: 5).
[0137] FIG. 14D shows the amino acid sequence of the light chain
variable region of the rat anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID
NO: 6).
[0138] FIG. 15A shows a nucleotide sequence encoding the heavy
chain variable region of the rat anti-FGFR2 antibody FR2-2nd_#023
(SEQ ID NO: 7).
[0139] FIG. 15B shows the amino acid sequence of the heavy chain
variable region of the rat anti-FGFR2 antibody FR2-2nd_#023 (SEQ ID
NO: 8).
[0140] FIG. 15C shows a nucleotide sequence encoding the light
chain variable region of the rat anti-FGFR2 antibody FR2-2nd_#023
(SEQ ID NO: 9).
[0141] FIG. 15D shows the amino acid sequence of the light chain
variable region of the rat anti-FGFR2 antibody FR2-2nd_#023 (SEQ ID
NO: 10).
[0142] FIG. 16 shows the nucleotide sequence of a DNA fragment
comprising a nucleotide sequence encoding the amino acid sequence
of a human .kappa. chain secretory signal sequence and a human
.kappa. chain constant region (SEQ ID NO: 11).
[0143] FIG. 17A shows a nucleotide sequence comprising a nucleotide
sequence (nucleotide positions 26 to 1411) encoding the heavy chain
of the mouse chimeric anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID NO:
14).
[0144] FIG. 17B shows the amino acid sequence of the heavy chain of
the mouse chimeric anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID NO:
15).
[0145] FIG. 17C shows a nucleotide sequence comprising a nucleotide
sequence (nucleotide positions 26 to 724) encoding the light chain
of the mouse chimeric anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID NO:
18).
[0146] FIG. 17D shows the amino acid sequence of the light chain of
the mouse chimeric anti-FGFR2 antibody FR2-2nd_#028 (SEQ ID NO:
19).
[0147] FIG. 18A shows a nucleotide sequence comprising a nucleotide
sequence (nucleotide positions 26 to 1423) encoding the heavy chain
of the mouse chimeric anti-FGFR2 antibody FR2-2nd_#023 (SEQ ID NO:
20).
[0148] FIG. 18B shows the amino acid sequence of the heavy chain of
the mouse chimeric anti-FGFR2 antibody FR2-2nd_#023 (SEQ ID NO:
21).
[0149] FIG. 18C shows a nucleotide sequence comprising a nucleotide
sequence (nucleotide positions 26 to 724) encoding the light chain
of the mouse chimeric anti-FGFR2 antibody FR2-2nd_#023 (SEQ ID NO:
22).
[0150] FIG. 18D shows the amino acid sequence of the light chain of
the mouse chimeric anti-FGFR2 antibody FR2-2nd_190 023 (SEQ ID NO:
23).
[0151] FIG. 19 shows the amino acid sequences of CDRH1 to CDRH3 and
CDRL1 to CDRL3 of the rat anti-human FGFR2 antibody FR2-2nd_#028
(SEQ ID NOs: 24 to 29).
[0152] FIG. 20 shows the amino acid sequences of CDRH1 to CDRH3 and
CDRL1 to CDRL3 of the rat anti-human FGFR2 antibody FR2-2nd_#023
(SEQ ID NOs: 30 to 35).
DESCRIPTION OF EMBODIMENTS
1. Definitions
[0153] In the present invention, the term "gene" means a nucleotide
comprising a nucleotide sequence encoding the amino acids of a
protein, or its complementary strand. The term "gene" is meant to
include, for example, a polynucleotide, an oligonucleotide, DNA,
mRNA, cDNA, and cRNA as the nucleotide comprising a nucleotide
sequence encoding the amino acids of a protein, or its
complementary strand. Such a gene may be a single-stranded,
double-stranded, or triple or more stranded nucleotide. The term
"gene" is also meant to include an association of DNA and RNA
strands, a mixture of ribonucleotides (RNAs) and
deoxyribonucleotides (DNAs) on one nucleotide strand, and a
double-stranded or triple or more stranded nucleotide comprising
such a nucleotide strand. Examples of the "FGFR2 gene" of the
present invention can include DNA, mRNA, cDNA, and cRNA comprising
a nucleotide sequence encoding the amino acid sequence of the FGFR2
protein.
[0154] In the present invention, the term "nucleotide" has the same
meaning as a "nucleic acid" and is also meant to include, for
example, DNA, RNA, a probe, an oligonucleotide, a polynucleotide,
and a primer. Such a nucleotide is a single-stranded,
double-stranded, or triple or more stranded nucleotide. The term
"nucleotide" is also meant to include an association of DNA and RNA
strands, a mixture of ribonucleotides (RNAs) and
deoxyribonucleotides (DNAs) on one nucleotide strand, and an
associate of two strands or three or more strands comprising such a
nucleotide strand.
[0155] In the present invention, the terms "polypeptide",
"peptide", and "protein" have the same meaning.
[0156] In the present invention, the term "antigen" has the same
meaning as "immunogen".
[0157] In the present invention, the term "cell" also includes, for
example, various cells derived from individual animals, subcultured
cells, primary cultured cells, cell lines, recombinant cells, and
microbial cells.
[0158] In the present invention, antibodies recognizing FGFR2,
FGFR2 IIIb, FGFR2 IIIc, FGFR3, FGFR4, and the like are also
referred to as an "anti-FGFR2 antibody", an "anti-FGFR2 IIIb
antibody", an "anti-FGFR2 IIIc antibody", an "anti-FGFR3 antibody",
and an "anti-FGFR4 antibody", respectively. These antibodies
include chimeric antibodies, humanized antibodies, human
antibodies, and the like.
[0159] In the present invention, the term "functional fragment of
the antibody" means an antibody fragment that exhibits at least a
portion of the functions exhibited by the original antibody.
Examples of the "functional fragment of the antibody" can include,
but are not limited to, Fab, F(ab')2, scFv, Fab', and single chain
immunoglobulin. Such a functional fragment of the antibody may be
obtained by treating a full-length molecule of the antibody protein
with an enzyme such as papain or pepsin, or may be a recombinant
protein produced in an appropriate host cell using a recombinant
gene.
[0160] In the present invention, the "site" to which an antibody
binds, i.e., the "site" recognized by an antibody, means a partial
peptide or partial conformation on an antigen bound or recognized
by the antibody. In the present invention, such a site is also
referred to as an epitope or an antibody binding site. Examples of
the site on the FGFR2 protein bound or recognized by the anti-FGFR2
antibody of the present invention can include a partial peptide or
partial conformation on the FGFR2 protein.
[0161] The heavy and light chains of an antibody molecule are known
to each have three complementarity determining regions (CDRs). The
complementarity determining regions are also called hypervariable
domains. These regions are located in the variable regions of the
antibody heavy and light chains. These sites have a particularly
highly variable primary structure and are usually separated at
three positions on the respective primary structures of heavy and
light chain polypeptide strands. In the present invention, the
complementarity determining regions of the antibody are referred to
as CDRH1, CDRH2, and CDRH3 from the amino terminus of the heavy
chain amino acid sequence for the complementarity determining
regions of the heavy chain; and as CDRL1, CDRL2, and CDRL3 from the
amino terminus of the light chain amino acid sequence for the
complementarity determining regions of the light chain. These sites
are proximal to each other on the three-dimensional structure and
determine specificity for the antigen to be bound.
[0162] In the present invention, the term "antibody mutant" means a
polypeptide that has an amino acid sequence derived from the amino
acid sequence of the original antibody by the substitution,
deletion, addition, and/or insertion (hereinafter, collectively
referred to as a "mutation") of amino acid(s) and binds to the
FGFR2 protein of the present invention. The number of mutated amino
acids in such an antibody mutant is 1 to 2, 1 to 3, 1 to 4, 1 to 5,
1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 1 to 20,
1 to 25, 1 to 30, 1 to 40, or 1 to 50. Such an antibody mutant is
also encompassed by the "antibody" of the present invention.
[0163] In the present invention, the term "several" in "1 to
several" refers to 3 to 10.
[0164] Examples of activities or properties exhibited by the
antibody of the present invention can include biological activities
or physicochemical properties and can specifically include various
biological activities, binding activity against an antigen or an
epitope, stability during production or storage, and thermal
stability.
[0165] In the present invention, the phrase "hybridizing under
stringent conditions" means hybridization under conditions
involving hybridization at 65.degree. C. in a solution containing
5.times.SSC, followed by washing at 65.degree. C. for 20 minutes in
an aqueous solution containing 2.times.SSC-0.1% SDS, at 65.degree.
C. for 20 minutes in an aqueous solution containing
0.5.times.SSC-0.1% SDS, and at 65.degree. C. for 20 minutes in an
aqueous solution containing 0.2.times.SSC-0.1% SDS, or
hybridization under conditions equivalent thereto. SSC means an
aqueous solution of 150 mM NaCl-15 mM sodium citrate, and
n.times.SSC means SSC with an n-fold concentration.
[0166] In the present invention, the term "cytotoxicity" refers to
some pathological change brought about to cells and means not only
direct trauma but any structural or functional damage to cells,
including DNA cleavage, formation of base dimers, chromosomal
break, damage to mitotic apparatus, and reduction in the activities
of various enzymes.
[0167] In the present invention, the term "cytotoxic activity"
means activity that causes the cytotoxicity mentioned above. In the
present invention, the term "antibody dependent cellular cytotoxic
activity", also called "ADCC activity", means the effect or
activity of damaging target cells such as tumor cells by NK cells
via antibodies.
[0168] In the present invention, the term "antibody dependent cell
phagocytosis activity", also called "ADCP activity", means the
effect or activity of englobing target cells such as tumor cells by
monocyte or macrophage cells via antibodies. This activity is also
referred to as "antibody dependent phagocytic effect or
activity".
[0169] In the present invention, the term "complement dependent
cytotoxic activity", also called "CDC activity", means the effect
or activity of damaging target cells such as tumor cells by
complement via antibodies.
[0170] In the present invention, the term "cancer" has the same
meaning as "tumor".
[0171] In the present invention, the term "immunohistochemistry
(IHC)" means a histological (histochemical) approach of detecting
an antigen in a tissue preparation. The term immunohistochemistry
is synonymous with an "immune antibody method" and has the same
meaning as "immunostaining".
[0172] In the present invention, "denatured" FGFR means a FGFR
molecule in a preparation fixed in formalin. The "denatured" FGFR
also refers to a FGFR molecule in a preparation fixed in formalin,
then treated with paraffin, and deparaffinized.
[0173] In the present invention, "non-denatured" FGFR means FGFR in
a sample that is not fixed in formalin. The "non-denatured" FGFR
also refers to a FGFR molecule in a preparation that is not fixed
in formalin.
2. Antigenic Protein
[0174] (2-1) Properties
[0175] FGFRs are receptor proteins that bind to fibroblast growth
factors (FGFs). In the present invention, FGFRs are derived from
vertebrates, preferably mammals, more preferably humans. Human FGFs
and FGFRs are classified into 22 FGFs (FGF1 to FGF14 and FGF16 to
FGF23) and 4 FGFRs (FGFR1 to FGFR4) having a tyrosine kinase
domain, respectively. These FGFRs are each composed of an
extracellular region comprising a ligand binding site composed of 2
or 3 immunoglobulin-like domains (IgD1 to IgD3), a single-pass
transmembrane region, and an intracellular region comprising the
tyrosine kinase domain. FGFR1, FGFR2, and FGFR3 each have two
splicing variants called IIIb and IIIc. These isoforms differ in
the sequence of approximately 50 amino acids in the latter half of
IgD3 and exhibit distinctive tissue distribution and ligand
specificity. FGFRs have the following activities: (1) binding to
FGFs; (2) this binding dimerizes the FGFRs; (3) this dimerization
phosphorylates the FGFRs at their particular tyrosine residues; (4)
this phosphorylation promotes the recruitment of adaptor proteins
such as FGFR substrate 2.alpha. (FRS2.alpha.); and (5) this
transduces signals generated by FGF stimulation to cells or tissues
expressing the FGFRs or activates signal transduction.
[0176] The FGFR2 protein according to the present invention has the
following properties:
[0177] (i) Binding to FGF.
[0178] The FGFR2 IIIb protein typically binds to one or two or more
FGFs selected from the group consisting of FGF1, FGF3, FGF7 (KGF),
FGF10, FGF22, and FGF23. The FGFR2 IIIb protein may bind to other
FGFs and may not bind to mutated forms of the FGFs included in the
above group.
[0179] The FGFR2 IIIc protein typically binds to one or two or more
FGFs selected from the group consisting of FGF1, FGF2, FGF4, FGF6,
FGF9, FGF17, FGF18, FGF21, and FGF23. The FGFR2 IIIc protein may
bind to other FGFs and may not bind to mutated forms of the FGFs
included in the above group.
[0180] (ii) Transducing signals generated by FGF stimulation into
FGFR2-expressing cells or tissues
[0181] Examples of the transduction of signals generated by FGF
stimulation can include, but are not particularly limited to, FGFR2
autophosphorylation, recruitment of FGFR substrates and promotion
thereof, and activation of signaling pathways such as MAPK, PI3K,
Akt, and extracellular signal-regulated kinase (ERK) pathways via
these events. Examples of the FGFR substrates can include FGFR
substrate 2.alpha. (FRS2.alpha.).
[0182] Testing methods for evaluating the activation of this signal
transduction and the inhibition thereof are not particularly
limited and can be arbitrarily selected from methods known in the
art. Examples thereof can include evaluation systems for ERK signal
transduction, and Elk1 luciferase reporter assay described
later.
[0183] (iii) The FGFR2 IIIb protein according to the present
invention comprises an amino acid sequence described in any one of
the following (a) to (d) (hereinafter, referred to as an "FGFR2
IIIb amino acid sequence"), consists of an amino acid sequence
comprising the FGFR2 IIIb amino acid sequence, or consists of the
FGFR2 IIIb amino acid sequence: [0184] (a) the amino acid sequence
represented by the amino acid sequence of NP 075259 published on
the database; [0185] (b) an amino acid sequence that exhibits 80%
or higher, 82% or higher, 84% or higher, 86% or higher, 88% or
higher, 90% or higher, 92% or higher, 94% or higher, 96% or higher,
98% or higher, or 99% or higher, sequence identity to the amino
acid sequence represented by the amino acid sequence of NP_075259
and is carried by a polypeptide having FGF binding activity; [0186]
(c) an amino acid sequence that is derived from the amino acid
sequence represented by the amino acid sequence of NP_075259 by the
substitution, deletion, addition, or insertion of 1 to 50, 1 to 45,
1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to
8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and
is carried by a polypeptide having FGF binding activity; and [0187]
(d) an amino acid sequence that is encoded by the nucleotide
sequence of a nucleotide hybridizing under stringent conditions to
a nucleotide having a nucleotide sequence complementary to a
nucleotide sequence encoding the amino acid sequence represented by
the amino acid sequence of NP_075259 and is carried by a
polypeptide having FGF binding activity.
[0188] The polypeptide described in any one of (b) to (d) may have
FGFR2 activities other than FGF binding activity.
[0189] The FGFR2 IIIc protein according to the present invention
comprises an amino acid sequence described in any one of the
following (a) to (d) (hereinafter, referred to as an "FGFR2 IIIc
amino acid sequence"), consists of an amino acid sequence
comprising the FGFR2 IIIc amino acid sequence, or consists of the
FGFR2 IIIc amino acid sequence: [0190] (a) an amino acid sequence
represented by NP_000132 published on the database; [0191] (b) an
amino acid sequence that exhibits 80% or higher, 82% or higher, 84%
or higher, 86% or higher, 88% or higher, 90% or higher, 92% or
higher, 94% or higher, 96% or higher, 98% or higher, or 99% or
higher, sequence identity to the amino acid sequence represented by
NP_000132 and is carried by a polypeptide having FGF binding
activity; [0192] (c) an amino acid sequence that is derived from
the amino acid sequence represented by NP 000132 by the
substitution, deletion, addition, or insertion of 1 to 50, 1 to 45,
1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to
8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1 amino acid(s) and
is carried by a polypeptide having FGF binding activity; and [0193]
(d) an amino acid sequence that is encoded by the nucleotide
sequence of a nucleotide hybridizing under stringent conditions to
a nucleotide having a nucleotide sequence complementary to a
nucleotide sequence encoding the amino acid sequence represented by
NP_000132 and is carried by a polypeptide having FGF binding
activity.
[0194] The polypeptide described in any one of (b) to (d) may have
FGFR2 activities other than FGF binding activity.
[0195] (iv) The FGFR2 protein of the present invention can be
obtained from FGFR2-expressing cells, tissues, or cancer tissues,
cells derived from the tissues, cultures of the cells, and the
like, of a vertebrate, preferably of a mammal, more preferably of a
rodent such as a mouse or a rat and a human, even more preferably
of a mouse, rat and a human.
[0196] Examples of normal tissues highly expressing FGFR2 can
include the brain, the large intestine, thyroid glands, the uterus,
the gallbladder, and the skin. Gene amplification is found in some
cancers highly expressing FGFR2, such as stomach cancer and breast
cancer, while overexpression is found in some cancers highly
expressing FGFR2, such as pancreatic cancer and ovarian cancer.
Examples of cultured cell lines highly expressing FGFR2 IIIb can
include stomach cancer cell lines and breast cancer cell lines.
Examples of cultured cell lines highly expressing FGFR2 IIIc can
include colorectal (cecal) cancer cell lines. Examples of cancer
tissues expressing FGFR2 IIIc can include tissues with uterine
cervix cancer and non-small cell lung cancer. Of these cancers,
uterine cervix cancer highly expresses FGFR2 IIIc.
[0197] The FGFR2 protein of the present invention may be a native
(non-recombinant) or recombinant protein. The FGFR2 protein is also
meant to include fusion products with another peptide or protein
such as a carrier or a tag. The FGFR2 protein is further meant to
include forms provided with chemical modification including the
addition of a polymer such as PEG and/or with biological
modification including sugar chain modification. Moreover, the
FGFR2 protein of the present invention is meant to include an FGFR2
protein fragment. An FGFR2 protein fragment possessing the
properties described above in (i) and/or (ii) is referred to as a
functional fragment of the FGFR2 protein.
[0198] (2-2) Antigen Gene
[0199] The FGFR2 IIIb gene according to the present invention
comprises a nucleotide sequence described in any one of the
following (a) to (c) (hereinafter, referred to as an "FGFR2 IIIb
gene sequence"), consists of a nucleotide sequence comprising the
FGFR2 gene sequence, or consists of the FGFR2 gene sequence: [0200]
(a) a nucleotide sequence encoding the amino acid sequence
represented by NP_075259; [0201] (b) a nucleotide sequence that
hybridizes under stringent conditions to a nucleotide consisting of
a nucleotide sequence complementary to the nucleotide sequence
encoding the amino acid sequence represented by NP_075259 and
encodes the amino acid sequence of a polypeptide having FGF binding
activity; and [0202] (c) a nucleotide sequence that encodes an
amino acid sequence derived from the amino acid sequence
represented by NP_075259 by the substitution, deletion, addition,
or insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to
20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or
2, or 1 amino acid(s) and encodes the amino acid sequence of a
polypeptide having FGF binding activity.
[0203] The polypeptide having the amino acid sequence encoded by
the nucleotide sequence (b) or (c) may have FGFR2 activities other
than FGF binding activity.
[0204] The FGFR2 IIIc gene according to the present invention
comprises a nucleotide sequence described in any one of the
following (a) to (c) (hereinafter, referred to as an "FGFR2 IIIc
gene sequence"), consists of a nucleotide sequence comprising the
FGFR2 gene sequence, or consists of the FGFR2 gene sequence: [0205]
(a) a nucleotide sequence encoding the amino acid sequence
represented by NP_000132; [0206] (b) a nucleotide sequence that
hybridizes under stringent conditions to a nucleotide consisting of
a nucleotide sequence complementary to the nucleotide sequence
encoding the amino acid sequence represented by NP_000132 and
encodes the amino acid sequence of a polypeptide having FGF binding
activity; and [0207] (c) a nucleotide sequence that encodes an
amino acid sequence derived from the amino acid sequence
represented by NP_000132 by the substitution, deletion, addition,
or insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to
20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or
2, or 1 amino acid(s) and encodes the amino acid sequence of a
polypeptide having FGF binding activity.
[0208] The polypeptide having the amino acid sequence encoded by
the nucleotide sequence (b) or (c) may have FGFR2 activities other
than FGF binding activity.
[0209] The expression and expression level of the FGFR2 gene may be
assayed with either an FGFR2 gene transcript or the FGFR2 protein
as an index. The former index can be determined by RT-PCR, Northern
blot hybridization, or the like, while the latter index can be
determined by, for example, immunoassay such as enzyme-linked
immunosorbent assay (hereinafter, referred to as "ELISA"), Western
blotting, or immunohistological staining.
[0210] (2-3) Preparation of an Antigenic Protein
[0211] The FGFR2 protein of the present invention can be prepared
by purification or isolation from animal tissues (including body
fluids), cells derived from the tissues, or cultures of the cells,
gene recombination, in vitro translation, chemical synthesis,
etc.
[0212] (2-3-1) Purification or Isolation of Non-Recombinant
FGFR2
[0213] Non-recombinant FGFR2 protein can be purified or isolated
from FGFR2-expressing cells, normal tissues, or cancer tissues, or
cells derived therefrom. Examples of FGFR2-expressing normal
tissues, cancer tissues, or cancer cells can include those
described in (iv) of paragraph (2-1), though the origin of the
non-recombinant FGFR2 protein is not limited thereto.
[0214] Purification or isolation from such tissues, cells, cell
cultures, or the like, can be performed by any combination of
approaches well known by those skilled in the art, such as
fractionation and chromatography. Such approaches include, but are
not limited to, salting out, gel filtration, ion-exchange
chromatography, affinity chromatography, hydrophobic
chromatography, normal-phase or reverse-phase chromatography, and
the like. A column for affinity chromatography can be prepared by
packing the column with an affinity gel cross-linked with an
anti-FGFR2 monoclonal antibody. A crude or partially purified
fraction containing the FGFR2 protein is applied to this column.
Subsequently, non-specifically adsorbed substances are removed with
sterilized phosphate-buffered saline (PBS), and a buffer solution
for elution can then be applied thereto to thereby selectively
recover the FGFR2 protein. The solution containing the FGFR2
protein can be subjected to gel filtration or to buffer replacement
and/or concentration using a concentrator such as Centriprep.
[0215] (2-3-2) Preparation of Recombinant FGFR2 Protein
[0216] The FGFR2 protein of the present invention can also be
prepared in a recombinant form. Specifically, host cells are
transfected with a gene encoding the amino acid sequence of the
FGFR2 protein or an FGFR2 protein fragment, and the FGFR2 protein
can be recovered from cultures of the cells. For example, the FGFR2
gene or its fragment is inserted into an expression vector.
Subsequently, prokaryotic or eukaryotic host cells are transfected
with the resulting recombinant vector, and the obtained recombinant
cells can be incubated to thereby express the FGFR2 protein. An
expression pattern known in the art, such as secretion expression,
intracellular expression of soluble forms, or expression in
inclusion body forms can be used. Also, the FGFR2 protein can be
expressed not only as a molecule having the same amino terminus (N
terminus) and/or carboxy terminus (C terminus) as native ones, but
also as a fusion protein with a secretory signal, an intracellular
localization signal, a tag for affinity purification, or a partner
peptide. The FGFR2 protein can be purified or isolated from such
recombinant cell cultures by an appropriate combination of methods
such as fractionation and chromatography described in (2-3-1).
[0217] The FGFR2 gene or its fragment can be prepared by a method
well known by those skilled in the art.
[0218] Examples thereof can include: polymerase chain reaction
(hereinafter, referred to as "PCR"; Saiki, R. K., et al., Science
(1988) 239, p. 487-489) with a cDNA library prepared from
FGFR2-expressing cells, tissues, or the like as a template using
one set of primers capable of specifically amplifying the sequence;
reverse transcription PCR (hereinafter, referred to as "RT-PCR")
with an mRNA fraction prepared from FGFR2-expressing cells,
tissues, or the like as a template using a primer capable of
reverse-transcribing the sequence and one set of primers capable of
specifically amplifying the sequence; expression cloning using
immunoassay; and cDNA cloning using the partial amino acid sequence
of purified FGFR2 protein.
