U.S. patent application number 10/390986 was filed with the patent office on 2003-09-04 for monoclonal antibody against connective tissue growth factor and medicinal uses thereof.
Invention is credited to Sakamoto, Shinji, Takigawa, Masaharu, Tamatani, Takuya, Tezuka, Katsunari.
Application Number | 20030166011 10/390986 |
Document ID | / |
Family ID | 26580389 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166011 |
Kind Code |
A1 |
Tamatani, Takuya ; et
al. |
September 4, 2003 |
Monoclonal antibody against connective tissue growth factor and
medicinal uses thereof
Abstract
A human monoclonal antibody useful for the treatment of various
diseases caused by human connective tissue growth factor (CTGF) and
preventing the onset of the above diseases; medicinal uses thereof;
and various monoclonal antibodies having various characteristics
against various mammalian CTGFs useful for detecting and assaying
CTGFs present in body fluids of mammals suffering from various
diseases.
Inventors: |
Tamatani, Takuya;
(Kanazawa-ku, JP) ; Tezuka, Katsunari;
(Kanazawa-ku, JP) ; Sakamoto, Shinji;
(Kanazawa-ku, JP) ; Takigawa, Masaharu;
(Okayama-shi, JP) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
26580389 |
Appl. No.: |
10/390986 |
Filed: |
March 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10390986 |
Mar 17, 2003 |
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09582337 |
Sep 18, 2000 |
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6562618 |
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09582337 |
Sep 18, 2000 |
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PCT/JP98/05697 |
Dec 16, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/335; 435/7.5; 435/7.92; 530/388.25 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 2317/76 20130101; C07K 2317/56 20130101; A61K 2039/505
20130101; Y10S 435/81 20130101; A01K 67/0275 20130101; C07K 14/475
20130101; A01K 2267/03 20130101; A01K 2267/01 20130101; C07K 16/22
20130101; C07K 2317/73 20130101; G01N 2800/347 20130101; A01K
2217/05 20130101 |
Class at
Publication: |
435/7.1 ;
530/388.25; 435/7.5; 435/335; 435/7.92 |
International
Class: |
G01N 033/53; C12N
005/06; G01N 033/537; G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 1997 |
JP |
JP9/367699 |
Dec 15, 1998 |
JP |
JP10/356183 |
Claims
1. A monoclonal antibody or a portion thereof, comprising a
property in any of (a) to (g) below: (a) reactive to human, mouse
and rat connective tissue growth factors (CTGFs); (b) reactive to
both human and mouse CTGFs but not reactive to rat CTGF; (c)
reactive to both mouse and rat CTGFs but not reactive to human
CTGF; (d) inhibiting binding of human CTGF to human kidney-derived
fibroblast cell line 293-T (ATCC CRL1573), or the binding of mouse
CTGF to said cell line 293-T; (e) inhibiting binding of human CTGF
to any cells of rat kidney-derived fibroblast cell line NRK-49F
(ATCC CRL-1570), human osteosarcoma-derived cell line MG-63 (ATCC
CRL-1427), or human lung-derived fibroblasts; (f) inhibiting cell
proliferation of rat kidney-derived fibroblast cell line NRK-49F
(ATCC CRL-1570)induced by a stimulus with human or mouse CTGF; or,
(g) inhibiting an increase of hydroxyproline in the kidney, wherein
said hydroxyproline level tends to be elevated.
2. The monoclonal antibody or a portion thereof according to claim
1, comprising a property in any of (a) to (c) below: (a) obtainable
by immunizing a mouse with human CTGF or a portion thereof, and
reactive to human, mouse and rat CTGFs; (b) obtainable by
immunizing a hamster with mouse CTGF or a portion thereof, and
reactive to human, mouse and rat CTGFs; or, (c) obtainable by
immunizing a rat with mouse CTGF or a portion thereof, and reactive
to human, mouse and rat CTGFs.
3. The monoclonal antibody or a portion thereof according to claim
1, comprising a property in any of (a) to (c) below: (a) obtainable
by immunizing a mouse with human CTGF or a portion thereof,
reactive to human, mouse and rat CTGFs and inhibiting binding of
human CTGF to human kidney-derived fibroblast cell line 293-T (ATCC
CRL1573); (b) obtainable by immunizing a rat with mouse CTGF or a
portion thereof, reactive to human, mouse and rat CTGFs and
inhibiting binding of mouse CTGF to human kidney-derived fibroblast
cell line 293-T (ATCC CRL1573); or, (c) obtainable by immunizing a
hamster with mouse CTGF or a portion thereof, and reactive to
human, mouse and rat CTGFs and inhibiting binding of mouse CTGF to
human kidney-derived fibroblast cell line 293-T (ATCC CRL1573).
4. The monoclonal antibody or a portion thereof according to claim
1, wherein said monoclonal antibody is produced by a hybridoma
identified by an international deposit accession No. FERM
BP-6208.
5. The monoclonal