[0219] (2-3-3) In Vitro Translation
[0220] The FGFR2 protein of the present invention can also be
prepared by in vitro translation. Such a translation method is not
particularly limited as long as the method employs a cell-free
translation system involving enzymes necessary for transcription
and translation, substrates, and energy substances. Examples
thereof can include a method using Rapid Translation System (RTS)
manufactured by Roche Diagnostics K. K.
[0221] (2-3-4) Chemical Synthesis
[0222] The FGFR2 protein of the present invention can also be
prepared by chemical synthesis. Examples of the chemical synthesis
method can include solid-phase peptide synthesis methods such as
Fmoc and Boc synthesis methods.
3. Antibody
[0223] (3-1) Antibody Classification
[0224] The antibodies of the present invention may be either
monoclonal or polyclonal antibodies. Examples of the monoclonal
antibody of the present invention can include non-human
animal-derived antibodies (non-human animal antibodies),
human-derived antibodies (human antibodies), chimeric antibodies,
and humanized antibodies.
[0225] Examples of a non-human animal antibody can include
antibodies derived from vertebrates such as mammals and birds.
Examples of a mammal-derived antibody can include rodent-derived
antibodies such as mouse antibodies and rat antibodies. Examples of
a bird-derived antibody can include chicken antibodies. Examples of
an anti-human FGFR2 rat monoclonal antibody can include
FR2-2nd_#023, and FR2-2nd_#028.
[0226] Examples of a chimeric antibody can include, but are not
limited to, an antibody comprising non-human animal
antibody-derived variable regions bound with human antibody (human
immunoglobulin) constant regions. Examples thereof can include:
mouse chimeric FR2-2nd_#023 (a nucleotide sequence encoding the
amino acid sequence of the heavy chain is described in nucleotide
positions 26 to 1423 of SEQ ID NO: 20 or FIG. 18A; the amino acid
sequence of the heavy chain is described in SEQ ID NO: 21 or FIG.
18B; a nucleotide sequence encoding the amino acid sequence of the
light chain is described in nucleotide positions 26 to 724 of SEQ
ID NO: 22 or FIG. 18C; and the amino acid sequence of the light
chain is described in SEQ ID NO: 23 or FIG. 18D) derived from rat
FR2-2nd_#023 (a nucleotide sequence encoding the amino acid
sequence of the heavy chain variable region is described in SEQ ID
NO: 7 or FIG. 15A; the amino acid sequence of the heavy chain
variable region is described in SEQ ID NO: 8 or FIG. 15B; a
nucleotide sequence encoding the amino acid sequence of the light
chain variable region is described in SEQ ID NO: 9 or FIG. 15C; and
the amino acid sequence of the light chain variable region is
described in SEQ ID NO: 10 or FIG. 15D) by the replacement of its
constant regions with mouse antibody constant regions; and mouse
chimeric FR2-2nd_#028 (a nucleotide sequence encoding the amino
acid sequence of the heavy chain is described in nucleotide
positions 26 to 1411 of SEQ ID NO: 14 or FIG. 17A; the amino acid
sequence of the heavy chain is described in SEQ ID NO: 15 or FIG.
17B; a nucleotide sequence encoding the amino acid sequence of the
light chain is described in nucleotide positions 26 to 724 of SEQ
ID NO: 18 or FIG. 17C; and the amino acid sequence of the light
chain is described in SEQ ID NO: 19 or FIG. 17D) derived from rat
FR2-2nd_#028 (a nucleotide sequence encoding the amino acid
sequence of the heavy chain variable region is described in SEQ ID
NO: 2 or FIG. 14A; the amino acid sequence of the heavy chain
variable region is described in SEQ ID NO: 3 or FIG. 14B; a
nucleotide sequence encoding the amino acid sequence of the light
chain variable region is described in SEQ ID NO: 5 or FIG. 14C; and
the amino acid sequence of the light chain variable region is
described in SEQ ID NO: 6 or FIG. 14D) by the replacement of its
constant regions with mouse antibody constant regions.
[0227] Examples of a humanized antibody can include, but are not
limited to, a human antibody (human immunoglobulin variable
regions) grafted with CDRs in the variable regions of a non-human
animal antibody, a human antibody grafted with the CDRs as well as
with partial sequences of framework regions of a non-human animal
antibody, and an antibody having human antibody amino acid(s)
substituted for one or two or more non-human animal
antibody-derived amino acid(s) in any of these humanized
antibodies.
[0228] A human antibody is not particularly limited as long as the
antibody recognizes the antigen of the present invention. Examples
thereof can include a human antibody binding to the same site, as
in the case of an antibody having the CDRs of the antibody of the
present invention, and a human antibody binding to the same site on
FGFR2 as in the case of the FR2-2nd_#023 antibody or the chimeric
antibody thereof or FR2-2nd_#028 or the chimeric antibody thereof
mentioned above.
[0229] The antibody according to the present invention may be
comprised of portions derived from a plurality of different
antibodies as long as the antibody has FGFR2 binding activity.
Examples of such an antibody can include an antibody comprising
heavy and/or light chains exchanged among a plurality of different
antibodies, an antibody comprising full-length heavy and/or light
chains exchanged among a plurality of different antibodies, an
antibody comprising variable or constant regions exchanged among a
plurality of different antibodies, and an antibody comprising all
or some CDRs exchanged among a plurality of different antibodies.
The heavy and light chain variable regions of the chimeric antibody
may be derived from different antibodies of the present invention.
CDRH1 to CDRH3 and CDRL1 to CDRL3 in the heavy and light chain
variable regions of the humanized antibody may be derived from two
or more different antibodies of the present invention. CDRH1 to
CDRH3 and CDRL1 to CDRL3 in the heavy and light chain variable
regions of the human antibody may be a combination of CDRs carried
by two or more different antibodies of the present invention.
[0230] Examples of the isotype of the monoclonal antibody of the
present invention can include, but are not particularly limited to,
IgG such as IgG1, IgG2, IgG3, and IgG4, IgM, IgA such as IgA1 and
IgA2, IgD, and IgE and can preferably include IgG and IgM. The
isotype and subclass of the monoclonal antibody can be determined
by, for example, an Ouchterlony test, ELISA, or radio immunoassay
(hereinafter, referred to as "RIA"). A commercially available kit
for identification (e.g., Mouse Typer Kit; Bio-Rad Laboratories,
Inc., and RAT MONOCLONAL ANTIBODY ISOTYPING TEST KIT: AbD Serotec)
may be used.
[0231] (3-2) Antibody Binding Specificity
[0232] The antibody of the present invention recognizes the FGFR2
protein. In other words, the antibody of the present invention
binds to the FGFR2 protein. Such an antibody is referred to as an
"anti-FGFR2 antibody". Preferably, the antibody of the present
invention specifically recognizes the FGFR2 protein. In other
words, preferably, the antibody of the present invention
specifically binds to the FGFR2 protein. More preferably, the
antibody of the present invention specifically binds to the FGFR2
IIIb protein and/or the FGFR2 IIIc protein. Even more preferably,
the antibody of the present invention specifically binds to the
immunoglobulin-like domain (hereinafter, referred to as "Ig-like
domain") of the FGFR2 IIIb protein and/or the FGFR2 IIIc protein.
Examples of such an Ig-like domain can include Ig-like domain 2 and
Ig-like domain 3.
[0233] According to an aspect, preferably, the antibody
specifically binds to the human FGFR2 IIIb protein and the human
FGFR2 IIIc protein.
[0234] According to another aspect, preferably, the antibody
specifically binds to the human FGFR2 IIIc protein, but does not
bind to the human FGFR2 IIIb protein.
[0235] According to an aspect, preferably, the antibody
specifically binds to both non-denatured human FGFR2 and denatured
FGFR2 in a preparation fixed in formalin. More preferably, the
antibody specifically binds to none of the following: non-denatured
human FGFR1 (IIIb and IIIc proteins), FGFR3 (IIIb and IIIc
proteins), and FGFR4, or denatured human FGFR1 (IIIb and IIIc
proteins), FGFR3 (IIIb and IIIc proteins), and FGFR4 in a
preparation fixed in formalin. For example, the binding specificity
for a non-denatured molecule of the FGFR family can be evaluated by
a method described in Example 3, and the binding specificity for a
denatured molecule of the FGFR family can be evaluated by a method
described in Example 4.
[0236] Thus, even more preferred examples of the antibody of the
present invention can include, but are not limited to: [0237] (A)
an antibody that specifically binds to non-denatured and denatured
human FGFR2 IIIc and human FGFR2 IIIb, but specifically binds to
none of the following: non-denatured and denatured human FGFR1,
human FGFR3, and human FGFR4; and [0238] (B) an antibody that
specifically binds to non-denatured and denatured human FGFR2 IIIc,
but specifically binds to neither non-denatured nor denatured human
FGFR2 IIIb and specifically binds to none of the following:
non-denatured and denatured human FGFR1, human FGFR3, and human
FGFR4.
[0239] In the present invention, the term "specific recognition",
i.e., "specific binding", means binding which is not non-specific
adsorption. Examples of criteria for determination of whether
binding is specific or not can include a dissociation constant
(hereinafter, referred to as "KD"). Preferably, the antibody of the
present invention has a KD value of 1.times.10.sup.-3 M or lower,
5.times.10.sup.-6 M or lower, 2.times.10.sup.-6 M or lower, or
1.times.10.sup.-6 M or lower, more preferably 5.times.10.sup.-7 M
or lower, 2.times.10.sup.-7 M or lower, or 1.times.10.sup.-7 M or
lower, even more preferably 5.times.10.sup.-8 M or lower,
2.times.10.sup.-8 M or lower, or 1.times.10.sup.-8 M or lower,
further more preferably 5.times.10.sup.-9 M or lower,
2.times.10.sup.-9 M or lower, or 1.times.10.sup.-9 M or lower, most
preferably 5.times.10.sup.-10 M or lower, 2.times.10.sup.-10 M or
lower, or 1.times.10.sup.-10 M or lower for the FGFR2 protein.
[0240] In the present invention, the term "selective" has the same
meaning as "specific".
[0241] In the present invention, the binding of the antibody to the
antigen can be assayed or determined by ELISA, RIA, surface plasmon
resonance (hereinafter, referred to as "SPR") analysis, or the
like. Examples of equipment used in the SPR analysis can include
BIAcore.TM. (manufactured by GE Healthcare Bio-Sciences Corp.),
ProteOn.TM. (manufactured by Bio-Rad Laboratories, Inc.),
SPR-Navi.TM. (manufactured by BioNavis Oy Ltd.), Spreeta.TM.
(manufactured by Texas Instruments Inc.), SPRi-Plex II.TM.
(manufactured by Horiba, Ltd.), and Autolab SPR.TM. (manufactured
by Metrohm Japan Ltd.). The binding of the antibody to the antigen
expressed on cell surface can be assayed by flow cytometry,
Cell-ELISA, or the like.
[0242] (3-3) Monoclonal Antibody
[0243] The present invention provides a monoclonal antibody. The
monoclonal antibody includes, for example, non-human animal-derived
monoclonal antibodies such as rat, mouse, rabbit, chicken, and fish
antibodies, chimeric antibodies, humanized antibodies, human
antibodies, functional fragments thereof, and modified forms of
these antibodies or functional fragments. Of them, examples of the
rat monoclonal antibody can include the FR2-2nd_#023, and
FR2-2nd_#028.
[0244] FR2-2nd_#023 is an anti-human FGFR2 rat monoclonal antibody
obtained by the method described in Example 1. The nucleotide
sequence of the heavy chain variable region is described in SEQ ID
NO: 7 (FIG. 15A), and its amino acid sequence is described in SEQ
ID NO: 8 (FIG. 15B). The nucleotide sequence of the light chain
variable region is described in SEQ ID NO: 9 (FIG. 15C), and its
amino acid sequence is described in SEQ ID NO: 10 (FIG. 15D). The
amino acid sequence of CDRH1 is described in SEQ ID NO: 30 (FIG.
20). The amino acid sequence of CDRH2 thereof is described in SEQ
ID NO: 31 (FIG. 20). The amino acid sequence of CDRH3 thereof is
described in SEQ ID NO: 32 (FIG. 20). The amino acid sequence of
CDRL1 thereof is described in SEQ ID NO: 33 (FIG. 20). The amino
acid sequence of CDRL2 thereof is described in SEQ ID NO: 34 (FIG.
20). The amino acid sequence of CDRL3 thereof is described in SEQ
ID NO: 35 (FIG. 20).
[0245] FR2-2nd_#028 is an anti-human FGFR2 rat monoclonal antibody
obtained by the method described in Example 1. The nucleotide
sequence of the heavy chain variable region is described in SEQ ID
NO: 2 (FIG. 14A), and its amino acid sequence is described in SEQ
ID NO: 3 (FIG. 14B). The nucleotide sequence of the light chain
variable region is described in SEQ ID NO: 5 (FIG. 14C), and its
amino acid sequence is described in SEQ ID NO: 6 (FIG. 14D). The
amino acid sequence of CDRH1 is described in SEQ ID NO: 24 (FIG.
19). The amino acid sequence of CDRH2 thereof is described in SEQ
ID NO: 25 (FIG. 19). The amino acid sequence of CDRH3 thereof is
described in SEQ ID NO: 26 (FIG. 19). The amino acid sequence of
CDRL1 thereof is described in SEQ ID NO: 27 (FIG. 19). The amino
acid sequence of CDRL2 thereof is described in SEQ ID NO: 28 (FIG.
19). The amino acid sequence of CDRL3 thereof is described in SEQ
ID NO: 29 (FIG. 19).
[0246] An antibody mutant of the present invention preferably
exhibits, for example, reduced sensitivity to protein degradation
or oxidation, an improved biological activity, an improved ability
to bind to the antigen, or physicochemical or functional properties
imparted thereto. Examples of such an antibody mutant can include
an antibody having an amino acid sequence derived from the amino
acid sequence of the original antibody by conservative amino acid
substitution. The conservative amino acid substitution is a
substitution that occurs in an amino acid group related to amino
acid side chains.
[0247] Preferred amino acid groups are as follows: an acidic group
including aspartic acid and glutamic acid; a basic group including
lysine, arginine, and histidine; a nonpolar group including
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, and tryptophan; and an uncharged polar family including
glycine, asparagine, glutamine, cysteine, serine, threonine, and
tyrosine. Other preferred amino acid groups are as follows: an
aliphatic hydroxy group including serine and threonine; an
amide-containing group including asparagine and glutamine; an
aliphatic group including alanine, valine, leucine, and isoleucine;
and an aromatic group including phenylalanine, tryptophan, and
tyrosine. Such amino acid substitution in the antibody mutant is
preferably performed without reducing the antigen binding activity
of the original antibody.
[0248] Aspartic acid contained in a protein is easily converted to
isoaspartic acid by isomerization when an amino acid linked thereto
on the C terminal side has a small side chain. On the other hand,
asparagine is easily converted to aspartic acid by deamidation and
may be further converted to isoaspartic acid by isomerization. The
progression of such isomerization or deamidation may influence the
stability of the protein. Accordingly, aspartic acid or asparagine
in the protein or, for example, an amino acid adjacent thereto, can
be substituted by a different amino acid in order to circumvent
such isomerization or deamidation. Preferably, an antibody mutant
having such amino acid substitution maintains the antigen binding
activity of the original antibody.
[0249] The present invention also encompasses, for example: an
antibody mutant having an amino acid sequence derived from the
amino acid sequence of antibodies of the present invention by
conservative amino acid substitution; and a mouse antibody, a rat
antibody, a chimeric antibody, a humanized antibody, or a human
antibody comprising a CDR having an amino acid sequence in which a
conservative amino acid mutation occurs in the amino acid sequence
of any of CDRH1 to CDRH3 and CDRL1 to CDRL3 derived from antibodies
of the present invention.
[0250] A mutant of the antibody of the present invention
encompasses a human FGFR2-binding antibody mutant comprising CDRH1
to CDRH3 and CDRL1 to CDRL3 having amino acid sequences derived
from the amino acid sequences of any one or two or more of CDRH1 to
CDRH3 and CDRL1 to CDRL3 derived from antibodies of the present
invention by the substitution of 1 to several, preferably 1 to 3,
more preferably 1 or 2, most preferably 1 amino acid(s) by
different amino acid(s).
[0251] An antibody mutant also includes an antibody having CDRH1 to
CDRH3 and CDRL1 to CDRL3 derived from a plurality of antibodies.
Examples of such a mutant can include an antibody mutant comprising
CDRH3 derived from a certain antibody and CDRH1, CDRH2, and CDRL1
to CDRL3 derived from another antibody.
[0252] The term "antibody" according to the present invention also
encompasses these antibody mutants.
[0253] The constant regions of the antibody of the present
invention are not particularly limited. Preferably, constant
regions derived from a human antibody are used in the antibody of
the present invention for the treatment or prevention of a disease
in a human. Examples of the heavy chain constant region of the
human antibody can include C.gamma.1, C.gamma.2, C.gamma.3,
C.gamma.4, C.mu., C.delta., C.alpha.1, C.alpha.2, and C.epsilon..
Examples of the light chain constant region of the human antibody
can include C.kappa. and C.lamda..
[0254] (3-4) Functional Fragments of the Antibody
[0255] According to one aspect, the present invention provides a
functional fragment of the anti-FGFR2 antibody of the present
invention. The functional fragment of the antibody means a fragment
that maintains at least a portion of the functions of the antibody.
Examples of such functions of the antibody can generally include
antigen binding activity.
[0256] The functional fragment of the antibody is not particularly
limited as long as the fragment of the antibody maintains at least
a portion of the activities of the antibody. Examples thereof can
include, but are not limited to, Fab, F(ab')2, Fv, single chain Fv
(scFv) comprising heavy and light chain Fvs linked via an
appropriate linker, diabodies, linear antibodies, multispecific
antibodies formed from antibody fragments, and Fab', which is a
monovalent fragment of antibody variable regions obtained by the
treatment of F(ab')2 under reducing conditions. The functional
fragment of the antibody of the present invention is also meant to
include a molecule comprising the fragment of the antibody of the
present invention as well as other portions, such as scFv retaining
a linker portion.
[0257] A molecule that is derived from the antibody protein by the
deletion of 1 to several or more amino acid(s) at its amino
terminus and/or carboxy terminus and maintains at least a portion
of the functions of the antibody is also encompassed in the meaning
of the functional fragment of the antibody. For example, the heavy
chain of an antibody produced by cultured mammalian cells is known
to lack a lysine residue at the carboxy terminus (Journal of
Chromatography A, 705: 129-134 (1995)). Also, the heavy chain of
such an antibody is known to lack two amino acid residues (glycine
and lysine) at the carboxy terminus and instead have an amidated
proline residue at the carboxy terminus (Analytical Biochemistry,
360: 75-83 (2007)). The deletion and the modification in these
heavy chain sequences, however, do not influence the ability of the
antibody to bind to the antigen or its effector functions
(complement activation, antibody dependent cytotoxic effects,
etc.). Such a modified form of the functional fragment of the
antibody is also encompassed by the antibody of the present
invention or the functional fragment thereof, or a modified form
(described later) of the antibody or functional fragment.
[0258] The antibody of the present invention or the functional
fragment thereof may be a multispecific antibody having specificity
for at least 2 types of different antigens. The multispecific
antibody is not limited to a bispecific antibody, which binds to 2
types of different antigens, and an antibody having specificity for
3 or more types of different antigens is also encompassed in the
meaning of the "multispecific antibody" of the present
invention.
[0259] The multispecific antibody of the present invention may be a
full-length antibody or a functional fragment thereof (e.g.,
bispecific F(ab')2 antibody). The bispecific antibody can also be
prepared by linking the heavy and light chains (HL pairs) of two
types of antibodies. Alternatively, the bispecific antibody may be
obtained by fusing two or more types of monoclonal
antibody-producing hybridomas to prepare bispecific
antibody-producing fusion cells (Millstein et al., Nature (1983)
305, p. 537-539). The multispecific antibody can also be prepared
in the same way as above.
[0260] According to one aspect, the antibody of the present
invention is a single chain antibody (single chain Fv; hereinafter,
referred to as "scFv"). The scFv is obtained by linking the heavy
and light chain V regions of the antibody via a polypeptide linker
(Pluckthun, The Pharmacology of Monoclonal Antibodies, 113,
Rosenburg and Moore, ed., Springer Verlag, New York, p. 269-315
(1994); and Nature Biotechnology (2005), 23, p. 1126-1136). Also,
bi-scFv comprising two scFvs linked via a polypeptide linker can be
used as a bispecific antibody. Alternatively, multi-scFv comprising
three or more scFvs may be used as a multispecific antibody.
[0261] The present invention includes a single chain immunoglobulin
comprising full-length heavy and light chain sequences of the
antibody linked via an appropriate linker (Lee, H-S, et al.,
Molecular Immunology (1999), 36, p. 61-71; and Shirrmann, T. et
al., mAbs (2010), 2 (1) p. 1-4). Such a single chain immunoglobulin
can be dimerized to thereby maintain a structure and activities
similar to those of the antibody, which is originally a tetramer.
Also, the antibody of the present invention may be an antibody that
has a single heavy chain variable region and has no light chain
region. Such an antibody, called a single domain antibody (sdAb) or
a nanobody, has been reported to maintain the ability to bind to an
antigen (Muyldemans S. et al., Protein Eng. (1994), 7 (9), 1129-35;
and Hamers-Casterman C. et al., Nature (1993), 363 (6428), 446-8).
These antibodies are also encompassed in the meaning of the
functional fragment of the antibody according to the present
invention.
[0262] The present invention also encompasses an antibody that
comprises a heavy or light chain comprising an amino acid sequence
having 80% or higher, 82% or higher, 84% or higher, 86% or higher,
88% or higher, 90% or higher, 92% or higher, 94% or higher, 96% or
higher, 98% or higher, or 99% or higher identity to the amino acid
sequence of the heavy or light chain, or heavy or light chain
variable region of the present invention and binds to hFGFR2, or a
functional fragment thereof. Such sequence identity is preferably
94% or higher, more preferably 96% or higher, even more preferably
98% or higher, most preferably 99% or higher.
[0263] The identity or homology between two types of amino acid
sequences can be determined using the default parameter of Blast
algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden,
Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and
David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25: 3389-3402). The Blast algorithm is also available, for example,
by Internet access at http://blast.ncbi.nlm.nih.gov/.
[0264] The present invention also encompasses an antibody that
comprises a heavy or light chain comprising an amino acid sequence
derived from the amino acid sequence of the heavy or light chain or
the heavy or light chain variable region of the antibody of the
present invention by the substitution, deletion, addition, or
insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25,
1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3,
1 or 2, or 1 amino acid(s) and binds to hFGFR2, or a functional
fragment thereof. Such an amino acid mutation is preferably
substitution. The number of mutated amino acids is preferably 1 to
5, more preferably 1 to 4, even more preferably 1 to 3, further
more preferably 1 or 2, most preferably 1.
[0265] The present invention also encompasses an antibody that
comprises a heavy or light chain comprising an amino acid sequence
encoded by the nucleotide sequence of nucleotide hybridizing under
stringent conditions to a nucleotide having a nucleotide sequence
complementary to a nucleotide sequence encoding the amino acid
sequence of the heavy or light chain or the heavy or light chain
variable region of the antibody of the present invention and binds
to hFGFR2, or a functional fragment thereof.
[0266] (3-5) Antibody Binding to the Epitope
[0267] An "antibody binding to the same site" as in the case of the
antibody provided by the present invention is also included in the
antibody of the present invention. The "antibody binding to the
same site" as in the case of a certain antibody, means another
antibody that binds to a site on an antigen molecule recognized by
the antibody. If a second antibody binds to a partial peptide or a
partial three-dimensional structure on an antigen molecule bound by
a first antibody, the first and second antibodies are considered to
bind to the same site. Alternatively, the first and second
antibodies are considered to bind to the same site by confirming
that the second antibody competes with the first antibody for
binding to the antigen, i.e., the second antibody interferes with
the binding of the first antibody to the antigen, even if the
peptide sequence or three-dimensional structure of the specific
binding site is not determined. When the first and second
antibodies bind to the same site and the first antibody has an
effect characteristic of one aspect of the antibody of the present
invention, such as an antitumor activity, the second antibody also
has an exceedingly high probability of having the same activity as
the first. Thus, if a second anti-FGFR2 antibody binds to a site
bound by a first anti-FGFR2 antibody, the first and second
antibodies are considered to bind to the same site on the FGFR2
protein. Alternatively, the first and second anti-FGFR2 antibodies
are considered to bind to the same site on the FGFR2 protein by
confirming that the second anti-FGFR2 antibody competes with the
first anti-FGFR2 antibody for binding to the FGFR2 protein.
[0268] The present invention also encompasses an antibody binding
to a site on the FGFR2 protein recognized by the monoclonal
antibody of the present invention.
[0269] The antibody binding site can be determined by a method well
known by those skilled in the art, such as immunoassay. For
example, a series of peptides are prepared by appropriately
sequentially cleaving the amino acid sequence of the antigen from
its C terminus or N terminus, and the reactivity of the antibody
thereto is studied to roughly determine a recognition site. Then,
shorter peptides are synthesized, and the reactivity of the
antibody to these peptides can be studied to thereby determine the
binding site. The antigen fragment peptides can be prepared using a
technique such as gene recombination or peptide synthesis.
[0270] When the antibody binds to or recognizes the partial
conformation of the antigen, the binding site for the antibody can
be determined by identifying amino acid residues on the antigen
adjacent to the antibody using X-ray structural analysis. For
example, the antibody or its fragment and the antigen or its
fragment can be bound to each other and crystallized, followed by
structural analysis to identify each amino acid residue on the
antigen having an interaction distance with the antibody. The
interaction distance is 8 angstroms or shorter, preferably 6
angstroms or shorter, more preferably 4 angstroms or shorter. One
or more such amino acid residues having an interaction distance
with the antibody can constitute a site (epitope) on the antigen to
which the antibody binds. Two or more such amino acid residues may
not be adjacent to each other on the primary sequence.
[0271] (3-6) Modified Form of the Antibody
[0272] The present invention provides a modified form of the
antibody or functional fragment thereof. The modified form of the
antibody of the present invention or the functional fragment
thereof means an antibody of the present invention or a functional
fragment thereof provided with chemical or biological modification.
The chemically modified form includes, for example, a form having
an amino acid skeleton conjugated with a chemical moiety, and a
form having a chemically modified N-linked or O-linked carbohydrate
chain. A biologically modified form includes, for example, a form
that has undergone post-translational modification (e.g., N-linked
or O-linked glycosylation, N-terminal or C-terminal processing,
deamidation, isomerization of aspartic acid, or oxidation of
methionine), and a form containing a methionine residue added to
the N-terminus by expression using prokaryotic host cells. Such a
modified form is also meant to include a form labeled to permit
detection or isolation of the antibody or the antigen of the
present invention, for example, an enzyme-labeled form, a
fluorescently labeled form, or an affinity-labeled form. Such a
modified form of the antibody of the present invention or the
functional fragment thereof is useful for improvement of the
stability or blood retention of the original antibody of the
present invention or the original functional fragment thereof,
reduction in antigenicity, detection or isolation of the antibody
or the antigen, etc.
[0273] Examples of a chemical moiety contained in the chemically
modified form can include water-soluble polymers such as
polyethylene glycol, ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, and polyvinyl alcohol.
[0274] Examples of a biologically modified form can include a form
modified by enzymatic treatment, cell treatment, or the like, a
form fused with another peptide, such as a tag, added by gene
recombination, and a form prepared from host cells expressing an
endogenous or exogenous sugar chain-modifying enzyme.
[0275] Such a modification may be made at an arbitrary position or
a desired position in the antibody or functional fragment thereof.
Alternatively, the same or two or more different modifications may
be made at one or two or more positions therein.
[0276] In the present invention, the term "modified form of the
antibody fragment" is also meant to include even a "fragment of the
modified form of the antibody".
[0277] In the present invention, a modified form of the antibody or
a modified form of the functional fragment thereof is also simply
referred to as an "antibody" or a "functional fragment of the
antibody".
4. Methods for Producing the Antibody
[0278] (4-1) Method Using a Hybridoma
[0279] In order to prepare the anti-FGFR2 antibody of the present
invention, anti-FGFR2 antibody-producing cells are isolated from
the spleens of animals immunized with the FGFR2 protein or its
soluble form according to the method of Kohler and Milstein (Kohler
and Milstein, Nature (1975), 256, p. 495-497; and Kennet, R. ed.,
Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)). The
cells are fused with myeloma cells to thereby establish hybridomas.
Monoclonal antibodies can be obtained from cultures of these
hybridomas.
[0280] (4-1-1) Preparation of an Antigen
[0281] An antigen for preparation of the anti-FGFR2 antibody can be
obtained according to, for example, the method for preparing a
native or recombinant FGFR2 protein described in other paragraphs
of the present specification. Examples of the antigen that may be
thus prepared can include the FGFR2 protein and an FGFR2 protein
fragment comprising a partial sequence with at least 6 consecutive
amino acids of the FGFR2 protein, and their derivatives further
comprising an arbitrary amino acid sequence or carrier added
thereto (hereinafter, collectively referred to as an "FGFR2
antigen").
[0282] The recombinant FGFR2 antigen can be prepared by
transfecting host cells with a gene comprising a nucleotide
sequence encoding the amino acid sequence of the FGFR2 antigen, and
recovering the antigen from cultures of the cells. Such a
recombinant antigen may be a fusion protein with another protein
such as an immunoglobulin Fc region. An FGFR2 antigen obtained in a
cell-free in vitro translation system from a gene comprising a
nucleotide sequence encoding the amino acid sequence of the FGFR2
antigen is also included in the recombinant FGFR2 antigen. The
non-recombinant FGFR2 antigen can be purified or isolated from
FGFR2-expressing normal tissues, cancer tissues, or cancer cells,
cultures of the cancer cells, or the like described in (iv) of
paragraph (2-1).
[0283] (4-1-2) Production of an Anti-FGFR2 Monoclonal Antibody
[0284] (a) Purification of the Antigen
[0285] This step is performed according to the method for preparing
the FGFR2 protein described above in (2-3).
[0286] (b) Step of Preparing an Antibody-Producing Cell
[0287] The antigen obtained in step (a) is mixed with an adjuvant
such as a complete or incomplete Freund's adjuvant or potassium
aluminum sulfate, and laboratory animals are immunized with the
resulting immunogen. Any laboratory animal used in a hybridoma
preparation method known in the art can be used without
limitations. Specifically, for example, mice, rats, goats, sheep,
cattle, or horses can be used. From the viewpoint of readily
available myeloma cells to be fused with isolated
antibody-producing cells, etc., the animals to be immunized are
preferably mice or rats.
[0288] The strain of mice or rats actually used is not particularly
limited. In the case of mice, for example, A, AKR, BALB/c,
BALB/cAnNCrj, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, FL, HTH,
HT1, LP, NZB, NZW, RF, R III, SJL, SWR, WB, or 129 can be used. In
the case of rats, for example, Wistar, Low, Lewis, Sprague-Dawley,
ACI, BN, or Fischer can be used.
[0289] Such mice and rats are available from laboratory animal
breeders or distributors, for example, CLEA Japan, Inc. or Charles
River Laboratories Japan Inc.
[0290] Of these mice and rats, a BALB/c mouse strain or Wistar and
Low rat strains are particularly preferred as animals to be
immunized in consideration of fusion compatibility with the myeloma
cells described later.
[0291] Also, in consideration of the homology between human and
mouse antigens, mice whose biological mechanism to remove
autoantibodies has been reduced, i.e., autoimmune disease mice, are
also preferably used.
[0292] In this context, these mice or rats are preferably 5 to 12
weeks old, more preferably 6 to 8 weeks old, at the time of
immunization.
[0293] The animals can be immunized with the FGFR2 protein using,
for example, the method of Weir, D. M., Handbook of Experimental
Immunology Vol. I. II. III., Blackwell Scientific Publications,
Oxford (1987), Kabat, E. A. and Mayer, M. M., Experimental
Immunochemistry, Charles C Thomas Publisher Spigfield, Ill.
(1964).
[0294] Examples of methods for determining antibody titers can
include, but are not limited to, immunoassay such as RIA and
ELISA.
[0295] Antibody-producing cells derived from spleen cells or
lymphocytes separated from the immunized animals, can be prepared
according to a method known in the art, for example, Kohler et al.,
Nature (1975) 256, p.495,; Kohler et al., Eur. J. Immnol. (1977) 6,
p.511,; Milstein et al., Nature (1977), 266, p.550; Walsh, Nature,
(1977) 266, p.495.
[0296] In the case of spleen cells, a general method can be
adopted, which involves chopping the spleens, filtering cells
through a stainless mesh, and then floating the resulting cells in
an Eagle's minimum essential medium (MEM) or the like, to separate
antibody-producing cells.
[0297] (c) Step of Preparing Myeloma
[0298] The myeloma cells used in cell fusion are not particularly
limited and can be selected appropriately for use from cell lines
known in the art. For example, a hypoxanthine-guanine
phosphoribosyl transferase (HGPRT)-deficient line, i.e.,
mouse-derived X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.Ul (P3U1),
X63-Ag8.653 (X63.653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7
(45.6TG), FO, S149/5XXO, or BU.1, rat-derived 210.RSY3.Ag.1.2.3
(Y3), or human-derived U266AR (SKO-007), GM1500-GTG-A12 (GM1500),
UC729-6, LICR-LOW-HMy2 (HMy2), or 8226AR/NIP4-1 (NP41), whose
screening procedures have already been established, is preferably
used in consideration of convenience in the selection of hybridomas
from fusion cells. These HGPRT-deficient lines are available from,
for example, American Type Culture Collection (ATCC).
[0299] These cell lines are subcultured in an appropriate medium,
for example, an 8-azaguanine medium [RPMI-1640 medium supplemented
with glutamine, 2-mercaptoethanol, gentamicin, and fetal bovine
serum (hereinafter, referred to as "FBS") and further supplemented
with 8-azaguanine], an Iscove's modified Dulbecco's medium
(hereinafter, referred to as "IMDM"), or a Dulbecco's modified
Eagle medium (hereinafter, referred to as "DMEM") and subcultured
in a normal medium [e.g., ASF104 medium (manufactured by Ajinomoto
Co., Inc.) containing 10% FBS] 3 to 4 days before cell fusion to
secure that the number of cells is equal to or greater than
2.times.10.sup.7 cells on the day of cell fusion.
[0300] (d) Fusing the Antibody-Producing Cell with a Myeloma
Cell
[0301] The antibody-producing cells can be fused with the myeloma
cells under conditions that prevent cell viability from being
exceedingly reduced, according to any method known in the art
(e.g., Weir, D. M., Handbook of Experimental Immunology Vol. I. II.
III., Blackwell Scientific Publications, Oxford (1987)). For
example, a chemical method which involves mixing antibody-producing
cells with myeloma cells in a high-concentration solution of a
polymer such as polyethylene glycol, or a physical method using
electric stimulation can be used.
[0302] (e) Screening for a Hybridoma Group Producing the Antibody
of Interest
[0303] A method for selection from the hybridomas obtained by the
cell fusion is not particularly limited, and a
hypoxanthine-aminopterin-thymidine (HAT) selection method (Kohler
et al., Nature (1975) 256, p.495; Milstein et al., Nature (1977)
266, p.550) is typically used. This method is effective for
obtaining hybridomas using an HGPRT-deficient myeloma cell line,
which cannot survive in the presence of aminopterin. Specifically,
unfused cells and hybridomas can be cultured in a HAT medium to
thereby allow only hybridomas resistant to aminopterin to
selectively live and grow.
[0304] (f) Obtaining a Single Cell Clone (Cloning)
[0305] The hybridomas can be cloned using any method known in the
art, for example, a methylcellulose, soft agarose, or limiting
dilution method (see e.g., Barbara, B. M. and Stanley, M. S.:
Selected Methods in Cellular Immunology, W. H. Freeman and Company,
San Francisco (1980)). The limiting dilution method is
preferred.
[0306] (g) Culturing the Hybridoma and Raising a
Hybridoma-Transplanted Animal
[0307] The selected hybridomas can be cultured to thereby produce
monoclonal antibodies. Preferably, the desired hybridomas are
cloned and then subjected to antibody production.
[0308] The monoclonal antibody produced by such a hybridoma can be
recovered from cultures of the hybridoma. Also, a recombinant
antibody can be recovered from cultures of cells transfected with
the monoclonal antibody gene. Alternatively, the hybridoma may be
injected intraperitoneally to mice of the same strain (e.g.,
BALB/cAnNCrj described above) or Nu/Nu mice and allowed to grow.
Then, the monoclonal antibody can be recovered from their
ascites.
[0309] (h) Assaying or Determining the Biological Activity of the
Monoclonal Antibody
[0310] Various biological tests can be selected and applied thereto
according to the purpose.
[0311] (4-2) Cell Immunization Method
[0312] Cells expressing the native FGFR2 protein, cells expressing
the recombinant FGFR2 protein or its fragment, or the like, can be
used as immunogens to thereby prepare an anti-FGFR2 antibody by the
hybridoma method described above.
[0313] Examples of cells expressing the native FGFR2 protein can
include FGFR2-expressing cells, cell lines derived from
FGFR2-expressing tissues or cancer, and cell lines derived from
cancer tissues in which switching from FGFR2 IIIb to FGFR2 IIIc
expression is seen. Cancers highly expressing FGFR2 include:
cancers found to have gene amplification, such as stomach cancer
and breast cancer; and cancers found to have overexpression, such
as pancreatic cancer and ovarian cancer. Examples of cultured cell
lines highly expressing FGFR2 IIIb can include stomach cancer cell
lines and breast cancer cell lines. Examples of cultured cell lines
highly expressing FGFR2 IIIc can include colorectal (cecal) cancer
cell lines. Examples of cancer tissues in which switching from
FGFR2 IIIb to FGFR2 IIIc expression is seen can include tissues of
prostate cancer, urinary bladder cancer, and breast cancer.
Examples of cancer tissues expressing FGFR2 IIIc can include
tissues of uterine cervix cancer and non-small cell lung cancer. Of
these cancers, uterine cervix cancer highly expresses FGFR2 IIIc.
Examples of normal tissues highly expressing FGFR2 can include the
brain, the large intestine, thyroid glands, the uterus, the
gallbladder, and the skin.
[0314] These FGFR2-expressing cells are used in an amount of
1.times.10.sup.5 to 1.times.10.sup.9 cells, preferably
1.times.10.sup.6 to 1.times.10.sup.8 cells, more preferably 0.5 to
2.times.10.sup.7 cells, even more preferably 1.times.10.sup.7
cells, per immunization shot. The number of cells used for
immunization can be changed according to the expression level of
the FGFR2 protein. The immunogens are generally administered
intraperitoneally and may be administered through an intradermal
route or the like. The hybridomas can be prepared by the
application of the method described in paragraph (4-1-2).
[0315] (4-3) Gene Recombination
[0316] In order to prepare the antibody of the present invention, a
nucleotide (heavy chain nucleotide) comprising a nucleotide
sequence encoding the amino acid sequence of its heavy chain and a
nucleotide (light chain nucleotide) comprising a nucleotide
sequence encoding the amino acid sequence of its light chain, or a
vector having an insert of the heavy chain nucleotide and a vector
having an insert of the light chain nucleotide are introduced into
host cells, and then the cells are cultured, and the antibody can
be recovered from the cultures. The heavy chain nucleotide and the
light chain nucleotide may be inserted in one vector.
[0317] Prokaryotic or eukaryotic cells can be used as the host
cells. In the case of using host eukaryotic cells, animal cells,
plant cells, or eukaryotic microbes can be used.
[0318] Examples of animal cells can include mammal-derived cells,
i.e., monkey-derived COS cells (Gluzman, Y. Cell (1981), 23, p.
175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No.
CRL-1658), a mouse NS0 cell line (ECACC), Chinese hamster ovary
cells (CHO cells, ATCC CCL-61), dihydrofolate reductase-deficient
lines thereof (CHO.sup.dhfr-; Urlaub, G. and Chasin, L. A. Proc.
Natl. Acad. Sci. U.S.A. (1980), 77, p. 4126-4220), CHOK1SV (Lonza
Biologics), cells derived from birds such as chickens, and cells
derived from insects.
[0319] Examples of eukaryotic microbes can include yeasts.
[0320] Examples of prokaryotic cells can include E. coli and
Bacillus subtilis.
[0321] A signal peptide for secretion of the antibody of the
present invention (monoclonal antibody derived from each animal,
rat antibody, mouse antibody, chimeric antibody, humanized
antibody, human antibody, etc.) is not limited to the secretory
signal of an antibody of the same species, the same type, and the
same subtype as the antibody of the present invention or to the
antibody of the present invention's own secretory signal. Any
secretory signal of an antibody of different type or subtype
therefrom or any secretory signal of a protein derived from a
different eukaryotic species therefrom or a prokaryotic species can
be selected and used.
[0322] (4-4) Methods for Designing and Preparing a Humanized
Antibody
[0323] Examples of the humanized antibody can include, but are not
limited to, a human-derived antibody having CDRs replaced with the
CDRs of a non-human animal antibody (see Nature (1986), 321, p.
522-525), a human antibody grafted with the CDR sequences and with
some amino acid residues of framework regions by CDR grafting (see
WO90/07861 and U.S. Pat. No. 6,972,323), and an antibody having
human antibody amino acid(s) replaced for one or two or more
non-human animal antibody-derived amino acid(s) in any of these
humanized antibodies.
[0324] (4-5) Methods for Preparing a Human Antibody
[0325] Further examples of the antibody of the present invention
can include a human antibody. The anti-FGFR2 human antibody means
an anti-FGFR2 antibody consisting of the amino acid sequence of a
human-derived antibody. The anti-FGFR2 human antibody can be
obtained by a method using human antibody-producing mice carrying
human genomic DNA fragments comprising human antibody heavy and
light chain genes (see e.g., Tomizuka, K. et al., Nature Genetics
(1997) 16, p.133-143; Tomizuka, K. et.al., Proc. Natl. Acad. Sci.
USA (2000) 97, p.722-727).
[0326] Specifically, such human antibody-producing animals may be
any of the recombinant animals that are obtained by disrupting the
endogenous immunoglobulin heavy and light chain gene loci of
non-human mammals and instead introducing thereto human
immunoglobulin heavy and light chain gene loci via yeast artificial
chromosome (YAC) vectors or the like, and recombinant animals that
are created by crossing these animals.
[0327] Alternatively, eukaryotic cells may be transfected with
cDNAs encoding the heavy and light chains, respectively, of such a
human antibody, preferably with vectors comprising the cDNAs, by a
gene recombination technique. The transfected cells producing a
recombinant human monoclonal antibody can be cultured. This
antibody can be obtained from the culture supernatant. In this
context, for example, eukaryotic cells, preferably mammalian cells
such as CHO cells, lymphocytes, or myelomas, can be used as the
hosts.
[0328] Also, a method for obtaining a phage display-derived human
antibody selected from a human antibody library (see e.g.,
Siriwardena, D.et.al., Opthalmology (2002) 109 (3), p.427-431) is
known. For example, a phage display method (Nature Biotechnology
(2005), 23, (9), p. 1105-1116) can be used, which involves allowing
the variable regions of a human antibody to be expressed as a
single chain antibody (scFv) on a phage surface and selecting a
phage binding to the antigen. The phage selected on the basis of
its ability to bind to the antigen can be subjected to gene
analysis to thereby determine DNA sequences encoding the variable
regions of the human antibody binding to the antigen. If the DNA
sequence of the scFv binding to the antigen is determined, an
expression vector having this sequence can be prepared and
introduced to appropriate hosts to allow them to express the human
antibody (e.g., Nature Biotechnology (2005) 23 (9),
p.1105-1116).
[0329] (4-6) Methods for Preparing a Functional Fragment of the
Antibody
[0330] The method for preparing a single chain antibody is well
known in the art (see e.g., U.S. Pat. Nos. 4,946,778, 5,260,203,
5,091,513, and 5,455,030). In this scFv, a heavy chain variable
region and a light chain variable region are linked via a linker
that prevents them from forming a conjugate, preferably a
polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci.
U.S.A. (1988), 85, p. 5879-5883). The heavy chain variable region
and the light chain variable region in the scFv may be derived from
the same antibody or may be derived from different antibodies.
[0331] For example, an arbitrary single chain peptide consisting of
12 to 19 residues is used as the polypeptide linker that links
these variable regions.
[0332] In order to obtain scFv-encoding DNA, of the sequences of
DNA encoding the heavy chain or heavy chain variable region of the
antibody and DNA encoding the light chain or light chain variable
region thereof, each DNA portion encoding the whole or desired
amino acid sequence is used as a template and amplified by PCR
using a primer pair flanking both ends of the template.
Subsequently, DNA encoding the polypeptide linker moiety is further
amplified in combination with a primer pair flanking both ends of
the DNA so that the obtained fragment can be linked at its ends to
the heavy and light chain DNAs, respectively.
[0333] The scFv-encoding DNA can be used to thereby prepare,
according to a routine method, an expression vector containing the
DNA and host cells transformed with the expression vector. In
addition, the host cells can be cultured, and the scFv can be
recovered from the cultures according to a routine method.
[0334] Also in order to obtain any other functional fragment of the
antibody, a gene encoding the functional fragment is obtained
according to the method described above and introduced into cells.
The functional fragment of interest can be recovered from cultures
of the cells.
[0335] The antibody of the present invention may be multimerized to
thereby enhance its affinity for the antigen. In this case,
antibodies of the same type may be multimerized, or a plurality of
antibodies recognizing a plurality of epitopes, respectively, of
the same antigen may be multimerized. Examples of methods for
multimerizing these antibodies can include the binding of two scFvs
to an IgG CH3 domain, the binding thereof to streptavidin, and the
introduction of a helix-turn-helix motif.
[0336] The antibody of the present invention may be a mixture of
plural types of anti-FGFR2 antibodies differing in amino acid
sequence, i.e., a polyclonal antibody. Examples of a polyclonal
antibody can include a mixture of plural types of antibodies
differing in a portion or the whole of CDRs. Such a polyclonal
antibody can be recovered from cultures of mixed-cultured different
antibody-producing cells (WO2004/061104). Alternatively, separately
prepared antibodies may be mixed. Antiserum, which is one aspect of
the polyclonal antibody, can be prepared by immunizing animals with
the desired antigen and recovering serum from the animals according
to a standard method.
[0337] Antibodies conjugated with various molecules such as
polyethylene glycol (PEG) can also be used as modified forms of the
antibody.
[0338] The antibody of the present invention may further be a
conjugate formed by these antibodies with other drugs
(immunoconjugates). Examples of such an antibody can include the
antibody conjugated with a radioactive material or a compound
having a pharmacological action (Nature Biotechnology (2005), 23,
p. 1137-1146).
[0339] (4-7) Purification of the Antibody
[0340] The obtained antibody can be purified until homogeneous.
Usual protein separation and purification methods can be used for
the separation and purification of the antibody.
[0341] The antibody can be separated and purified by appropriately
selected or combined approach(es), for example, chromatography
columns, filters, ultrafiltration, salting out, dialysis,
preparative polyacrylamide gel electrophoresis, and/or isoelectric
focusing (e.g., Antibodies: A Laboratory Manual. Ed Harlow and
David Lane, Cold Spring Harbor Laboratory (1988)) though the
separation and purification method is not limited thereto.
[0342] Examples of chromatography include affinity chromatography,
ion-exchange chromatography, hydrophobic chromatography, gel
filtration, reverse-phase chromatography, and adsorption
chromatography.
[0343] These chromatography approaches can be performed using
liquid-phase chromatography such as HPLC or FPLC.
[0344] Examples of columns used in affinity chromatography can
include protein A, protein G, and antigen columns.
[0345] Examples of the protein A columns include Protein A Ceramic
HyperD (manufactured by Pall Corp.), POROS (manufactured by Applied
Biosystems, Inc.), and Sepharose F. F. (manufactured by GE
Healthcare Bio-Sciences Corp.).
[0346] Also, the antibody may be purified using its binding
activity against the antigen using an antigen-immobilized
carrier.
[0347] (4-8) Nucleotides Encoding the Antibody, Recombinant
Vectors, and Recombinant Cells
[0348] The present invention provides a nucleotide(s) encoding the
antibody of the present invention or a functional fragment thereof,
or a modified form of the antibody or functional fragment
(hereinafter, this nucleotide is referred to as an "antibody
gene"), a recombinant vector having an insert of the gene, a cell
comprising the gene or the vector (hereinafter, this cell is
referred to as an "antibody gene-transfected cell"), and a cell
producing the antibody of the present invention or a functional
fragment thereof, or a modified form of the antibody or functional
fragment (hereinafter, this cell is referred to as an
"antibody-producing cell").
[0349] Preferably, the antibody gene of the present invention
comprises a nucleotide sequence described in any one of the
following (a) to (e) (hereinafter, referred to as an "antibody gene
sequence"), consists of a nucleotide sequence comprising the
antibody gene sequence, or consists of the antibody gene sequence:
[0350] (a) a combination of a nucleotide sequence encoding the
heavy chain amino acid sequence of the rat FR2-2nd_#023 antibody or
the mouse chimeric antibody thereof or the rat FR2-2nd_#028
antibody or the mouse chimeric antibody thereof and a nucleotide
sequence encoding the light chain amino acid sequence thereof;
[0351] (b) a combination of a nucleotide sequence encoding the
amino acid sequence of a heavy chain comprising CDRH1 to CDRH3 of
any of the antibodies described in (a) and a nucleotide sequence
encoding the amino acid sequence of a light chain comprising CDRL1
to CDRL3 of any of thereof; [0352] (c) a combination of a
nucleotide sequence encoding a heavy chain amino acid sequence
comprising the amino acid sequence of the heavy chain variable
region of any of the antibodies described in (a) and a nucleotide
sequence encoding a light chain amino acid sequence comprising the
amino acid sequence of the light chain variable region of any of
thereof; [0353] (d) a nucleotide sequence that hybridizes under
stringent conditions to a nucleotide consisting of a nucleotide
sequence complementary to any one of the nucleotide sequences (a)
to (c) and encodes the amino acid sequence of an antibody binding
to FGFR2; and [0354] (e) a nucleotide sequence that encodes an
amino acid sequence derived from any one of the amino acid
sequences (a) to (c) by the substitution, deletion, addition, or
insertion of 1 to 50, 1 to 45, 1 to 40, 1 to 30, 1 to 25, 1 to 20,
1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2,
or 1 amino acid(s) and encodes the amino acid sequence of an
antibody binding to FGFR2.
[0355] However, the antibody gene of the present invention is not
limited to those described in (a) to (e).
[0356] The present invention provides, as described in paragraph
(4-3), a method for producing the antibody of the present invention
or a functional fragment thereof, or a modified form of the
antibody or functional fragment, comprising the steps of: culturing
the antibody gene-transfected cell of the present invention and
recovering the antibody, the functional fragment, or the modified
form from the cultures. The antibody or functional fragment
thereof, or the modified form of the antibody or functional
fragment obtained by this production method is also included in the
present invention.
5. Composition for Diagnosis
[0357] The present invention provides a composition for testing or
diagnosis (hereinafter, collectively referred to as a "composition
for diagnosis") comprising the anti-FGFR2 antibody of the present
invention or a functional fragment thereof, or a modified form of
the antibody or functional fragment.
[0358] The composition for diagnosis of the present invention is
useful in the testing or diagnosis of FGFR2-related diseases such
as cancer or of FGFR2 expression. In the present invention, the
testing or the diagnosis includes, for example, the determination
or measurement of a risk of developing a disease, the determination
of the presence or absence of a disease, the measurement of the
degree of progression or exacerbation of a disease, the measurement
or determination of the effect of drug therapy using the
pharmaceutical composition comprising the anti-FGFR2 antibody or
the like, the measurement or determination of the effect of therapy
other than drug therapy, the measurement of a risk of recurrence of
a disease, and the determination of the presence or absence of
recurrence of a disease. However, the testing or diagnosis
according to the present invention is not limited to these, and any
approach can be used.
[0359] The composition for diagnosis of the present invention is
useful in the identification of a recipient individual for the
antibody of the present invention or the functional fragment
thereof, or a modified form of the antibody or functional fragment,
a composition comprising the same, or a pharmaceutical composition
comprising the same.
[0360] The composition for diagnosis can comprise a pH buffer, an
osmoregulator, salts, a stabilizer, an antiseptic, a color
developer, a sensitizer, an aggregation inhibitor, and the
like.
[0361] The present invention also provides a method for testing or
diagnosing FGFR2-related diseases such as cancer, use of the
antibody of the present invention for preparing a composition for
diagnosis of the diseases, and use of the antibody of the present
invention for testing or diagnosing the diseases. The present
invention also encompasses a kit for testing or diagnosis
comprising the antibody of the present invention.
[0362] The desirable testing or diagnosis method involving the
antibody of the present invention is sandwich ELISA. Any usual
detection method using antibodies, such as ELISA, RIA,
enzyme-linked immunospot (ELISPOT) assay, dot blotting, Ouchterlony
test, counterimmunoelectrophoresis (CIE), chemiluminescent
immunoassay (CLIA), or flow cytometry (FCM), may be used. The
antibodies can be labeled by a method using biotin or by any other
labeling method that can be carried out in biochemical analysis
using, for example, a label such as HRP, alkaline phosphatase, a
fluorophore (e.g., FITC), or a radioisotope. A chromogenic
substrate such as TMB (3, 3', 5, 5'-tetramethylbenzidine), BCIP
(5-bromo-4-chloro-3-indolyl phosphate), p-NPP (p-nitrophenyl
phosphate), OPD (o-Phenylenediamine), ABTS
(3-Ethylbenzothiazoline-6-sulfonic acid), and SuperSignal ELISA
Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.), a
fluorescent substrate QuantaBlu.TM. Fluorogenic Peroxidase
Substrate (Thermo Fisher Scientific Inc.), and a chemiluminescent
substrate can be used in detection using enzymatic labeling.
Samples derived from humans or non-human animals as well as
artificially treated samples such as recombinant proteins can be
subjected to this assay. Examples of test samples derived from
individual organisms can include, but are not limited to, blood,
synovial fluids, ascites, lymph, cerebrospinal fluids, tissue
homogenate supernatants, and tissue sections.
[0363] The sandwich ELISA kit for testing or diagnosis comprising
the antibody of the present invention may comprise a solution of
FGFR2 protein standards, a coloring reagent, a buffer solution for
dilution, an antibody for solid phase, an antibody for detection,
and a washing solution, and the like. Preferably, the amount of the
antibody bound to the antigen can be measured by the application of
a method such as an absorbance, fluorescence, luminescence, or
radioisotope (RI) method. Preferably, an absorbance plate reader, a
fluorescence plate reader, a luminescence plate reader, an RI
liquid scintillation counter, or the like is used in the
measurement.
[0364] Such a composition, etc. of the present invention can be
used not only in these immunohistological tests but in Western
blotting or dot blot which involves preparing soluble proteins
according to a routine method from cells, tissues, or an organ in a
sample, or a portion thereof, and reacting the soluble proteins
with a labeled antibody to confirm the presence or absence of FGFR2
in the soluble proteins.
[0365] The present invention provides an antibody useful for
immunohistochemistry (IHC) analysis or a functional fragment
thereof, and a modified form of the antibody or functional
fragment, and a composition comprising the same. Such a composition
is also encompassed by the "composition for diagnosis" of the
present invention.
[0366] The immunohistochemistry is not particularly limited as long
as this approach involves reacting a tissue section with an
antigen-binding antibody (primary antibody) and detecting the
primary antibody bound with the antigen.
[0367] Preferably, the tissue section is fixed in formalin and then
treated with paraffin. The paraffin-treated tissue section is
deparaffinized, followed by antigen retrieval treatment and
nonspecific reaction inhibition treatment. Examples of methods for
the antigen retrieval treatment (hereinafter, also simply referred
to as "retrieval") can include heat treatment and enzymatic
treatment.
[0368] The heat treatment is usually performed under preferred
conditions involving a temperature of 80 to 110.degree. C., pH 7 to
12, and a treatment time ranging from 1 to 300 minutes, more
preferably a temperature of 90 to 100.degree. C., pH 8 to 10, and a
treatment time of 20 to 60 minutes. A buffer solution, more
preferably, a buffer solution containing EDTA (examples thereof
include, but are not limited to, a 10 mM Tris buffer solution
containing 1 mM EDTA) or the like can be used in pH adjustment.
Examples of commercially available buffer solutions can include
Bond Epitope Retrieval Solution 2 (manufactured by Leica Biosystems
Nussloch GmbH; pH 9, EDTA-containing). The retrieval by heat
treatment can be preferably used in the detection of an antigen
using the rat FR2-2nd_#023 antibody, the mouse chimeric
FR2-2nd_#023 antibody, or an antibody having a structure and/or
binding specificity similar thereto.
[0369] The enzymatic treatment is usually performed under preferred
conditions involving a temperature of 10 to 50.degree. C. and a
treatment time ranging from 1 to 120 minutes, more preferably a
temperature of 20 to 38.degree. C. and a treatment time ranging
from 5 to 10 minutes. The enzyme is not particularly limited as
long as the enzyme is protease. For example, proteinase or
peptidase such as trypsin can be used. Examples of commercially
available proteinase can include Enzyme Proteinase K (IHC)
(manufactured by Leica Biosystems Nussloch GmbH) and DAKO
Proteinase K RTU (manufactured by DAKO/Agilent Technologies, Inc.).
The retrieval by enzymatic treatment can be preferably used in the
detection of an antigen using the rat FR2-2nd_#028 antibody, the
mouse chimeric FR2-2nd_#028 antibody, or an antibody having a
structure and/or binding specificity similar thereto.
[0370] A method for inactivating an endogenous enzyme having the
same or similar catalytic activity as an enzyme used in color
development is usually used as the nonspecific reaction inhibition
treatment. For color development through peroxidase reaction,
endogenous peroxidase present in tissues is preferably inhibited in
advance using H.sub.2O.sub.2 or the like. A solvent such as water
or methanol can be used for H.sub.2O.sub.2. The concentration of
H.sub.2O.sub.2 is 0.1 to 3%, preferably 0.3 to 3%. The
H.sub.2O.sub.2 solution can be supplemented with sodium azide.
Also, a blocking method using serum or casein can be used as the
nonspecific reaction inhibition treatment. Tissues can be treated
with serum or casein before the primary antibody reaction.
Alternatively, serum or casein may be contained in a solvent for
diluting the primary antibody.
[0371] The reaction conditions for the primary antibody are not
particularly limited and involve a temperature of 20 to 50.degree.
C., preferably 25 to 42.degree. C., more preferably 37.degree. C.
The reaction time is 5 minutes to all night and all day, preferably
10 minutes to 6 hours, more preferably 30 minutes to 2 hours.
[0372] Preferably, an antibody (secondary antibody) capable of
being visualized and binding to the primary antibody can be used in
the detection of the primary antibody. Preferably, the secondary
antibody can be visualized by use of a method involving binding an
enzyme such as peroxidase or alkaline phosphatase to the secondary
antibody or adding biotin or the like to the secondary antibody and
binding thereto streptavidin or the like conjugated with the
enzyme, followed by reaction with a chromogenic substrate
compatible with the enzyme. Examples of the method involving
binding an enzyme to the secondary antibody can include a method
using a reagent comprising a dextrin polymer or an amino acid
polymer to which multiple molecules of the enzyme and the secondary
antibody are attached (polymer method). A chromogenic substrate
such as DAB can be used in the method involving reacting a
biotinylated secondary antibody with peroxidase-labeled
streptavidin (LSAB method). Also, a secondary antibody labeled with
a fluorescent dye or the like can be used. After treatment with the
fluorescently labeled secondary antibody, positive cells are
detected using a fluorescence microscope.
[0373] A smear method involves separating isolated cells into
cellular components and fluid components by application to glass or
centrifugation in a centrifuge and immunostaining the cellular
components. Specifically, the cellular components can be applied
onto a glass slide, fixed in an ethanol solution, a 10% formalin
solution, or the like, and then immunostained in the same way as in
the tissue section.
[0374] A freeze embedding method involves embedding isolated
tissues in an OCT compound or the like, then rapidly freezing the
embedded tissues in liquid nitrogen or the like, and slicing the
frozen tissues using a cryostat to prepare a slide preparation.
This preparation can be fixed in a 10% formalin solution, an
ethanol solution, or the like and then immunostained in the same
way as in the tissue section.
[0375] The immunohistochemistry procedure can be performed
automatically using an immunological apparatus programmed with a
reaction solution, reaction conditions, the number of washing runs,
etc.
[0376] For diagnostic imaging, an antibody is labeled with a
pharmaceutically acceptable radionuclide or luminescent material
and administered to a test subject, and images can be taken using a
diagnostic imaging technique such as PET/CT to determine or test
the presence of FGFR2.
[0377] The antibody or functional fragment thereof, or a modified
form of the antibody or functional fragment comprised in the
composition for diagnosis of the present invention is preferably an
antibody binding to FGFR2, i.e., an antibody having FGFR2
selectivity or a functional fragment thereof, or a modified form of
the antibody or functional fragment, more preferably an antibody
having selectivity for both human FGFR2 IIIb and human FGFR2 IIIc
or functional fragment thereof or a modified form of the antibody
or functional fragment. More preferably, according to another
aspect, the antibody or functional fragment thereof, or a modified
form of the antibody or functional fragment contained in the
composition for diagnosis of the present invention has selectivity
for human FGFR2 IIIc.
[0378] Examples of the antibody having selectivity for both human
FGFR2 IIIb and human FGFR2 IIIc can include an antibody comprising
a heavy chain comprising the heavy chain CDRH1 to CDRH3 of the rat
FR2-2nd_#023 antibody and a light chain comprising the light chain
CDRL1 to CDRL3 thereof, an antibody comprising the heavy and light
chain variable regions of the rat FR2-2nd_#023 antibody, and an
antibody comprising the heavy and light chains of the rat
FR2-2nd_#023 antibody. Examples of such an antibody can include,
but are not limited to, the rat FR2-2nd_#023 antibody and the mouse
chimeric FR2-2nd_#023 antibody.
[0379] Examples of the antibody having selectivity for human FGFR2
IIIc can include an antibody comprising a heavy chain comprising
the heavy chain CDRH1 to CDRH3 of the rat FR2-2nd_#028 antibody and
a light chain comprising the light chain CDRL1 to CDRL3 thereof, an
antibody comprising the heavy and light chain variable regions of
the rat FR2-2nd_#028 antibody, and an antibody comprising the heavy
and light chains of the rat FR2-2nd_#028 antibody. Examples of such
an antibody can include, but are not limited to, the rat
FR2-2nd_#028 antibody and the mouse chimeric FR2-2nd_#028
antibody.
[0380] According to a preferred aspect, the composition for
diagnosis of the present invention is for detection or assay of
FGFR2, more preferably for detection or assay of human FGFR2 IIIb
and/or human FGFR2 IIIc, even more preferably for detection or
assay of human FGFR2 IIIb and human FGFR2 IIIc or of human FGFR2
IIIc.
[0381] The present invention provides a method for detecting or
assaying human FGFR2 IIIc in a test sample.
[0382] Alternatively, human FGFR2 IIIc in a test sample can be
detected or assayed by: (i) detecting or assaying human FGFR2 IIIb
and human FGFR2 IIIc in the test sample; (ii) detecting or assaying
human FGFR2 IIIb in the sample; and (iii) comparing the results of
detection or assay in step (i) with the results of detection or
assay in step (ii) or subtracting the results of detection or assay
in step (ii) from the results of detection or assay in step (i).
Such a method for detecting or assaying human FGFR2 IIIc is also
encompassed in the present invention.
[0383] Examples of the antibody or antigen binding fragment
thereof, or the composition used in the detection or assay of human
FGFR2 IIIb and human FGFR2 IIIc in step (i) include, but are not
limited to, the rat FR2-2nd_#023 antibody, the mouse chimeric
FR2-2nd_#023 antibody, and a composition comprising the same. The
composition may contain an additional antibody.
[0384] Examples of the antibody or antigen binding fragment
thereof, or the composition used in the detection or assay of human
FGFR2 IIIb in step (ii) include an antibody specifically binding to
human FGFR2 IIIb or an antigen binding fragment thereof (e.g., a
rat FR2-10 antibody or a chimeric antibody thereof
(WO2013/154206)), and a composition comprising the same.
[0385] The present invention provides a method for detecting or
assaying human FGFR2 IIIb in a test sample.
[0386] Alternatively, human FGFR2 IIIb in a test sample can be
detected or assayed by: (i) detecting or assaying human FGFR2 IIIb
and human FGFR2 IIIc in the test sample; (ii) detecting or assaying
human FGFR2 IIIc in the sample; and (iii) comparing the results of
detection or assay in step (i) with the results of detection or
assay in step (ii) or subtracting the results of detection or assay
in step (ii) from the results of detection or assay in step (i).
Such a method for detecting or assaying human FGFR2 IIIb is also
encompassed in the present invention.
[0387] Examples of the antibody or antigen binding fragment
thereof, or the composition used in the detection or assay of human
FGFR2 IIIb and human FGFR2 IIIc in step (i) include, but are not
limited to, the rat FR2-2nd_#023 antibody, the mouse chimeric
FR2-2nd_#023 antibody, and a composition comprising the same. The
composition may contain an additional antibody.
[0388] Examples of the antibody or antigen binding fragment
thereof, or the composition used in the detection or assay of human
FGFR2 IIIc in step (ii) include, but are not limited to, the rat
FR2-2nd_#028 antibody, the mouse chimeric FR2-2nd_#028 antibody,
and a composition comprising the same.
[0389] The composition for diagnosis of the present invention can
be used in these detection or assay methods. The present invention
also encompasses such an assay method and a composition for
diagnosis which are intended for diagnosis or testing of human
FGFR2-positive cancer, preferably human FGFR2 IIIb- and/or human
FGFR2 IIIc-positive cancer, more preferably human FGFR2
IIIc-positive cancer or human FGFR2 IIIb and human FGFR2
IIIc-positive cancer.
[0390] The present invention also encompasses a method for
identifying a recipient individual for the pharmaceutical
composition of the present invention. This identification method
involves assaying human FGFR2 in a sample derived from the
individual. The individual can be determined to be positive when
human FGFR2 is detected in the sample or when human FGFR2 is
detected in a larger amount than that of human FGFR2 detected in a
sample derived from a healthy individual. The human FGFR2 in the
identification method is preferably human FGFR2 IIIb and/or human
FGFR2 IIIc, more preferably human FGFR2 IIIb and human FGFR2 IIIc,
or human FGFR2 IIIc.
[0391] The composition for diagnosis of the present invention can
be used in this method.
[0392] According to a preferred aspect, the individual in the
identification method has cancer or is at risk thereof.
[0393] According to one aspect, the pharmaceutical composition of
the present invention can be administered to an individual
determined to be positive by the identification method.
6. Pharmaceutical Composition
[0394] The present invention provides a pharmaceutical composition
comprising an anti-FGFR2 antibody or a functional fragment thereof,
or a modified form of the antibody or functional fragment
(WO2013/154206, WO2015/053407, etc.).
[0395] Examples of the antibody for the pharmaceutical composition
specifically binding to human FGFR2 can include HuGAL-FR21 MAb,
BAY-1179470, BAY-1187982, FGFR2/FGFR4 dual targeting antibody-drug
conjugate (Novartis International AG), and antibodies described in
WO2013/154206 (including hFR2-14 H19/L and hFR2-14 H12/L1). These
antibodies can be preferably used in the treatment or prevention of
human FGFR2-positive diseases. Examples of the antibody for the
pharmaceutical composition specifically binding to human FGFR2
IIIc, particularly, the antibody for the pharmaceutical composition
specifically binding to human FGFR2 IIIc and human FGFR2 IIIb, can
include antibodies described in WO2013/154206 (including hFR2-14
H19/L1 and hFR2-14 H12/L1). These antibodies can be preferably used
in the treatment or prevention of human FGFR2 IIIc-positive
diseases, particularly, human FGFR2 IIIc- and human FGFR2
IIIb-positive diseases.
[0396] The pharmaceutical composition of the present invention is
useful in the treatment or prevention of various diseases that are
initiated or exacerbated by abnormal or increased FGFR2 signals due
to overexpression of FGFR2 or its ligand or FGFR2 mutations or gene
amplification, or by isoform switching of FGFR2 (hereinafter, these
diseases are referred to as "FGFR2-related diseases"),
particularly, various cancers.
[0397] Examples of causes of the initiation or exacerbation of such
cancers to be treated or prevented can include single nucleotide
polymorphism (SNP) in an intron of the FGFR2 gene, high expression
of FGFR2, missense mutations that constitutively activate FGFR2,
amplification or overexpression of the FGFR2 gene, and switching
from FGFR2 IIIb to FGFR2 IIIc.
[0398] Examples of such cancer types can include breast cancer,
endometrial cancer, ovary cancer, lung cancer (e.g., non-small cell
lung cancer), stomach cancer, prostate cancer, kidney cancer, liver
cancer, pancreatic cancer, colorectal cancer, esophageal cancer,
urinary bladder cancer, uterine cervix cancer, blood cancer,
lymphoma, and malignant melanoma. Preferred examples thereof can
include these cancers expressing the FGFR2 protein.
[0399] In the present invention, the treatment or prevention of a
disease includes, but is not limited to, the prevention of the
onset of the disease, preferably the disease in an individual
expressing the FGFR2 protein, the suppression or inhibition of
exacerbation or progression thereof, the alleviation of one or two
or more symptoms exhibited by an individual affected with the
disease, the suppression or remission of exacerbation or
progression thereof, the treatment or prevention of a secondary
disease, etc.
[0400] The pharmaceutical composition of the present invention can
comprise a therapeutically or prophylactically effective amount of
the anti-FGFR2 antibody or the functional fragment of the antibody
and a pharmaceutically acceptable diluent, vehicle, solubilizer,
emulsifier, preservative, and/or additive.
[0401] The "therapeutically or prophylactically effective amount"
means an amount that exhibits therapeutic or prophylactic effects
on a particular disease by means of a particular dosage form and
administration route and has the same meaning as a
"pharmacologically effective amount".
[0402] The pharmaceutical composition of the present invention may
comprise materials for changing, maintaining, or retaining pH,
osmotic pressure, viscosity, transparency, color, tonicity,
sterility, or the stability, solubility, sustained release,
absorbability, permeability, dosage form, strength, properties,
shape, etc., of the composition or the antibody comprised therein
(hereinafter, referred to as "pharmaceutical materials"). The
pharmaceutical materials are not particularly limited as long as
the materials are pharmacologically acceptable. For example, no or
low toxicity is a property preferably possessed by these
pharmaceutical materials.
[0403] Examples of the pharmaceutical materials can include amino
acids, antimicrobial agents, antioxidants, buffers, fillers,
chelating agents, complexing agents, bulking agents,
monosaccharides, disaccharides, carbohydrates, coloring agents,
corrigents, diluents, emulsifiers, hydrophilic polymers,
antiseptics, solvents, sugar alcohols, suspending agents,
surfactants, stability enhancers, elasticity enhancers, transport
agents, diluents, excipients, and/or pharmaceutical additives. The
amount of these materials added is 0.001 to 1000 times, preferably
0.01 to 100 times, more preferably 0.1 to 10 times the weight of
the anti-FGFR2 antibody or functional fragment thereof, or a
modified form of the antibody or functional fragment.
[0404] An immunoliposome comprising the anti-FGFR2 antibody or
functional fragment thereof, or a modified form of the antibody or
functional fragment encapsulated in a liposome, or a modified
antibody form comprising the antibody conjugated with a liposome
(U.S. Pat. No. 6,214,388, etc.) is also included in the
pharmaceutical composition of the present invention.
[0405] The excipients or vehicles are not particularly limited as
long as they are liquid or solid materials usually used in
injectable water, saline, artificial cerebrospinal fluids, and
other preparations for oral or parenteral administration. Examples
of saline can include neutral saline and serum albumin-containing
saline.
[0406] Examples of buffers can include a Tris buffer adjusted to
bring the final pH of the pharmaceutical composition to 7.0 to 8.5,
an acetate buffer adjusted to bring the final pH thereof to 4.0 to
5.5, a citrate buffer adjusted to bring the final pH thereof to 5.0
to 8.0, and a histidine buffer adjusted to bring the final pH
thereof to 5.0 to 8.0.
[0407] The pharmaceutical composition of the present invention is a
solid, a liquid, a suspension, or the like. Another example of the
pharmaceutical composition of the present invention can include
freeze-dried preparations. The freeze-dried preparations can be
formed using an excipient such as sucrose.
[0408] The administration route of the pharmaceutical composition
of the present invention may be any of enteral administration,
local administration, and parenteral administration. Examples
thereof can include intravenous administration, intraarterial
administration, intramuscular administration, intradermal
administration, hypodermic administration, intraperitoneal
administration, transdermal administration, intraosseous
administration, intraarticular administration, and the like.
[0409] The composition of a pharmaceutical composition can be
determined according to the administration method, the binding
affinity of the antibody for the FGFR2 protein, etc. The anti-FGFR2
antibody of the present invention or a functional fragment thereof,
or a modified form of the antibody or functional fragment having
higher affinity (lower KD value) for the FGFR2 protein can exhibit
its pharmaceutical efficacy at a lower dose.
[0410] The dose of the anti-FGFR2 antibody of the present invention
is not limited as long as the dose is a pharmacologically effective
amount. The dose can be appropriately determined according to the
species of an individual, the type of disease, symptoms, sex, age,
pre-existing conditions, the binding affinity of the antibody for
the FGFR2 protein or its biological activity, and other factors. A
dose of usually 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg,
can be administered once every day to every 180 days or twice or
three or more times a day.
[0411] Examples of the form of the pharmaceutical composition can
include injections (including freeze-dried preparations and drops),
suppositories, transnasal absorption preparations, transdermal
absorption preparations, sublingual formulations, capsules,
tablets, ointments, granules, aerosols, pills, powders,
suspensions, emulsions, eye drops, and biological implant
formulations.
[0412] The pharmaceutical composition comprising the anti-FGFR2
antibody or functional fragment thereof, or a modified form of the
antibody or functional fragment as an active ingredient can be
administered concurrently with or separately from an additional
drug. For example, the pharmaceutical composition comprising the
anti-FGFR2 antibody or functional fragment thereof as an active
ingredient may be administered after administration of the
additional drug, or the additional drug may be administered after
administration of the pharmaceutical composition. Alternatively,
the pharmaceutical composition and the additional drug may be
administered concurrently. Examples of the additional drug can
include various anticancer agents such as chemotherapeutics and
radiation therapy. These use approaches are collectively referred
to as "combined use of the additional drug" with the antibody of
the present invention. The present invention also encompasses a
pharmaceutical composition comprising the antibody of the present
invention or a functional fragment thereof, or a modified form of
the antibody or functional fragment and further comprising an
additional drug.
[0413] The present invention provides a method for treating or
preventing FGFR-related diseases such as cancer, use of the
antibody of the present invention for preparing a pharmaceutical
composition for treatment or prevention of the diseases, and use of
the antibody of the present invention for treating or preventing
the diseases. The present invention also encompasses a kit for
treatment or prevention comprising the antibody of the present
invention.
7. Reagent and Kit
[0414] The antibody of the present invention or functional fragment
thereof, or a modified form of the antibody or a functional
fragment is also useful as a reagent and a kit. Such a reagent and
a kit are used for testing or diagnosis as mentioned above, for
research, and for any other use.
EXAMPLES
[0415] Hereinafter, the present invention will be described further
specifically with reference to the Examples. However, the present
invention is not intended to be limited to them.
[0416] Procedures related to gene manipulation in the Examples
below were performed according to the methods described in
"Molecular Cloning" (Sambrook, J., Fritsch, E. F. and Maniatis, T.,
Cold Spring Harbor Laboratory Press, 1989) or the methods described
in other experimental manuals used by those skilled in the art, or
using commercially available reagents or kits according to the
instruction manuals, unless otherwise specified.
Example 1
Preparation of a Rat Anti-Human FGFR2 Antibody
[0417] 1)-1 Immunization
[0418] Female WKY/Izm rats (Japan SLC, Inc.) were used in
immunization. A mixture of an antigenic protein Recombinant Human
FGFR2.beta. (IIIc)/Fc Chimera (manufactured by R&D Systems,
Inc.) and Freund's Complete Adjuvant (manufactured by Wako Pure
Chemicals Industries, Ltd.) was administered to the tail base of
each WKY/Izm rat. The lymph node and the spleen were collected from
the rat and used in hybridoma preparation.
[0419] 1)-2 Hybridoma Preparation
[0420] The lymph node cells or the spleen cells were electrically
fused with mouse myeloma SP2/0-Ag14 cells (ATCC: CRL-1581) using
LF301-Cell Fusion Unit (manufactured by BEX Co., Ltd.). The fused
cells were diluted with ClonaCell-HY Selection Medium D
(manufactured by StemCell Technologies Inc.) and cultured.
Hybridoma colonies that appeared were recovered to prepare
monoclonal hybridomas. Each hybridoma colony thus recovered was
cultured, and the obtained hybridoma culture supernatant was used
to screen for an anti-FGFR2 antibody-producing hybridoma.
[0421] 1)-3 Construction of an Expression Vector for Screening for
Antigen-Binding Antibody
[0422] 1)-3-1 Construction of Human FGFR2 IIIb and FGFR2 IIIc
Expression Vectors (pcDNA-DEST40-FGFR2 IIIb and pcDNA-DEST40-FGFR2
IIIc)
[0423] cDNAs encoding a human FGFR2 IIIb variant protein (amino
acid sequence of positions 1 to 822 of isoform 2(NP_075259)) and a
human FGFR2 IIIc variant protein (amino acid sequence of positions
1 to 821 of isoform 1 (NP_000132)) were cloned into pcDNA-DEST40
vectors to construct vectors pcDNA-DEST40-FGFR2 IIIb and
pcDNA-DEST40-FGFR2 IIIc for expression of each variant protein,
respectively.
[0424] 1)-3-2 Construction of Human FGFR1 IIIc, Human FGFR3 IIIb,
Human FGFR3 IIIc, and Human FGFR4 Expression Vectors
[0425] cDNAs encoding a human FGFR1 IIIc variant protein (amino
acid sequence of positions 1 to 822 of isoform 1 (NP_075598)), a
human FGFR3 IIIb variant protein (amino acid sequence of positions
1 to 808 of isoform 3 (NP_001156685)), a human FGFR3 IIIc variant
protein (amino acid sequence of positions 1 to 806 of isoform 1
(NP_000133)), and a human FGFR4 protein (amino acid sequence of
positions 1 to 802 of isoform 1 (NP_002002)) were cloned into
pcDNA-DEST40 vectors to construct vectors pcDNA-DEST40-FGFR1 IIIc,
pcDNA-DEST40-FGFR3 IIIb, pcDNA-DEST40-FGFR3 IIIc, and
pcDNA-DEST40-FGFR4 for expression of each variant protein,
respectively.
[0426] 1)-3-3 Construction of a Human FGFR1 IIIb Expression Vector
(pcDNA-DEST40-FGFR1 IIIb)
[0427] A cDNA encoding a human FGFR1 IIIb variant protein (protein
comprising the amino acid sequence of the FGFR1 IIIb domain
(AAB19502) between an amino acid sequence of positions 1 to 310 and
an amino acid sequence of positions 359 to 820 of isoform 2
(NP_056934)) was cloned into a pcDNA-DEST40 vector to construct
pcDNA-DEST40-FGFR1 IIIb.
[0428] 1)-4 Antibody Screening by Cell-ELISA
[0429] 1)-4-1 Preparation of an Antigen Gene-Expressing Cell for
Cell-ELISA
[0430] HEK293 cells were adjusted to 7.5.times.10.sup.3 cells/ml in
a DMEM medium containing 10% FBS. pcDNA-DEST40-FGFR2 IIIc or a
control pcDNA-DEST40 was transfected thereto using Lipofectamine
2000 (manufactured by Life Technologies Corp.). The resulting cells
were dispensed in an amount of 50 .mu.l/well to a 96-well half area
plate (manufactured by Corning Inc.) and cultured overnight at
37.degree. C. under 5% CO.sub.2 conditions in a DMEM medium
containing 10% FBS. The obtained transfected cells were used in the
attached state in Cell-ELISA.
[0431] 1)-4-2 Cell-ELISA
[0432] After removal of the culture supernatant from the expression
vector-transfected HEK293 cells prepared in Example 1)-4-1, each
hybridoma culture supernatant was added to the pcDNA-DEST40-FGFR2
IIIc- or pcDNA-DEST40-transfected HEK293 cells, and the plate was
left standing at 4.degree. C. for 1 hour. The cells in the wells
were washed once with PBS containing 5% FBS. Then, Anti-Rat
IgG-Peroxidase antibody produced in rabbit (manufactured by
Sigma-Aldrich Corp.) diluted 500-fold with PBS containing 5% FBS
was added thereto, and the plate was left standing at 4.degree. C.
for 1 hour. The cells in the wells were washed 6 times with PBS
containing 5% FBS. Then, an OPD chromogenic solution (OPD solution
(o-phenylenediamine dihydrochloride (manufactured by Wako Pure
Chemicals Industries, Ltd.) and H.sub.2O.sub.2 dissolved at
concentrations of 0.4 mg/ml and 0.6% (v/v), respectively, in 0.05 M
trisodium citrate and 0.1 M disodium hydrogen phosphate
dodecahydrate, pH 4.5)) was added thereto at a concentration of 25
.mu.l/well. Color reaction was performed with occasional stirring
and stopped by the addition of 1 M HCl at a concentration of 25
.mu.l/well. Then, the absorbance was measured at 490 nm using a
plate reader (ENVISION; PerkinElmer, Inc.). In order to select a
hybridoma producing an antibody specifically binding to FGFR2
expressed on cell membrane surface, hybridomas that yielded a
culture supernatant exhibiting higher absorbance for the
pcDNA-DEST40-FGFR2 IIIc expression vector-transfected HEK293 cells
compared with the control pcDNA-DEST40-transfected HEK293 cells
were selected as anti-FGFR2 antibody production-positive
hybridomas.
[0433] 1)-5 Antibody Screening by Flow Cytometry
[0434] 1)-5-1 Preparation of an Antigen Gene-Expressing Cell for
Flow Cytometry Analysis
[0435] HEK293T cells were inoculated at a density of
5.times.10.sup.4 cells/cm.sup.2 to a 225-cm.sup.2 flask
(manufactured by Sumitomo Bakelite Co., Ltd.) and cultured
overnight at 37.degree. C. under 5% CO.sub.2 conditions in a DMEM
medium containing 10% FBS. On the next day, the pcDNA-DEST40-FGFR2
IIIc, the pcDNA-DEST40-FGFR2 IIIb or a control pcDNA-DEST40 was
transfected to the HEK293T cells using Lipofectamine 2000, and the
cells were further cultured overnight at 37.degree. C. under 5%
CO.sub.2 conditions. On the next day, the expression
vector-transfected HEK293T cells were treated with TrypLE Express
(manufactured by Life Technologies Corp.), washed with DMEM
containing 10% FBS, and then suspended in PBS containing 5% FBS.
The obtained cell suspension was used in flow cytometry
analysis.
[0436] 1)-5-2 Flow Cytometry Analysis
[0437] The pcDNA-DEST40-FGFR2 IIIc and pcDNA-DEST40-FGFR2 IIIb
binding specificity of the antibody produced by each hybridoma
determined to be positive by Cell-ELISA in Example 1)-4 was further
confirmed by flow cytometry. Each HEK293T cell suspension prepared
in Example 1)-5-1 was centrifuged to remove a supernatant. Then,
the pcDNA-DEST40-FGFR2 IIIc-transfected HEK293T cells, the
pcDNA-DEST40-FGFR2 IIIb-transfected HEK293T cells, or the
pcDNA-DEST40-transfected HEK293T cells were suspended by the
addition of the hybridoma culture supernatant and left standing at
4.degree. C. for 1 hour. The cells were washed once with PBS
containing 5% FBS, then suspended by the addition of Anti-Rat IgG
FITC conjugate (manufactured by Sigma-Aldrich Corp.) diluted
500-fold with PBS containing 5% FBS, and left standing at 4.degree.
C. for 1 hour. The cells were washed 3 times with PBS containing 5%
FBS and then resuspended in PBS containing 5% FBS and 2 .mu.g/ml
7-aminoactinomycin D (manufactured by Molecular Probes, Inc.),
followed by detection using a flow cytometer (FC500; manufactured
by Beckman Coulter Inc.). The data was analyzed using Flowjo
(manufactured by Tree Star Inc.). After removal of
7-aminoactinomycin D-positive dead cells by gating, the FITC
fluorescence intensity of live cells was plotted to a histogram.
Hybridomas that yielded a sample exhibiting a shift to stronger
fluorescence intensity in the histogram of the pcDNA-DEST40-FGFR2
IIIc- or pcDNA-DEST40-FGFR2 IIIb-transfected HEK293T cells compared
with the fluorescence intensity histogram of the control
pcDNA-DEST40-transfected HEK293T cells were selected such that rat
FR2-2nd_#028 was obtained as a hybridoma producing an antibody
specifically binding to FGFR2 IIIc and rat FR2-2nd_#023 was
obtained as a hybridoma producing an antibody binding to both FGFR2
IIIb and FGFR2 IIIc.
[0438] 1)-6 Isotyping of the Antibody
[0439] The rat FR2-2nd_#028 and FR2-2nd_#023 produced by the
anti-FGFR2 antibody-producing hybridomas were isotyped using Rat
monoclonal isotyping test kit (manufactured by AbD Serotec). As a
result, their isotypes were confirmed to be IgG2b and .kappa.
chains for the rat FR2-2nd_#028 and IgG1 and .kappa. chains for the
rat FR2-2nd_#023.
[0440] 1)-7 Preparation of a Monoclonal Antibody
[0441] Each rat anti-human FGFR2 monoclonal antibody was purified
from the hybridoma culture supernatant.
[0442] First, the hybridoma producing the FR2-2nd_#023 or the
FR2-2nd_#028 antibody was grown into a sufficient amount with
ClonaCell-HY Selection Medium E. Then, the medium was replaced with
Hybridoma SFM (Life Technologies Corp.) supplemented with 20% of
Ultra Low IgG FBS (Life Technologies Corp.), followed by culture
for 7 days. This culture supernatant was recovered and sterilized
through a 0.45-.mu.m filter.
[0443] The antibody was purified using Hitrap protein G HP
(manufactured by GE Healthcare Bio-Sciences Corp.) according to the
description of the attached manual. The recovered culture
supernatant was added to a column, which was then washed with a
binding buffer (0.02 M sodium phosphate (pH 7.0)), followed by
elution with 0.1 M glycine (pH 2.7). The eluted antibody solution
was neutralized, then buffer-replaced with PBS using PD-10 SX
G-25(M) 30ST column (manufactured by GE Healthcare Bio-Sciences
Corp.), and concentrated using Amicon-Ultra 4 centrifugal filter
(Merck Millipore).
[0444] The concentration of the antibody was determined by the
measurement of absorbance (O.D. 280 nm) using Gene Spec I
(manufactured by Hitachi, Ltd.). Specifically, the peak area of the
absorbance (O.D. 280 nm) of the antibody solution was measured, and
the concentration was calculated according to the following
expression: Antibody sample concentration (mg/ml)=(Peak area of the
antibody sample)/(Peak area of a standard (human
IgG1)).times.Concentration (mg/ml) of the standard.times.Dilution
ratio of the sample. Also, the concentration of endotoxin contained
in the obtained antibody was measured using The Endosafe-PTS
Portable Test System (Charles River Laboratories Japan Inc.) and
confirmed to be 1 EU/mg or lower. The antibody was used in the
subsequent experiments.
Example 2
Cloning of the Rat Antibody FR2-2nd_#028 and the Rat Antibody
FR2-2nd_#023
[0445] 2)-1 Cloning of the Rat Antibody FR2-2nd_#028
[0446] 2)-1-1 Preparation of Total RNA from the Hybridoma Producing
Rat Antibody FR2-2nd_#028
[0447] In order to amplify cDNAs encoding the variable regions of
the rat antibody FR2-2nd_#028, total RNA was prepared from the
hybridoma producing the rat antibody 2nd_#28 using TRIzol Reagent
(Ambion/Thermo Fisher Scientific Inc.).
[0448] 2)-1-2 Synthesis of cDNA (5'-RACE-Ready cDNA)
[0449] cDNAs (5'-RACE-Ready cDNAs) were synthesized using 1 .mu.g
of the total RNA prepared in Example 2)-1-1 and SMARTer RACE cDNA
Amplification Kit (Clontech Laboratories, Inc.).
[0450] 2)-1-3 5'-RACE PCR Amplification and Sequencing of cDNA
Encoding the Heavy Chain Variable Region of the Rat Antibody
FR2-2nd_#028
[0451] The primers used for PCR amplification of the variable
region-encoding cDNA of the heavy chain gene of the rat antibody
FR2-2nd_#028 were oligonucleotides having the sequences of UPM
(Universal Primer A Mix; attached to SMARTer RACE cDNA
Amplification Kit) and 5'-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3' (RG2AR3:
SEQ ID NO: 1). The UPM used was attached to SMARTer RACE cDNA
Amplification Kit (Clontech Laboratories, Inc.), while RG2AR3 was
designed from the sequences of rat heavy chain constant regions in
the database.
[0452] cDNA encoding the heavy chain variable region of the rat
antibody FR2-2nd_#028 was amplified by 5'-RACE PCR using this
primer set and the cDNA (5'-RACE-Ready cDNA) synthesized in Example
2)-1-2 as a template. This PCR was carried out on the Touchdown PCR
program according to the manual of SMARTer RACE cDNA Amplification
Kit (Clontech Laboratories, Inc.) using polymerase KOD-Plus-
(Toyobo Co., Ltd.).
[0453] The heavy chain variable region-encoding cDNA amplified by
5'-RACE PCR was purified using MinElute PCR Purification Kit
(Qiagen N. V.) and then cloned using Zero Blunt TOPO PCR Cloning
Kit (Invitrogen Corp.). The cloned heavy chain variable
region-encoding cDNA was analyzed by sequencing.
[0454] The sequencing primers used an oligonucleotide having the
sequence 5'-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3' (RG2AR3: SEQ ID NO: 1)
designed from the sequences of rat heavy chain constant regions in
the database, and NUP (Nested Universal Primer A; attached to
SMARTer RACE cDNA Amplification Kit).
[0455] The sequencing analysis was carried out using a gene
sequence analyzer ("ABI PRISM 3700 DNA Analyzer; Applied
Biosystems, Inc." or "Applied Biosystems 3730x1 Analyzer; Applied
Biosystems, Inc."). GeneAmp 9700 (Applied Biosystems, Inc.) was
used in sequencing reaction.
[0456] The determined nucleotide sequence of the cDNA encoding the
heavy chain variable region of the rat antibody FR2-2nd_#028 is
shown in SEQ ID NO: 2 (FIG. 14A), and the amino acid sequence
thereof is shown in SEQ ID NO: 3 (FIG. 14B).
[0457] 2)-1-4 5'-RACE PCR Amplification and Sequencing of cDNA
Encoding the Light Chain Variable Region of the Rat Antibody
FR2-2nd_#028
[0458] The primers used for PCR amplification of the variable
region-encoding cDNA of the light chain gene of the rat antibody
FR2-2nd_#028 were oligonucleotides having the sequences of UPM
(Universal Primer A Mix; attached to SMARTer RACE cDNA
Amplification Kit) and 5'-TCAGTAACACTGTCCAGGACACCATCTC-3' (RKR5:
SEQ ID NO: 4). The UPM used was attached to SMARTer RACE cDNA
Amplification Kit (Clontech Laboratories, Inc.), while RKR5 was
designed from the sequences of rat light chain constant regions in
the database.
[0459] cDNA encoding the light chain variable region of the rat
antibody FR2-2nd_#028 was amplified by 5'-RACE PCR using this
primer set and the cDNA (5'-RACE-Ready cDNA) synthesized in Example
2)-1-2 as a template. This PCR was carried out on the Touchdown PCR
program according to the manual of SMARTer RACE cDNA Amplification
Kit (Clontech Laboratories, Inc.) using polymerase KOD-Plus-
(Toyobo Co., Ltd.).
[0460] The light chain variable region-encoding cDNA amplified by
5'-RACE PCR was purified using MinElute PCR Purification Kit
(Qiagen N. V.) and then cloned using Zero Blunt TOPO PCR Cloning
Kit (Invitrogen Corp.). The cloned light chain variable
region-encoding cDNA was analyzed by sequencing.
[0461] The sequencing primers used were an oligonucleotide having
the sequence 5'-TCAGTAACACTGTCCAGGACACCATCTC-3' (RKR5: SEQ ID NO:
4) designed from the sequences of rat light chain constant regions
in the database, and NUP (Nested Universal Primer A; attached to
SMARTer RACE cDNA Amplification Kit).
[0462] The sequencing analysis was carried out using a gene
sequence analyzer ("ABI PRISM 3700 DNA Analyzer; Applied
Biosystems, Inc." or "Applied Biosystems 3730x1 Analyzer; Applied
Biosystems, Inc."). GeneAmp 9700 (Applied Biosystems, Inc.) was
used in sequencing reaction.
[0463] The determined nucleotide sequence of the cDNA encoding the
light chain variable region of the rat antibody FR2-2nd_#028 is
shown in SEQ ID NO: 5 (FIG. 14C), and the amino acid sequence
thereof is shown in SEQ ID NO: 6 (FIG. 14D).
[0464] 2)-2 Cloning of the Rat Antibody FR2-2nd_#023
[0465] 2)-2-1 Preparation of Total RNA from the Hybridoma Producing
the Rat Antibody FR2-2nd_#023
[0466] In order to amplify cDNAs encoding the variable regions of
the rat antibody FR2-2nd_#023, total RNA was prepared from the
hybridoma producing the rat antibody 2nd_#023 in the same way as in
Example 2)-1-1.
[0467] 2)-2-2 Synthesis of cDNA (5'-RACE-Ready cDNA)
[0468] cDNAs (5'-RACE-Ready cDNAs) were synthesized in the same way
as in Example 2)-1-2 using 1 .mu.g of the total RNA prepared in
Example 2)-2-1.
[0469] 2)-2-3 5'-RACE PCR Amplification and Sequencing of cDNA
Encoding the Heavy Chain Variable Region of the Rat Antibody
FR2-2nd_#023
[0470] cDNA encoding the heavy chain variable region of the rat
antibody FR2-2nd_#023 was amplified using the cDNA (5'-RACE-Ready
cDNA) synthesized in Example 2)-2-2 as a template, and sequenced,
in the same way as in Example 2)-1-3.
[0471] The determined nucleotide sequence of the cDNA encoding the
heavy chain variable region of the rat antibody FR2-2nd_#023 is
shown in SEQ ID NO: 7 (FIG. 15A), and the amino acid sequence
thereof is shown in SEQ ID NO: 8 (FIG. 15B).
[0472] 2)-2-4 5'-RACE PCR Amplification and Sequencing of cDNA
Encoding the Light Chain Variable Region of the Rat Antibody
FR2-2nd_#023
[0473] cDNA encoding the light chain variable region of the rat
antibody FR2-2nd_#023 was amplified using the cDNA (5'-RACE-Ready
cDNA) synthesized in Example 2)-2-2 as a template, and sequenced,
in the same way as in Example 2)-1-4.
[0474] The determined nucleotide sequence of the cDNA encoding the
light chain variable region of the rat antibody FR2-2nd_#023 is
shown in SEQ ID NO: 9 (FIG. 15C), and the amino acid sequence
thereof is shown in SEQ ID NO: 10 (FIG. 15D).
Example 3
Preparation of the Mouse IgG1 Chimeric FR2-2nd_#028 and Mouse IgG1
Chimeric FR2-2nd_#023
[0475] 3)-1 Construction of the Expression Vector pCMA-LK
[0476] A plasmid pcDNA3.3-TOPO/LacZ (Invitrogen Corp.) was digested
with restriction enzymes Xbal and Pmel. The obtained fragment of
approximately 5.4 kb was ligated with a DNA fragment comprising a
nucleotide sequence (shown in SEQ ID NO: 11 (FIG. 16)) encoding a
human .kappa. chain secretory signal and a human .kappa. chain
constant region using In-Fusion Advantage PCR cloning kit (Clontech
Laboratories, Inc.) to prepare pcDNA3.3/LK.
[0477] PCR was performed with pcDNA3.3/LK as a template using a
primer set shown below. The obtained fragment of approximately 3.8
kb was phosphorylated and then self-ligated to construct an
expression vector pCMA-LK having a signal sequence and the
nucleotide sequence encoding the human .kappa. chain constant
region, downstream of the CMV promoter.
TABLE-US-00001 Primer set (3.3-F1: SEQ ID NO: 12)
5'-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3' (3.3-R1: SEQ ID NO: 13)
5'-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3'
[0478] 3)-2 Construction of the Mouse Chimeric FR2-2nd_#028 Heavy
Chain Expression Vector
[0479] A DNA fragment comprising a nucleotide sequence (shown in
SEQ ID NO: 14) encoding the heavy chain of mouse chimeric
FR2-2nd_#028 was synthesized (GeneScript Gene Synthesis Service).
The DNA fragment comprising a nucleotide sequence encoding the
heavy chain of mouse chimeric FR2-2nd_#028 was amplified using the
synthesized DNA fragment as a template, KOD-Plus- (Toyobo Co.,
Ltd.), and a primer set shown below, and inserted to the site from
which the DNA sequence encoding the .kappa. chain secretory signal
and the human .kappa. chain constant region was removed by the
digestion of the expression vector pCMA-LK with restriction enzymes
XbaI and PmeI, using In-Fusion HD PCR cloning kit (Clontech
Laboratories, Inc.) to construct a chimeric 2nd_#028 heavy chain
expression vector. The obtained expression vector was designated as
"pCMA/2nd_#28H".
TABLE-US-00002 Primer set (CM-inf-F: SEQ ID NO: 16)
5'-CCAGCCTCCGGACTCTAGAGCCACC-3' (CM-inf-R: SEQ ID NO: 17)
5'-AGTTAGCCTCCCCCGTTTAAACTC-3'
[0480] The amino acid sequence of the heavy chain of the mouse
chimeric FR2-2nd_#028 is shown in SEQ ID NO: 15.
[0481] 3)-3 Construction of the Mouse Chimeric 2nd_#028 Light Chain
Expression Vector
[0482] A DNA fragment comprising a nucleotide sequence (shown in
SEQ ID NO: 18) encoding the light chain of the mouse chimeric
FR2-2nd_#028 was synthesized (GeneScript Artificial Gene Synthesis
Service). A chimeric 2nd_#28 light chain expression vector was
constructed in the same way as in Example 3)-2. The obtained
expression vector was designated as "pCMA/2nd_#28L".
[0483] The amino acid sequence of the light chain of the mouse
chimeric FR2-2nd_#028 is shown in SEQ ID NO: 19.
[0484] 3)-4 Construction of the Mouse Chimeric 2nd_#023 Heavy Chain
Expression Vector
[0485] A DNA fragment comprising a nucleotide sequence (shown in
SEQ ID NO: 20) encoding the heavy chain of mouse chimeric
FR2-2nd_#023 was synthesized (GeneScript Artificial Gene Synthesis
Service). A mouse chimeric 2nd_#023 heavy chain expression vector
was constructed in the same way as in Example 3)-2. The obtained
expression vector was designated as "pCMA/2nd_#23H".
[0486] The amino acid sequence of the heavy chain of the mouse
chimeric FR2-2nd_#023 is shown in SEQ ID NO: 21.
[0487] 3)-5 Construction of the Mouse Chimeric 2nd_#023 Light Chain
Expression Vector
[0488] A DNA fragment comprising a sequence (shown in SEQ ID NO:
22) encoding the light chain of the mouse chimeric FR2-2nd_#023 was
synthesized (GeneScript Artificial Gene Synthesis Service). A mouse
chimeric 2nd_#023 light chain expression vector was constructed in
the same way as in Example 3)-2. The obtained expression vector was
designated as "pCMA/2nd_#23L".
[0489] The amino acid sequence of the light chain of the mouse
chimeric FR2-2nd_#023 is shown in SEQ ID NO: 23.
[0490] 3)-6 Production of the Mouse Chimeric FR2-2nd_#028 and Mouse
Chimeric FR2-2nd_#023
[0491] FreeStyle 293F cells (Invitrogen Corp.) were subcultured and
cultured according to the manual. 1.2.times.10.sup.9 FreeStyle 293F
cells (Invitrogen Corp.) in the logarithmic growth phase were
inoculated to a 3-L Fernbach Erlenmeyer Flask (Corning Inc.),
adjusted to 2.0.times.10.sup.6 cells/ml by dilution with FreeStyle
293 expression medium (Invitrogen Corp.), and then shake-cultured
at 90 rpm at 37.degree. C. for 1 hour in an 8% CO.sub.2 incubator.
1.8 mg of polyethyleneimine (Polysciences #24765) was dissolved in
20 ml of Opti-Pro SFM medium (Invitrogen Corp.). Next, each H chain
expression vector (0.24 mg) and each L chain expression vector
(0.36 mg) prepared using NucleoBond Xtra (Takara Bio Inc.) were
added to 20 ml of Opti-Pro SFM medium (Invitrogen Corp.). 20 ml of
the expression vector/Opti-Pro SFM mixed solution was added to 20
ml of the polyethyleneimine/Opti-Pro SFM mixed solution, and the
mixture was gently stirred, left for 5 minutes, and then added to
the FreeStyle 293F cells. The cells were shake-cultured at 90 rpm
at 37.degree. C. for 4 hours in an 8% CO.sub.2 incubator. Then, 600
ml of EX-CELL VPRO medium (SAFC Biosciences), 18 ml of GlutaMAX I
(GIBCO/Thermo Fisher Scientific Inc.), and 30 ml of Yeastolate
Ultrafiltrate (GIBCO/Thermo Fisher Scientific Inc.) were added
thereto. The cells were shake-cultured at 90 rpm at 37.degree. C.
for 7 days in an 8% CO.sub.2 incubator, and the obtained culture
supernatant was filtered through Disposable Capsule Filter
(Advantec #CCS-045-E1H).
[0492] The chimeric antibody of the rat antibody FR2-2nd_#028
obtained by the combination of pCMA/2nd_#28H and pCMA/2nd_#28L was
designated as "mouse chimeric FR2-2nd_#028", and the chimeric
antibody of the rat antibody FR2-2nd_#023 obtained by the
combination of pCMA/2nd_#23H and pCMA/2nd_#23L was designated as
"mouse chimeric FR2-2nd_#023".
[0493] 3)-7 Purification of the Mouse Chimeric FR2-2nd_#023 and
Mouse Chimeric FR2-2nd_#028
[0494] Each culture supernatant obtained in Example 3)-6 was
purified by one step using rProtein A affinity chromatography (at 4
to 6.degree. C.). A buffer replacement step after the rProtein A
affinity chromatography purification was carried out at 4 to
6.degree. C. First, the culture supernatant was applied to
MabSelect SuRe (GE Healthcare Bio-Sciences Corp.) equilibrated with
PBS. After entry of the whole culture solution into the column, the
column was washed with PBS in an amount at least twice the column
volume. Next, antibody-containing fractions were collected by
elution with a 2 M arginine hydrochloride solution (pH 4.0). The
fractions were buffer-replaced with HBSor (25 mM histidine and 5%
sorbitol, pH 6.0) by dialysis (Thermo Fisher Scientific Inc.,
Slide-A-Lyzer Dialysis Cassette). The fractions were concentrated
and adjusted to an IgG concentration of 5 mg/ml using Centrifugal
UF Filter Device VIVASPIN 20 (molecular weight cutoff: UF10K,
Sartorius Japan K. K., at 4.degree. C.). Finally, the concentrate
was filtered through Minisart-Plus filter (Sartorius Japan K. K.)
and used as a purified sample.
[0495] 3)-8 Analysis of the Mouse Chimeric FR2-2nd_#028 and Mouse
Chimeric FR2-2nd_#028 for Binding to Each Molecule of FGFR Family
by Flow Cytometry Analysis
[0496] 3)-8-1 Preparation of an Antigen Gene-Expressing Cell for
Flow Cytometry Analysis
[0497] HEK293T cells were adjusted to 7.5.times.10.sup.3 cells/ml
in a DMEM medium containing 10% FBS. The cells were transfected
with the FGFR1 IIIb, FGFR1 IIIc, FGFR2 IIIb, FGFR2 IIIc, FGFR3
IIIb, FGFR3 IIIc, or FGFR4 expression vector constructed in
Examples 1)-3-1, 1)-3-2, and 1)-3-3 or an empty vector, i.e.,
pcDNA-DEST40-FGFR1 IIIb, pcDNA-DEST40-FGFR1 IIIc,
pcDNA-DEST40-FGFR2 IIIb, pcDNA-DEST40-FGFR2 IIIc,
pcDNA-DEST40-FGFR3 IIIb, pcDNA-DEST40-FGFR3 IIIc,
pcDNA-DEST40-FGFR4, or pcDNA-DEST40, using Lipofectamine 2000
(manufactured by Life Technologies Corp.) and cultured at
37.degree. C. under 5% CO.sub.2 conditions in a DMEM medium
containing 10% FBS. On the next day, the expression
vector-transfected HEK293T cells were treated with TrypLE Express
(manufactured by Life Technologies Corp.), washed with DMEM
containing 10% FBS, and then suspended in PBS containing 5% FBS.
The obtained cell suspension was used in flow cytometry
analysis.
[0498] 3) -8-2
[0499] The binding specificity of the mouse chimeric FR2-2nd_#028
and the mouse chimeric FR2-2nd_#023 prepared in Example 3)-7 for
non-denatured molecules of the FGFR family was confirmed by flow
cytometry. Each HEK293T cell suspension prepared in Example 3)-8-1
was centrifuged to remove a supernatant. Then, the
pcDNA-DEST40-FGFR1 IIIb-, pcDNA-DEST40-FGFR1 IIIc-,
pcDNA-DEST40-FGFR2 IIIb-, pcDNA-DEST40-FGFR2 IIIc-,
pcDNA-DEST40-FGFR3 IIIb-, pcDNA-DEST40-FGFR3 IIIc-,
pcDNA-DEST40-FGFR4-, or pcDNA-DEST40-transfected HEK293T cells were
suspended by the addition of a negative control mouse IgG1
(manufactured by R&D Systems, Inc.) or the mouse chimeric
FR2-2nd_#028 or the mouse chimeric FR2-2nd_#023 adjusted to 5
.mu.g/ml, and left standing at 4.degree. C. for 1 hour. The cells
were washed once with PBS containing 5% FBS, then suspended by the
addition of Fluorescein-Conjugated Goat IgG Fraction to Mouse IgG
(manufactured by MP Biomedicals, Inc.) diluted 500-fold with PBS
containing 5% FBS, and left standing at 4.degree. C. for 1 hour.
The cells were washed 3 times with PBS containing 5% FBS and then
resuspended in PBS containing 5% FBS and 2 .mu.g/ml
7-aminoactinomycin D (manufactured by Molecular Probes, Inc.),
followed by detection using a flow cytometer (FC500; manufactured
by Beckman Coulter Inc.). The data was analyzed using Flowjo
(manufactured by Tree Star Inc.). After removal of
7-aminoactinomycin D-positive dead cells by gating, the average
fluorescence intensity (MFI) of the FITC of live cells was
calculated (FIG. 1). As compared with the fluorescence intensity
histogram of the control pcDNA-DEST40-transfected HEK293T cells,
the mouse chimeric FR2-2nd_#028 exhibited specific binding to the
FGFR2 IIIc-expressing HEK293T cells and no binding to the HEK293T
cells expressing FGFR2 IIIb or the other members of the FGFR
family. Also, FR2-2nd_#023 exhibited specific binding to the FGFR2
IIIb- or FGFR2 IIIc-expressing HEK293T cells and no binding to the
HEK293T cells expressing the other members of the FGFR family.
Example 4
Immunostaining
[0500] 4)-1 Preparation of a Sample for Immunostaining
[0501] 4)-1-1 Preparation of a Cell Line Expressing Each Molecule
of FGFR Family
[0502] Cell line 293.alpha. cells, which were HEK293 cells stably
transfected with integrin .alpha.v and integrin .beta.3 expression
vectors, were adjusted to 6.times.10.sup.6 cells/225-cm.sup.2 flask
(manufactured by Sumitomo Bakelite Co., Ltd.) in a DMEM medium
containing 10% FBS and cultured overnight at 37.degree. C. under 5%
CO.sub.2 conditions. The cells were transfected with the FGFR1
IIIb, FGFR1 IIIc, FGFR2 IIIb, FGFR2 IIIc, FGFR3 IIIb, FGFR3 IIIc,
or FGFR4 expression vector constructed in Examples 1)-3-1, 1)-3-2,
and 1)-3-3 or an empty vector, i.e., pcDNA-DEST40-FGFR1 IIIb,
pcDNA-DEST40-FGFR1 IIIc, pcDNA-DEST40-FGFR2 IIIb,
pcDNA-DEST40-FGFR2 IIIc, pcDNA-DEST40-FGFR3 IIIb,
pcDNA-DEST40-FGFR3 IIIc, pcDNA-DEST40-FGFR4, or pcDNA-DEST40 using
FuGENE 6 (manufactured by Roche Diagnostics K. K.) and cultured for
two nights at 37.degree. C. under 5% CO.sub.2 conditions. The
obtained cells were recovered using TrypLE Express (manufactured by
Life Technologies Corp.) and centrifuged to obtain a pellet, which
was then washed once with PBS and centrifuged. The resulting pellet
was fixed in 20% neutral buffered formalin.
[0503] 4)-1-2 Preparation of a FGFR2-Expressing Cancer Cell
Line
[0504] A human stomach cancer line SNU-16 and a human colorectal
cancer line NCI-H716 (purchased from ATCC) cultured in RPMI
containing 10% FBS were each recovered and centrifuged to obtain a
pellet, which was then fixed in 20% neutral buffered formalin. A
human stomach cancer line KATO III (purchased from ATCC) cultured
in DMEM containing 10% FBS was recovered and centrifuged to obtain
a pellet, which was then fixed in 20% neutral buffered
formalin.
[0505] 4)-1-3 Preparation of a Tumor Sample of a FGFR2-Expressing
Cancer Cell Line Xenograft Model
[0506] 5.times.10.sup.6 cells of SNU-16 were suspended in 50%
Matrigel (manufactured by Nippon Becton Dickinson Company, Ltd.)
and subcutaneously transplanted to the axillary region of each nude
mice (CAnN.Cg-Foxn1.sup.nu/Cr1Cr1j, purchased from Charles River
Laboratories Japan Inc.). Twenty days after transplantation, tumor
was recovered and fixed in Mildform (manufactured by Wako Pure
Chemicals Industries, Ltd.).
[0507] 3.times.10.sup.5 cells of KATO III were suspended in 100%
Matrigel (manufactured by Nippon Becton Dickinson Company, Ltd.)
and subcutaneously transplanted to the axillary region of each SCID
mouse (CB17/1cr-Prkdc.sup.scid/Cr1Cr1j, purchased from Charles
River Laboratories Japan Inc.). Thirty days after transplantation,
tumor was recovered and fixed in Mildform (manufactured by Wako
Pure Chemicals Industries, Ltd.).
[0508] 2.5.times.10.sup.6 cells of NCI-H716 were suspended in 100%
Matrigel (manufactured by Nippon Becton Dickinson Company, Ltd.)
and subcutaneously transplanted to the axillary region of each nude
mice (CAnN.Cg-Foxn1.sup.nu/Cr1Cr1j, purchased from Charles River
Laboratories Japan Inc.). Twenty-one days after transplantation,
tumor was recovered and fixed in Mildform (manufactured by Wako
Pure Chemicals Industries, Ltd.).
[0509] 4)-2 Paraffin Embedding and Sectioning
[0510] Paraffin embedding and sectioning are general approaches,
and any tool or instrument can be used without particular
limitations.
[0511] The cells of each line prepared in Examples 4)-1-1 and
4)-1-2 were recovered into a 15-mL tube and centrifuged at 1500 rpm
for 5 minutes to remove a supernatant. 3 mL of 20% neutral buffered
formalin (manufactured by Wako Pure Chemicals Industries, Ltd.) was
layered over the cell pellet and left standing at room temperature
for 30 minutes or longer for fixation. Then, 5 mL of chloroform was
added thereto. Immediately thereafter, the tube was centrifuged at
1000 rpm for 10 minutes, and the formalin layer was immediately
removed. Then, the cell pellet formed between the formalin layer
and the chloroform layer was recovered. The cell pellet was put in
a nylon mesh bag, which was then placed in a cassette for tissue
preparation (Unicassette Standard, manufactured by Sakura Finetek
Japan Co., Ltd.). The cell pellet, together with the cassette, was
dipped in ethanol to wash off the chloroform. Each xenograft tissue
prepared in Example 4)-1-3 was fixed in Mildform (purchased from
Wako Pure Chemicals Industries, Ltd.), then trimmed at the cutout
portion, and placed in a cassette.
[0512] The cell pellet and the xenograft tissue were
paraffin-embedded by a conventional method.
[0513] Dehydration, delipidation, and paraffin impregnation were
performed using an automatic fixation and embedding apparatus
(Tissue-Tek VIP5 Jr.; manufactured by Sakura Finetek Japan Co.,
Ltd.). The cassette was taken out of the automatic fixation and
embedding apparatus and transferred to the paraffin bath of a
paraffin-embedded block preparation apparatus (Tissue-Tek TEC Plus;
manufactured by Sakura Finetek Japan Co., Ltd.). A small amount of
melted paraffin was injected into an embedding dish loaded to this
apparatus. The cell pellet or the tissue was separated with
tweezers from the cassette container or the nylon mesh taken out of
the paraffin bath, and loaded into the paraffin in this embedding
dish. Subsequently, a cassette was placed as an embedding frame on
the embedding dish, and melted paraffin was poured over the cell
pellet or the tissue within the cassette. The embedding dish
containing the embedding frame integrated with the cells or the
tissue was placed on a cooling unit and cooled. After
solidification of paraffin, the embedded block was taken out of the
embedding dish and subjected to sectioning. The sectioning was
performed by the slicing of the embedded block thus prepared into
sections with a thickness of 3 .mu.m using a microtome (IVS-410;
manufactured by Sakura Finetek Japan Co., Ltd.). Each section thus
obtained was applied to an antistripping glass slide (Platinum;
manufactured by Matsunami Glass Ind., Ltd.). The glass slide was
dried overnight on a paraffin stretcher (manufactured by Sakura
Finetek Japan Co., Ltd.) at 50.degree. C., accommodated in a slide
case, and stored in a desiccator.
[0514] 4)-3 Staining
[0515] 4)-3-1 Staining Using Commercially Available Antibody 18601
(Anti-Human K-sam Rabbit IgG Affinity Purify, Manufactured by
Immuno-Biological Laboratories Co., Ltd.)
[0516] Each sample was stained using an automatic staining
apparatus (Discovery Ultra; manufactured by Ventana Medical
Systems, Inc.). The reaction temperature during the staining
process was set to 37.degree. C., unless otherwise specified. The
sample was deparaffinized by 3 incubation runs each involving
68.degree. C. for 4 minutes using fresh EZ buffer (manufactured by
Ventana Medical Systems, Inc.), and then washed with EZ buffer.
Antigen retrieval was carried out by 4 runs each involving
95.degree. C. using fresh CC1 buffer (manufactured by Ventana
Medical Systems, Inc.) for a total of 52 minutes. The sample was
washed 4 times with a reaction buffer (manufactured by Ventana
Medical Systems, Inc.). Protein Block serum free (manufactured by
DAKO/Agilent Technologies, Inc.) was added thereto, and the sample
was incubated for 16 minutes and washed 3 times with a reaction
buffer. 18601 was diluted to 5 .mu.g/mL with an antibody diluent
dedicated to Discovery (manufactured by Ventana Medical Systems,
Inc.), and reacted with the sample for 1 hour. After washing 3
times with a reaction buffer, Inhibitor CM (ChromoMap kit;
manufactured by Ventana Medical Systems, Inc.) was reacted
therewith for 8 minutes, and the sample was then washed twice with
a reaction buffer. UMap anti-Rb HRP (manufactured by Ventana
Medical Systems, Inc.) was reacted therewith for 32 minutes, and
the sample was washed 4 times with a reaction buffer. DAB CM
(ChromoMap kit; manufactured by Ventana Medical Systems, Inc.) was
reacted therewith for 4 minutes. Then, H2O2CM (ChromoMap kit;
manufactured by Ventana Medical Systems, Inc.) was added thereto,
followed by reaction with 8 minutes. The sample was washed once
with a reaction buffer. Copper-CM (ChromoMap kit; manufactured by
Ventana Medical Systems, Inc.) was reacted therewith for 4 minutes,
and the sample was washed once with a reaction buffer. Hematoxylin
II counter stain reagent (manufactured by Ventana Medical Systems,
Inc.) was reacted therewith for 4 minutes, and the sample was
washed twice with a reaction buffer. Bluing Reagent (lithium
carbonate reagent, manufactured by Ventana Medical Systems, Inc.)
was reacted therewith for 4 minutes, and the sample was washed once
with a reaction buffer.
[0517] 4)-3-2 Staining Using Rat FR2-2nd_#028 Antibody
[0518] Each sample was deparaffinized according to a standard
method by passing the sample through 4 baths of 100% xylene and 3
baths of 100% ethanol for 5 minutes each, followed by washing with
ion-exchanged water. Then, the sample was stained using an
automatic staining apparatus (Dako Autostainer Link 48;
manufactured by DAKO/Agilent Technologies, Inc.). After washing
once with EnVision FLEX WASH BUFFER (manufactured by DAKO/Agilent
Technologies, Inc.), the sample was incubated with DAKO Proteinase
K RTU (manufactured by DAKO/Agilent Technologies, Inc.) at room
temperature for 6 minutes and washed three times with EnVision FLEX
WASH BUFFER. Peroxidase Block 3% H2O2 (manufactured by DAKO/Agilent
Technologies, Inc.) was added thereto, and the sample was incubated
for 5 minutes and washed twice with EnVision FLEX WASH BUFFER.
Protein Block serum free (manufactured by DAKO/Agilent
Technologies, Inc.) was added thereto, and the sample was incubated
for 30 minutes and washed once with EnVision FLEX WASH BUFFER. The
rat FR2-2nd_#028 was diluted to 15 .mu.g/mL with DAKO REAL Antibody
Diluent and reacted with the sample for 1 hour. After washing 3
times with EnVision FLEX WASH BUFFER, Histofine simple stain mouse
MAX-PRO (Rat) #414311 (manufactured by Nichirei Biosciences Inc.)
was added thereto, and the sample was incubated for 30 minutes and
then washed twice with EnVision FLEX WASH BUFFER.
[0519] DAKO Liquid DAB+Substrate Chromogen System was added
thereto, and the sample was incubated for a total of 10 minutes and
then washed once with EnVision FLEX WASH BUFFER. EnVision FLEX
Hematoxylin was added thereto, and the sample was incubated for 5
minutes and then washed a total of 3 times with EnVision FLEX WASH
BUFFER and ion-exchanged water.
[0520] 4)-3-3 Staining Using Mouse Chimeric FR2-2nd_#028
Antibody
[0521] Each sample was stained using an automatic staining
apparatus (LEICA BOND-III; manufactured by Leica Biosystems
Nussloch GmbH). For deparaffinization, the sample was heated to
72.degree. C., then Bond Dewax Solution (manufactured by Leica
Biosystems Nussloch GmbH) was added thereto, and the sample was
incubated for 30 seconds, followed by addition of ethanol and
washing 4 times with Bond Wash Solution (manufactured by Leica
Biosystems Nussloch GmbH). Enzyme Proteinase K (IHC) (manufactured
by Leica Biosystems Nussloch GmbH) diluted 200-fold was added
thereto, and the sample was incubated at 37.degree. C. for 5
minutes. The sample was washed 7 times with Bond Wash Solution. The
mouse chimeric FR2-2nd_#028 was diluted to 15 .mu.g/mL and reacted
with the sample for 30 minutes.
[0522] The sample was washed 3 times with Bond Wash Solution. Post
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes.
[0523] The sample was washed 4 times with Bond Wash Solution.
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes. The sample was washed 4
times with Bond Wash Solution. Peroxide Block reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
5 minutes. The sample was washed a total of 4 times with Bond Wash
Solution and ion-exchanged water. DAB reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
10 minutes. The sample was washed 4 times with ion-exchanged water.
Hematoxylin (Bond Polymer System/Leica IHC Refine kit, manufactured
by Leica Biosystems Nussloch GmbH) was added thereto, and the
sample was incubated for 5 minutes and then washed a total of 4
times with ion-exchanged water and Bond Wash Solution.
[0524] 4)-3-4 Staining Using Rat FR2-2nd_#023 or Mouse Chimeric
FR2-2nd_#023 Antibodies
[0525] Each sample was stained using an automatic staining
apparatus (LEICA BOND-III; manufactured by Leica Biosystems
Nussloch GmbH). For deparaffinization, the sample was heated to
72.degree. C., then Bond Dewax Solution (manufactured by Leica
Biosystems Nussloch GmbH) was added thereto, and the sample was
incubated for 30 seconds, followed by addition of ethanol and
washing 4 times with Bond Wash Solution (manufactured by Leica
Biosystems Nussloch GmbH). Bond Epitope Retrieval Solution 2
(manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated at 100.degree. C. for 20 minutes (rat
FR2-2nd_#023) or 40 minutes (mouse chimeric FR2-2nd_#023). The
sample was washed 7 times with Bond Wash Solution. Each antibody
was diluted to 15 .mu.g/mL and reacted with the sample for 30
minutes.
[0526] The sample was washed 3 times with Bond Wash Solution. Post
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes.
[0527] The sample was washed 4 times with Bond Wash Solution.
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes. The sample was washed 4
times with Bond Wash Solution. Peroxide Block reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
5 minutes. The sample was washed a total of 4 times with Bond Wash
Solution and ion-exchanged water. DAB reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
10 minutes. The sample was washed 4 times with ion-exchanged water.
Hematoxylin (Bond Polymer System/Leica IHC Refine kit, manufactured
by Leica Biosystems Nussloch GmbH) was added thereto, and the
sample was incubated for 5 minutes and then washed a total of 4
times with ion-exchanged water and Bond Wash Solution.
[0528] 4)-3-5 Staining Using ab58201 Antibody (Anti-FGFR2 Antibody,
Abcam plc.)
[0529] Each sample was stained using an automatic staining
apparatus (LEICA BOND-III; manufactured by Leica Biosystems
Nussloch GmbH). For deparaffinization, the sample was heated to
72.degree. C., then Bond Dewax Solution (manufactured by Leica
Biosystems Nussloch GmbH) was added thereto, and the sample was
incubated for 30 seconds, followed by addition of ethanol and
washing 4 times with Bond Wash Solution (manufactured by Leica
Biosystems Nussloch GmbH). Bond Epitope Retrieval Solution 2
(manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated at 100.degree. C. for 20 minutes. The
sample was washed 7 times with Bond Wash Solution. ab58201 was
diluted to 2 .mu.g/mL and reacted with the sample for 30
minutes.
[0530] The sample was washed 3 times with Bond Wash Solution. Post
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes.
[0531] The sample was washed 4 times with Bond Wash Solution.
Primary reagent (Bond Polymer System/Leica IHC Refine kit,
manufactured by Leica Biosystems Nussloch GmbH) was added thereto,
and the sample was incubated for 8 minutes. The sample was washed 4
times with Bond Wash Solution. Peroxide Block reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
5 minutes. The sample was washed a total of 4 times with Bond Wash
Solution and ion-exchanged water. DAB reagent (Bond Polymer
System/Leica IHC Refine kit, manufactured by Leica Biosystems
Nussloch GmbH) was added thereto, and the sample was incubated for
10 minutes. The sample was washed 4 times with ion-exchanged water.
Hematoxylin (Bond Polymer System/Leica IHC Refine kit, manufactured
by Leica Biosystems Nussloch GmbH) was added thereto, and the
sample was incubated for 5 minutes and then washed a total of 4
times with ion-exchanged water and Bond Wash Solution.
[0532] 4)-4 Evaluation of Stained Preparation: Binding Specificity
for Each Denatured Molecule of FGFR Family
[0533] The preparations completely stained in Example 4)-3 were
dehydrated with ethanol series, cleared with xylene series, and
then mounted on glass covers together with mounting agents. The
preparations were observed under an optical microscope and
evaluated for brown stains representing positive reaction
products.
[0534] The commercially available antibody 18601 exhibited positive
stains on cells forced to express FGFR2 IIIb or FGFR2 IIIc, but
exhibited no positive stain on cells forced to express FGFR1 IIIb,
FGFR1 IIIc, FGFR3 IIIb, FGFR3 IIIc, and FGFR4, which are other
members of the FGFR family, or empty vector-transfected cells
(FIGS. 2A to 2D). Furthermore, clear positive stains were observed
on many cells in the blocks of SNU-16 cells (FIG. 3-(A)), KATO III
cells (FIG. 3-(C)), and NCI-H716 cells (FIG. 3-(B)), demonstrating
that these cell lines expressed the FGFR2 protein.
[0535] As shown in FIGS. 4A to 4D and FIGS. 5A to 5D, the rat
FR2-2nd_#028 and the mouse chimeric FR2-2nd_#028 exhibited very
strong stains only on some cells in the blocks of cells forced to
express FGFR2 IIIc, but exhibited no positive stain on other
forcedly expressing cells or empty vector-transfected cells. Thus,
it was concluded that the rat FR2-2nd_#028 and the mouse chimeric
FR2-2nd_#028 are capable of specifically staining FGFR2 IIIc. Also,
the rat FR2-2nd_#028 and the mouse chimeric FR2-2nd_#028 exhibited
clear positive stains on many NCI-H716 cells (FIGS. 6-(B) and
7-(B)) confirmed to express the FGFR2 IIIc protein, but exhibited
no positive stain on SNU-16 cells (FIGS. 6-(A) and 7-(A)) and KATO
III cells (FIGS. 6-(C) and 7-(C)) expressing no FGFR2 IIIc protein.
From these results, the rat FR2-2nd_#028 and the mouse chimeric
FR2-2nd_#028 were confirmed to also exhibit positive reaction with
cancer cells endogenously having the FGFR2 IIIc protein. As shown
in FIG. 8, the rat FR2-2nd_#028 exhibited clear positive stains on
many cells in the xenograft tumors derived from NCI-H716 cells.
From these results, the rat FR2-2nd_#028 was confirmed to also
react with FGFR2 IIIc in the xenograft tumors.
[0536] As shown in FIGS. 9A to 9D and FIGS. 10A to 10D, the rat
FR2-2nd_#023 and mouse chimeric FR2-2nd_#023 antibodies exhibited
no positive stain on cells forced to express FGFR1 IIIb, FGFR1
IIIc, FGFR3 IIIb, FGFR3 IIIc, and FGFR4, which are other members of
the FGFR family. Thus, it was concluded that the rat FR2-2nd_#023
and mouse chimeric FR2-2nd_#023 antibodies are capable of
specifically staining FGFR2 IIIb or FGFR2 IIIc. As shown in FIGS.
11 and 12, the rat FR2-2nd_#023 and the mouse chimeric FR2-2nd_#023
exhibited clear positive stains on many cells in SNU-16 cells,
NCI-H716 cells, and KATO III cells confirmed to express the FGFR2
protein. From these results, the rat FR2-2nd_#023 and the mouse
chimeric FR2-2nd_#023 were confirmed to also exhibit positive
reaction with cancer cells endogenously having the FGFR2 IIIb or
FGFR2 IIIc protein.
[0537] The commercially available antibody 18601, a polyclonal
antibody, was found to have lot-to-lot variation in staining. On
the other hand, the rat FR2-2nd_#023, the mouse chimeric
FR2-2nd_#023, the rat FR2-2nd_#028, and the mouse chimeric
FR2-2nd_#028 are capable of staining with little variation due to
being recombinant monoclonal antibodies. Particularly, the rat
FR2-2nd_#023 and the mouse chimeric FR2-2nd_#023 rarely varied. As
for stained states, 18601 offered a low contrast in positive images
and a poor S/N ratio, as compared with the rat FR2-2nd_#023 and the
mouse chimeric FR2-2nd_#023.
[0538] On the other hand, as shown in FIGS. 13A to 13D, ab58201
exhibited clear positive stains on many cells forced to express
FGFR1 IIIb, FGFR1 IIIc, FGFR3 IIIb, and FGFR3 IIIc, which are other
members of the FGFR family, and was therefore confirmed to be
unsuitable for the expression analysis of FGFR2 alone.
INDUSTRIAL APPLICABILITY
[0539] Use of the antibody provided by the present invention
enables the testing or diagnosis of various cancers.
Sequence Listing Free Text
[0540] SEQ ID NO: 1: Nucleotide sequence of a primer RG2AR3 [0541]
SEQ ID NO: 2: Nucleotide sequence encoding the heavy chain variable
region of a rat anti-FGFR2 antibody FR2-2nd_#028 (FIG. 14A) [0542]
SEQ ID NO: 3: Amino acid sequence of the heavy chain variable
region of the rat anti-FGFR2 antibody FR2-2nd_#028 (FIG. 14B)
[0543] SEQ ID NO: 4: Nucleotide sequence of a primer RKR5 [0544]
SEQ ID NO: 5: Nucleotide sequence encoding the light chain variable
region of the rat anti-FGFR2 antibody FR2-2nd_#028 (FIG. 14C)
[0545] SEQ ID NO: 6: Amino acid sequence of the light chain
variable region of the rat anti-FGFR2 antibody FR2-2nd_#028 (FIG.
14D) [0546] SEQ ID NO: 7: Nucleotide sequence encoding the heavy
chain variable region of a rat anti-FGFR2 antibody FR2-2nd_#023
(FIG. 15A) [0547] SEQ ID NO: 8: Amino acid sequence of the heavy
chain variable region of the rat anti-FGFR2 antibody FR2-2nd_#023
(FIG. 15B) [0548] SEQ ID NO: 9: Nucleotide sequence encoding the
light chain variable region of the rat anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 15C) [0549] SEQ ID NO: 10: Amino acid sequence
of the light chain variable region of the rat anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 15D) [0550] SEQ ID NO: 11: DNA fragment
comprising a DNA sequence encoding the amino acids of a human
.kappa. chain secretory signal sequence and a human .kappa. chain
constant region (FIG. 16) [0551] SEQ ID NO: 12: Nucleotide sequence
of a primer 3.3-F1 [0552] SEQ ID NO: 13: Nucleotide sequence of
primer 3.3-R1 [0553] SEQ ID NO: 14: Nucleotide sequence comprising
a nucleotide sequence (nucleotide positions 26 to 1141) encoding
the heavy chain of a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028 (FIG. 17A) [0554] SEQ ID NO: 15: Amino acid sequence
of the heavy chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028 (FIG. 17B) [0555] SEQ ID NO: 16: Nucleotide sequence
of a primer CM-inf-F [0556] SEQ ID NO: 17: Nucleotide sequence of a
primer CM-inf-R [0557] SEQ ID NO: 18: Nucleotide sequence
comprising a nucleotide sequence (nucleotide positions 26 to 724)
encoding the light chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028 (FIG. 17C) [0558] SEQ ID NO: 19: Amino acid sequence
of the light chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#028 (FIG. 17D) [0559] SEQ ID NO: 20: Nucleotide sequence
comprising a nucleotide sequence (nucleotide positions 26 to 1423)
encoding the heavy chain of a mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 18A) [0560] SEQ ID NO: 21: Amino acid sequence
of the heavy chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 18B) [0561] SEQ ID NO: 22: Nucleotide sequence
comprising a nucleotide sequence (nucleotide positions 26 to 724)
encoding the light chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 18C) [0562] SEQ ID NO: 23: Amino acid sequence
of the light chain of the mouse chimeric anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 18D) [0563] SEQ ID NO: 24: Amino acid sequence
of the heavy chain CDR1 of the rat anti-FGFR2 antibody FR2-2nd_#028
(FIG. 19) [0564] SEQ ID NO: 25: Amino acid sequence of the heavy
chain CDR2 of the rat anti-FGFR2 antibody FR2-2nd_#028 (FIG. 19)
[0565] SEQ ID NO: 26: Amino acid sequence of the heavy chain CDR3
of the rat anti-FGFR2 antibody FR2-2nd_#028 (FIG. 19) [0566] SEQ ID
NO: 27: Amino acid sequence of the light chain CDR1 of the rat
anti-FGFR2 antibody FR2-2nd_#028 (FIG. 19) [0567] SEQ ID NO: 28:
Amino acid sequence of the light chain CDR2 of the rat anti-FGFR2
antibody FR2-2nd_#028 (FIG. 19) [0568] SEQ ID NO: 29: Amino acid
sequence of the light chain CDR3 of the rat anti-FGFR2 antibody
FR2-2nd_#028 (FIG. 19) [0569] SEQ ID NO: 30: Amino acid sequence of
the heavy chain CDR1 of the rat anti-FGFR2 antibody FR2-2nd_#023
(FIG. 20) [0570] SEQ ID NO: 31: Amino acid sequence of the heavy
chain CDR2 of the rat anti-FGFR2 antibody FR2-2nd_#023 (FIG. 20)
[0571] SEQ ID NO: 32: Amino acid sequence of the heavy chain CDR3
of the rat anti-FGFR2 antibody FR2-2nd_#023 (FIG. 20) [0572] SEQ ID
NO: 33: Amino acid sequence of the light chain CDR1 of the rat
anti-FGFR2 antibody FR2-2nd_#023 (FIG. 20) [0573] SEQ ID NO: 34:
Amino acid sequence of the light chain CDR2 of the rat anti-FGFR2
antibody FR2-2nd_#023 (FIG. 20) [0574] SEQ ID NO: 35: Amino acid
sequence of the light chain CDR3 of the rat anti-FGFR2 antibody
FR2-2nd_#023 (FIG. 20)
Sequence CWU 1
1
35129DNAArtificial Sequenceprimer 1ctccagagtt ccaggtcacg gtgactggc
292357DNARattus norvegicusCDS(1)..(357) 2gag gtg caa ctg gtg gag
tct ggt gga ggc tta gtg cag cct gga agg 48Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15tcc ctg aaa ctc tcc
tgt gca gcc tca gga ttc act ttc agt gac tat 96Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30ggc atg gcc tgg
gtc cgc cag gct cca acg aag ggg ctg gag tgg gtc 144Gly Met Ala Trp
Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val 35 40 45gca acc att
agt tat gat ggt agt agc act tac tat cga gac tcc gtg 192Ala Thr Ile
Ser Tyr Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60aag ggc
cgt ttc act atc tcc aga gaa aat gca aaa agc acc cta tcc 240Lys Gly
Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Ser Thr Leu Ser65 70 75
80ctg caa atg gac agt ctg agg tct gag gac acg gcc act tat tac tgt
288Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95aca aga cat ccg act tat tac tat ata atg gat gcc tgg ggt caa
gga 336Thr Arg His Pro Thr Tyr Tyr Tyr Ile Met Asp Ala Trp Gly Gln
Gly 100 105 110gct tca gtc act gtc tcc tca 357Ala Ser Val Thr Val
Ser Ser 1153119PRTRattus norvegicus 3Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Gly Met Ala Trp Val
Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val 35 40 45Ala Thr Ile Ser
Tyr Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Glu Asn Ala Lys Ser Thr Leu Ser65 70 75 80Leu
Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90
95Thr Arg His Pro Thr Tyr Tyr Tyr Ile Met Asp Ala Trp Gly Gln Gly
100 105 110Ala Ser Val Thr Val Ser Ser 115428DNAArtificial
Sequenceprimer 4tcagtaacac tgtccaggac accatctc 285324DNARattus
exulansCDS(1)..(324) 5gac act gta ctg acc cag tct cct gct ttg gct
gtg tct cta ggg cag 48Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala
Val Ser Leu Gly Gln1 5 10 15agg gtc acc atc tct tgt agg gcc agc aaa
agt gtc agt aca ttt atg 96Arg Val Thr Ile Ser Cys Arg Ala Ser Lys
Ser Val Ser Thr Phe Met 20 25 30aac tgg tac caa cag aaa tcg gga cag
caa ccc aaa ctc ctg atc tat 144Asn Trp Tyr Gln Gln Lys Ser Gly Gln
Gln Pro Lys Leu Leu Ile Tyr 35 40 45aga gca tcc aac cta gaa tct gga
gtc cct tcc agg ttc agt ggg agt 192Arg Ala Ser Asn Leu Glu Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60ggg tct ggg aca gac ttt acc
ctc acc ata gat cct gtg gag gct gat 240Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Asp Pro Val Glu Ala Asp65 70 75 80gac ata gca aac tat
tac tgt cag cag agt aat gaa ctt cct ctc acg 288Asp Ile Ala Asn Tyr
Tyr Cys Gln Gln Ser Asn Glu Leu Pro Leu Thr 85 90 95ttc ggt tct ggg
acc aag ctg gag atc aaa cgg gct 324Phe Gly Ser Gly Thr Lys Leu Glu
Ile Lys Arg Ala 100 1056108PRTRattus exulans 6Asp Thr Val Leu Thr
Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln1 5 10 15Arg Val Thr Ile
Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Phe Met 20 25 30Asn Trp Tyr
Gln Gln Lys Ser Gly Gln Gln Pro Lys Leu Leu Ile Tyr 35 40 45Arg Ala
Ser Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp65 70 75
80Asp Ile Ala Asn Tyr Tyr Cys Gln Gln Ser Asn Glu Leu Pro Leu Thr
85 90 95Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Ala 100
1057369DNARattus norvegicusCDS(1)..(369) 7gag gtg cag ctg gtg gag
tct ggg ggc ggc tta gtg cag cct gga agg 48Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10 15tcc atg aaa ctc tcc
tgt gca gcc tca gga ctc act ttc agt aac tat 96Ser Met Lys Leu Ser
Cys Ala Ala Ser Gly Leu Thr Phe Ser Asn Tyr 20 25 30ggc atg gcc tgg
gtc cgc cag gct cca aag aag ggt ctg gag tgg gtc 144Gly Met Ala Trp
Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val 35 40 45gca ttc att
agt cat gat ggt ggt agc tct tac tat cga gac tcc gtg 192Ala Phe Ile
Ser His Asp Gly Gly Ser Ser Tyr Tyr Arg Asp Ser Val 50 55 60gag ggc
cga ttc att atc tcc aga gat aat gcg aaa agc acc cta tcc 240Glu Gly
Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Ser65 70 75
80ctg caa atg gac agt ctg agg tct gag gac acg gcc act tat tac tgt
288Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95aca aca gcc ggg gac tac tac agc gac aat gac tgg tac ttt gac
ttc 336Thr Thr Ala Gly Asp Tyr Tyr Ser Asp Asn Asp Trp Tyr Phe Asp
Phe 100 105 110tgg ggc cca gga atc atg gtc acc gtg tcc tca 369Trp
Gly Pro Gly Ile Met Val Thr Val Ser Ser 115 1208123PRTRattus
norvegicus 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe
Ser Asn Tyr 20 25 30Gly Met Ala Trp Val Arg Gln Ala Pro Lys Lys Gly
Leu Glu Trp Val 35 40 45Ala Phe Ile Ser His Asp Gly Gly Ser Ser Tyr
Tyr Arg Asp Ser Val 50 55 60Glu Gly Arg Phe Ile Ile Ser Arg Asp Asn
Ala Lys Ser Thr Leu Ser65 70 75 80Leu Gln Met Asp Ser Leu Arg Ser
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Thr Ala Gly Asp Tyr Tyr
Ser Asp Asn Asp Trp Tyr Phe Asp Phe 100 105 110Trp Gly Pro Gly Ile
Met Val Thr Val Ser Ser 115 1209324DNARattus
norvegicusCDS(1)..(324) 9gac atc cag atg acc cag tct cct tca ctc
ctg tca gca tct gtg gga 48Asp Ile Gln Met Thr Gln Ser Pro Ser Leu
Leu Ser Ala Ser Val Gly1 5 10 15gac aga gtc act ctc agc tgc aaa gca
agt cag agt att tac aac agt 96Asp Arg Val Thr Leu Ser Cys Lys Ala
Ser Gln Ser Ile Tyr Asn Ser 20 25 30tta gcc tgg tat cag caa aaa ctt
gga gaa gct ccc aaa ctc ctc ata 144Leu Ala Trp Tyr Gln Gln Lys Leu
Gly Glu Ala Pro Lys Leu Leu Ile 35 40 45tat gat gca gac agt ttg caa
acg ggc atc cca tca agg ttc agt ggc 192Tyr Asp Ala Asp Ser Leu Gln
Thr Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60agt gga tct ggt aca gat
tac aca ctc acc atc agc agc ctg cag cct 240Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80gaa gat gtt gcc
aca tat ttc tgc cag aag tat tat agc ggg tgg acg 288Glu Asp Val Ala
Thr Tyr Phe Cys Gln Lys Tyr Tyr Ser Gly Trp Thr 85 90 95ttc ggt gga
ggc acc aag ctg gaa ttg aaa cgg gct 324Phe Gly Gly Gly Thr Lys Leu
Glu Leu Lys Arg Ala 100 10510108PRTRattus norvegicus 10Asp Ile Gln
Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Leu Ser Cys Lys Ala Ser Gln Ser Ile Tyr Asn Ser 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Leu Leu Ile 35 40
45Tyr Asp Ala Asp Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Val Ala Thr Tyr Phe Cys Gln Lys Tyr Tyr Ser
Gly Trp Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala
100 10511449DNAHomo sapiens 11gcctccggac tctagagcca ccatggtgct
gcagacccag gtgttcatct ccctgctgct 60gtggatctcc ggcgcgtacg gcgatatcgt
gatgattaaa cgtacggtgg ccgccccctc 120cgtgttcatc ttccccccct
ccgacgagca gctgaagtcc ggcaccgcct ccgtggtgtg 180cctgctgaat
aacttctacc ccagagaggc caaggtgcag tggaaggtgg acaacgccct
240gcagtccggg aactcccagg agagcgtgac cgagcaggac agcaaggaca
gcacctacag 300cctgagcagc accctgaccc tgagcaaagc cgactacgag
aagcacaagg tgtacgcctg 360cgaggtgacc caccagggcc tgagctcccc
cgtcaccaag agcttcaaca ggggggagtg 420ttaggggccc gtttaaacgg gggaggcta
4491230DNAArtificial Sequenceprimer 12tataccgtcg acctctagct
agagcttggc 301330DNAArtificial Sequenceprimer 13gctatggcag
ggcctgccgc cccgacgttg 30141436DNAArtificial
SequencechimericCDS(26)..(1411) 14ccagcctccg gactctagag ccacc atg
aaa cac ctg tgg ttc ttc ctg ctg 52 Met Lys His Leu Trp Phe Phe Leu
Leu 1 5ctg gtc gcc gca cct aga tgg gtc ctg agt gaa gtc cag ctg gtc
gaa 100Leu Val Ala Ala Pro Arg Trp Val Leu Ser Glu Val Gln Leu Val
Glu10 15 20 25agc ggg ggg ggc ctg gtg cag cca gga cga tcc ctg aag
ctg tct tgc 148Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Lys
Leu Ser Cys 30 35 40gcc gct agt ggc ttc acc ttt tcc gac tat ggg atg
gca tgg gtg cga 196Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Gly Met
Ala Trp Val Arg 45 50 55cag gcc cct acc aaa gga ctg gag tgg gtg gcc
aca atc tct tac gac 244Gln Ala Pro Thr Lys Gly Leu Glu Trp Val Ala
Thr Ile Ser Tyr Asp 60 65 70ggc agc tcc act tac tat agg gat agt gtg
aag ggg cgg ttc acc att 292Gly Ser Ser Thr Tyr Tyr Arg Asp Ser Val
Lys Gly Arg Phe Thr Ile 75 80 85tca aga gag aat gct aaa tct aca ctg
agt ctg cag atg gac tca ctg 340Ser Arg Glu Asn Ala Lys Ser Thr Leu
Ser Leu Gln Met Asp Ser Leu90 95 100 105cga agc gaa gat act gca acc
tac tat tgc acc cgg cac cct aca tac 388Arg Ser Glu Asp Thr Ala Thr
Tyr Tyr Cys Thr Arg His Pro Thr Tyr 110 115 120tat tac atc atg gac
gct tgg gga cag gga gca agc gtc acc gtg tct 436Tyr Tyr Ile Met Asp
Ala Trp Gly Gln Gly Ala Ser Val Thr Val Ser 125 130 135agt gcc aag
acc aca ccc cct agc gtg tat cca ctg gct cca gga tcc 484Ser Ala Lys
Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser 140 145 150gca
gca cag acc aat tct atg gtg aca ctg gga tgt ctg gtc aag ggc 532Ala
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly 155 160
165tac ttc cct gag cca gtc aca gtg act tgg aac agc ggg tcc ctg tca
580Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu
Ser170 175 180 185agc gga gtg cac act ttt ccc gcc gtc ctg cag agc
gat ctg tac acc 628Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Asp Leu Tyr Thr 190 195 200ctg tcc tct agt gtc act gtg ccc tca agc
acc tgg cct agc gag acc 676Leu Ser Ser Ser Val Thr Val Pro Ser Ser
Thr Trp Pro Ser Glu Thr 205 210 215gtg aca tgc aat gtc gcc cat cca
gct tcc tct aca aag gtg gac aag 724Val Thr Cys Asn Val Ala His Pro
Ala Ser Ser Thr Lys Val Asp Lys 220 225 230aaa atc gtc ccc cgg gat
tgc ggc tgt aaa cca tgc att tgt act gtc 772Lys Ile Val Pro Arg Asp
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val 235 240 245ccc gaa gtg agt
tca gtc ttc atc ttt cca ccc aag ccc aaa gac gtg 820Pro Glu Val Ser
Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val250 255 260 265ctg
act att acc ctg aca cct aag gtc acc tgt gtg gtc gtg gat atc 868Leu
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile 270 275
280agc aaa gac gat ccc gag gtg cag ttc tcc tgg ttt gtc gac gat gtc
916Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val
285 290 295gaa gtg cac aca gca cag act cag cct agg gag gaa cag ttc
aac agc 964Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe
Asn Ser 300 305 310aca ttt cgc tct gtg agt gag ctg cca att atg cat
cag gac tgg ctg 1012Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His
Gln Asp Trp Leu 315 320 325aat ggc aag gaa ttc aaa tgc aga gtg aac
tcc gct gca ttt ccc gct 1060Asn Gly Lys Glu Phe Lys Cys Arg Val Asn
Ser Ala Ala Phe Pro Ala330 335 340 345cct atc gag aag act att tct
aag acc aaa ggg agg cct aaa gca cca 1108Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly Arg Pro Lys Ala Pro 350 355 360cag gtg tat acc atc
cct cca ccc aag gaa cag atg gcc aag gat aaa 1156Gln Val Tyr Thr Ile
Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys 365 370 375gtg agc ctg
aca tgt atg atc act gac ttc ttt cca gag gat att aca 1204Val Ser Leu
Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr 380 385 390gtg
gaa tgg cag tgg aat ggg cag cct gcc gag aac tac aag aat aca 1252Val
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr 395 400
405cag cca att atg gac act gat gga tca tat ttc gtg tac agc aag ctg
1300Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys
Leu410 415 420 425aac gtc cag aaa tct aat tgg gaa gct gga aac act
ttt acc tgt agt 1348Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr
Phe Thr Cys Ser 430 435 440gtg ctg cac gag ggc ctg cat aac cac cat
acc gaa aag tca ctg agc 1396Val Leu His Glu Gly Leu His Asn His His
Thr Glu Lys Ser Leu Ser 445 450 455cat tcc ccc ggc aaa tgagtttaaa
cgggggaggc taact 1436His Ser Pro Gly Lys 46015462PRTArtificial
SequenceSynthetic Construct 15Met Lys His Leu Trp Phe Phe Leu Leu
Leu Val Ala Ala Pro Arg Trp1 5 10 15Val Leu Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 20 25 30Pro Gly Arg Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45Ser Asp Tyr Gly Met Ala
Trp Val Arg Gln Ala Pro Thr Lys Gly Leu 50 55 60Glu Trp Val Ala Thr
Ile Ser Tyr Asp Gly Ser Ser Thr Tyr Tyr Arg65 70 75 80Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Ser 85 90 95Thr Leu
Ser Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr 100 105
110Tyr Tyr Cys Thr Arg His Pro Thr Tyr Tyr Tyr Ile Met Asp Ala Trp
115 120 125Gly Gln Gly Ala Ser Val Thr Val Ser Ser Ala Lys Thr Thr
Pro Pro 130 135 140Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln
Thr Asn Ser Met145 150 155 160Val Thr Leu Gly Cys Leu Val Lys Gly
Tyr Phe Pro Glu Pro Val Thr 165 170 175Val Thr Trp Asn Ser Gly Ser
Leu Ser Ser Gly Val His Thr Phe Pro 180 185 190Ala Val Leu Gln Ser
Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 195 200 205Pro Ser Ser
Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 210 215 220Pro
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys225 230
235 240Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
Phe 245 250 255Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr
Leu Thr Pro 260 265 270Lys Val Thr Cys Val Val Val Asp Ile Ser Lys
Asp Asp Pro Glu Val 275 280 285Gln Phe Ser Trp Phe Val Asp Asp Val
Glu Val His Thr Ala Gln Thr 290 295 300Gln Pro Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Ser Val Ser Glu305 310 315 320Leu Pro Ile Met
His Gln Asp
Trp Leu Asn Gly Lys Glu Phe Lys Cys 325 330 335Arg Val Asn Ser Ala
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350Lys Thr Lys
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 355 360 365Pro
Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 370 375
380Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
Gly385 390 395 400Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile
Met Asp Thr Asp 405 410 415Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
Val Gln Lys Ser Asn Trp 420 425 430Glu Ala Gly Asn Thr Phe Thr Cys
Ser Val Leu His Glu Gly Leu His 435 440 445Asn His His Thr Glu Lys
Ser Leu Ser His Ser Pro Gly Lys 450 455 4601625DNAArtificial
Sequenceprimer 16ccagcctccg gactctagag ccacc 251724DNAArtificial
Sequenceprimer 17agttagcctc ccccgtttaa actc 2418749DNAArtificial
SequencechimericCDS(26)..(724) 18ccagcctccg gactctagag ccacc atg
gtg ctg cag acc cag gtg ttc att 52 Met Val Leu Gln Thr Gln Val Phe
Ile 1 5tca ctg ctg ctg tgg att tca ggc gct tac ggc gat act gtg ctg
acc 100Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Asp Thr Val Leu
Thr10 15 20 25cag agc ccc gct ctg gct gtg agc ctg ggc cag cgg gtc
aca atc tcc 148Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln Arg Val
Thr Ile Ser 30 35 40tgc aga gca tca aag agc gtg tcc act ttc atg aac
tgg tac cag cag 196Cys Arg Ala Ser Lys Ser Val Ser Thr Phe Met Asn
Trp Tyr Gln Gln 45 50 55aag tcc ggc cag cag cca aaa ctg ctg atc tac
agg gcc agc aat ctg 244Lys Ser Gly Gln Gln Pro Lys Leu Leu Ile Tyr
Arg Ala Ser Asn Leu 60 65 70gag tcc ggg gtg ccc tct cgc ttc tct gga
agt ggc tca ggg acc gac 292Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp 75 80 85ttt acc ctg aca atc gat cct gtc gaa
gca gac gat att gcc aac tac 340Phe Thr Leu Thr Ile Asp Pro Val Glu
Ala Asp Asp Ile Ala Asn Tyr90 95 100 105tat tgc cag cag tct aat gag
ctg cca ctg acc ttc gga agt ggc aca 388Tyr Cys Gln Gln Ser Asn Glu
Leu Pro Leu Thr Phe Gly Ser Gly Thr 110 115 120aag ctg gaa atc aaa
cgg gcc gac gcc gct ccc aca gtg agc att ttt 436Lys Leu Glu Ile Lys
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe 125 130 135ccc cct agc
tcc gag cag ctg acc agt ggc ggg gct tca gtg gtc tgt 484Pro Pro Ser
Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys 140 145 150ttc
ctg aac aat ttt tac cct aaa gac atc aac gtg aag tgg aaa att 532Phe
Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile 155 160
165gat ggg agc gaa cgg cag aac gga gtc ctg aat tcc tgg act gac cag
580Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln170 175 180 185gat tct aag gac agt acc tat tca atg tct agt act
ctg acc ctg aca 628Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr
Leu Thr Leu Thr 190 195 200aaa gat gag tac gaa cga cac aat tct tat
aca tgc gag gcc act cat 676Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
Thr Cys Glu Ala Thr His 205 210 215aag act agc acc tcc ccc atc gtg
aaa agc ttt aac aga aat gaa tgt 724Lys Thr Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 220 225 230tgagtttaaa cgggggaggc
taact 74919233PRTArtificial SequenceSynthetic Construct 19Met Val
Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser1 5 10 15Gly
Ala Tyr Gly Asp Thr Val Leu Thr Gln Ser Pro Ala Leu Ala Val 20 25
30Ser Leu Gly Gln Arg Val Thr Ile Ser Cys Arg Ala Ser Lys Ser Val
35 40 45Ser Thr Phe Met Asn Trp Tyr Gln Gln Lys Ser Gly Gln Gln Pro
Lys 50 55 60Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Val Pro
Ser Arg65 70 75 80Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Asp Pro 85 90 95Val Glu Ala Asp Asp Ile Ala Asn Tyr Tyr Cys
Gln Gln Ser Asn Glu 100 105 110Leu Pro Leu Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg Ala 115 120 125Asp Ala Ala Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Glu Gln Leu 130 135 140Thr Ser Gly Gly Ala
Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro145 150 155 160Lys Asp
Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn 165 170
175Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
180 185 190Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
Arg His 195 200 205Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser
Thr Ser Pro Ile 210 215 220Val Lys Ser Phe Asn Arg Asn Glu Cys225
230201448DNAArtificial SequencechimericCDS(26)..(1423) 20ccagcctccg
gactctagag ccacc atg aaa cat ctg tgg ttc ttc ctg ctg 52 Met Lys His
Leu Trp Phe Phe Leu Leu 1 5ctg gtc gcc gct cct cgg tgg gtc ctg agt
gaa gtg cag ctg gtc gaa 100Leu Val Ala Ala Pro Arg Trp Val Leu Ser
Glu Val Gln Leu Val Glu10 15 20 25tct ggg ggg ggc ctg gtg cag ccc
ggc aga tcc atg aag ctg tct tgc 148Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg Ser Met Lys Leu Ser Cys 30 35 40gcc gct agt gga ctg acc ttc
agc aat tat ggc atg gca tgg gtg agg 196Ala Ala Ser Gly Leu Thr Phe
Ser Asn Tyr Gly Met Ala Trp Val Arg 45 50 55cag gcc cct aag aaa gga
ctg gag tgg gtg gct ttc atc agc cac gac 244Gln Ala Pro Lys Lys Gly
Leu Glu Trp Val Ala Phe Ile Ser His Asp 60 65 70ggc ggg agc tcc tac
tat cgc gat agt gtg gaa ggc cgg ttt atc att 292Gly Gly Ser Ser Tyr
Tyr Arg Asp Ser Val Glu Gly Arg Phe Ile Ile 75 80 85tca aga gac aat
gca aag tct aca ctg agt ctg cag atg gac tca ctg 340Ser Arg Asp Asn
Ala Lys Ser Thr Leu Ser Leu Gln Met Asp Ser Leu90 95 100 105cga agc
gag gat aca gct act tac tat tgc acc aca gca ggc gac tac 388Arg Ser
Glu Asp Thr Ala Thr Tyr Tyr Cys Thr Thr Ala Gly Asp Tyr 110 115
120tat tcc gac aac gat tgg tac ttc gat ttt tgg gga cca ggc atc atg
436Tyr Ser Asp Asn Asp Trp Tyr Phe Asp Phe Trp Gly Pro Gly Ile Met
125 130 135gtc acc gtg tct agt gcc aag act acc ccc cct tct gtg tat
cca ctg 484Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
Pro Leu 140 145 150gct cca gga tcc gca gca cag acc aat tct atg gtg
aca ctg ggg tgt 532Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val
Thr Leu Gly Cys 155 160 165ctg gtc aaa gga tac ttc cct gag cca gtc
acc gtg aca tgg aac agc 580Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
Thr Val Thr Trp Asn Ser170 175 180 185ggc tcc ctg tca agc gga gtg
cac acc ttt cca gca gtc ctg cag tcc 628Gly Ser Leu Ser Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser 190 195 200gat ctg tac aca ctg
tcc tct agt gtc act gtg ccc tca agc acc tgg 676Asp Leu Tyr Thr Leu
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp 205 210 215cct tct gag
act gtg acc tgc aat gtc gcc cat cca gct tcc tct act 724Pro Ser Glu
Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr 220 225 230aag
gtg gac aag aaa atc gtc ccc agg gat tgc ggc tgt aaa cca tgc 772Lys
Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys 235 240
245att tgt acc gtc ccc gaa gtg agt tca gtc ttc atc ttt cca ccc aag
820Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro
Lys250 255 260 265ccc aaa gac gtg ctg aca att act ctg acc cct aag
gtc aca tgt gtg 868Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys
Val Thr Cys Val 270 275 280gtc gtg gac atc agc aaa gac gat ccc gag
gtg cag ttc tcc tgg ttt 916Val Val Asp Ile Ser Lys Asp Asp Pro Glu
Val Gln Phe Ser Trp Phe 285 290 295gtc gac gat gtc gaa gtg cac acc
gcc cag aca cag cct agg gag gaa 964Val Asp Asp Val Glu Val His Thr
Ala Gln Thr Gln Pro Arg Glu Glu 300 305 310cag ttc aac agc acc ttt
cgc tct gtg agt gag ctg cca att atg cat 1012Gln Phe Asn Ser Thr Phe
Arg Ser Val Ser Glu Leu Pro Ile Met His 315 320 325cag gac tgg ctg
aat ggg aag gaa ttc aaa tgc cga gtg aac agc gct 1060Gln Asp Trp Leu
Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala330 335 340 345gca
ttt ccc gcc cct atc gag aag act att agc aag acc aaa gga cgg 1108Ala
Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg 350 355
360cct aaa gca cca cag gtg tat aca atc cct cca ccc aag gaa cag atg
1156Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met
365 370 375gcc aag gat aaa gtg agc ctg aca tgt atg atc act gac ttc
ttt cct 1204Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe
Phe Pro 380 385 390gag gat att act gtg gaa tgg cag tgg aat ggc cag
cct gcc gag aac 1252Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln
Pro Ala Glu Asn 395 400 405tac aag aat aca cag cca att atg gac act
gat ggg tca tac ttc gtg 1300Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
Asp Gly Ser Tyr Phe Val410 415 420 425tat agc aag ctg aac gtc cag
aaa tct aat tgg gaa gct ggg aac acc 1348Tyr Ser Lys Leu Asn Val Gln
Lys Ser Asn Trp Glu Ala Gly Asn Thr 430 435 440ttc acc tgt agt gtg
ctg cac gag gga ctg cat aac cac cat acc gaa 1396Phe Thr Cys Ser Val
Leu His Glu Gly Leu His Asn His His Thr Glu 445 450 455aag tca ctg
agc cat tcc ccc ggc aaa tgagtttaaa cgggggaggc taact 1448Lys Ser Leu
Ser His Ser Pro Gly Lys 460 46521466PRTArtificial SequenceSynthetic
Construct 21Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro
Arg Trp1 5 10 15Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln 20 25 30Pro Gly Arg Ser Met Lys Leu Ser Cys Ala Ala Ser
Gly Leu Thr Phe 35 40 45Ser Asn Tyr Gly Met Ala Trp Val Arg Gln Ala
Pro Lys Lys Gly Leu 50 55 60Glu Trp Val Ala Phe Ile Ser His Asp Gly
Gly Ser Ser Tyr Tyr Arg65 70 75 80Asp Ser Val Glu Gly Arg Phe Ile
Ile Ser Arg Asp Asn Ala Lys Ser 85 90 95Thr Leu Ser Leu Gln Met Asp
Ser Leu Arg Ser Glu Asp Thr Ala Thr 100 105 110Tyr Tyr Cys Thr Thr
Ala Gly Asp Tyr Tyr Ser Asp Asn Asp Trp Tyr 115 120 125Phe Asp Phe
Trp Gly Pro Gly Ile Met Val Thr Val Ser Ser Ala Lys 130 135 140Thr
Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln145 150
155 160Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe
Pro 165 170 175Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
Ser Gly Val 180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu
Tyr Thr Leu Ser Ser 195 200 205Ser Val Thr Val Pro Ser Ser Thr Trp
Pro Ser Glu Thr Val Thr Cys 210 215 220Asn Val Ala His Pro Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Val225 230 235 240Pro Arg Asp Cys
Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 245 250 255Ser Ser
Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 260 265
270Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp
275 280 285Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
Val His 290 295 300Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg305 310 315 320Ser Val Ser Glu Leu Pro Ile Met His
Gln Asp Trp Leu Asn Gly Lys 325 330 335Glu Phe Lys Cys Arg Val Asn
Ser Ala Ala Phe Pro Ala Pro Ile Glu 340 345 350Lys Thr Ile Ser Lys
Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 355 360 365Thr Ile Pro
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 370 375 380Thr
Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp385 390
395 400Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
Ile 405 410 415Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu
Asn Val Gln 420 425 430Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr
Cys Ser Val Leu His 435 440 445Glu Gly Leu His Asn His His Thr Glu
Lys Ser Leu Ser His Ser Pro 450 455 460Gly Lys46522749DNAArtificial
SequencechimericCDS(26)..(724) 22ccagcctccg gactctagag ccacc atg
gtg ctg cag act cag gtg ttc att 52 Met Val Leu Gln Thr Gln Val Phe
Ile 1 5tca ctg ctg ctg tgg att agc ggc gca tac ggc gac att cag atg
acc 100Ser Leu Leu Leu Trp Ile Ser Gly Ala Tyr Gly Asp Ile Gln Met
Thr10 15 20 25cag agc ccc tca ctg ctg tcc gca tct gtg ggc gac agg
gtc act ctg 148Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Leu 30 35 40agc tgc aag gct agt cag tca atc tac aac tcc ctg
gca tgg tat cag 196Ser Cys Lys Ala Ser Gln Ser Ile Tyr Asn Ser Leu
Ala Trp Tyr Gln 45 50 55cag aag ctg ggg gag gca cca aaa ctg ctg atc
tac gac gcc gat agc 244Gln Lys Leu Gly Glu Ala Pro Lys Leu Leu Ile
Tyr Asp Ala Asp Ser 60 65 70ctg cag acc gga att cca tcc cgc ttc agc
gga tcc gga tct gga aca 292Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser
Gly Ser Gly Ser Gly Thr 75 80 85gac tac acc ctg aca atc agc tcc ctg
cag ccc gaa gat gtg gct acc 340Asp Tyr Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Val Ala Thr90 95 100 105tat ttc tgc cag aag tac tat
tcc ggg tgg acc ttt ggc ggg gga aca 388Tyr Phe Cys Gln Lys Tyr Tyr
Ser Gly Trp Thr Phe Gly Gly Gly Thr 110 115 120aag ctg gag ctg aaa
cga gcc gat gcc gct cct aca gtc agc att ttt 436Lys Leu Glu Leu Lys
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe 125 130 135ccc cct tct
agt gaa cag ctg act agt ggc ggg gct tca gtg gtc tgt 484Pro Pro Ser
Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys 140 145 150ttc
ctg aac aat ttt tac cca aaa gac atc aac gtg aag tgg aaa att 532Phe
Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile 155 160
165gat gga tct gag aga cag aac ggc gtc ctg aat agt tgg act gac cag
580Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp
Gln170 175 180 185gat agc aag gac tcc acc tat tct atg tca agc act
ctg acc ctg aca 628Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr
Leu Thr Leu Thr 190 195 200aaa gat gag tac gaa cgg cac aat tct tat
aca tgc gag gcc act cat 676Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
Thr Cys Glu Ala Thr His 205 210 215aag act agt acc tca cct att gtg
aaa agc ttc aac aga aat gaa tgt 724Lys Thr Ser Thr Ser Pro Ile Val
Lys Ser Phe Asn Arg Asn Glu Cys 220 225 230tgagtttaaa cgggggaggc
taact 74923233PRTArtificial SequenceSynthetic Construct 23Met Val
Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser1 5
10 15Gly Ala Tyr Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu
Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Leu Ser Cys Lys Ala Ser
Gln Ser 35 40 45Ile Tyr Asn Ser Leu Ala Trp Tyr Gln Gln Lys Leu Gly
Glu Ala Pro 50 55 60Lys Leu Leu Ile Tyr Asp Ala Asp Ser Leu Gln Thr
Gly Ile Pro Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp Val Ala Thr
Tyr Phe Cys Gln Lys Tyr Tyr 100 105 110Ser Gly Trp Thr Phe Gly Gly
Gly Thr Lys Leu Glu Leu Lys Arg Ala 115 120 125Asp Ala Ala Pro Thr
Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu 130 135 140Thr Ser Gly
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro145 150 155
160Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
165 170 175Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
Thr Tyr 180 185 190Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu
Tyr Glu Arg His 195 200 205Asn Ser Tyr Thr Cys Glu Ala Thr His Lys
Thr Ser Thr Ser Pro Ile 210 215 220Val Lys Ser Phe Asn Arg Asn Glu
Cys225 2302410PRTRattus norvegicus 24Gly Phe Thr Phe Ser Asp Tyr
Gly Met Ala1 5 102517PRTRattus norvegicus 25Thr Ile Ser Tyr Asp Gly
Ser Ser Thr Tyr Tyr Arg Asp Ser Val Lys1 5 10 15Gly2610PRTRattus
norvegicus 26His Pro Thr Tyr Tyr Tyr Ile Met Asp Ala1 5
102711PRTRattus norvegicus 27Arg Ala Ser Lys Ser Val Ser Thr Phe
Met Asn1 5 10287PRTRattus norvegicus 28Arg Ala Ser Asn Leu Glu Ser1
5299PRTRattus norvegicus 29Gln Gln Ser Asn Glu Leu Pro Leu Thr1
53010PRTRattus norvegicus 30Gly Leu Thr Phe Ser Asn Tyr Gly Met
Ala1 5 103117PRTRattus norvegicus 31Phe Ile Ser His Asp Gly Gly Ser
Ser Tyr Tyr Arg Asp Ser Val Glu1 5 10 15Gly3214PRTRattus norvegicus
32Ala Gly Asp Tyr Tyr Ser Asp Asn Asp Trp Tyr Phe Asp Phe1 5
103311PRTRattus norvegicus 33Lys Ala Ser Gln Ser Ile Tyr Asn Ser
Leu Ala1 5 10347PRTRattus norvegicus 34Asp Ala Asp Ser Leu Gln Thr1
5358PRTRattus norvegicus 35Gln Lys Tyr Tyr Ser Gly Trp Thr1 5
* * * * *
References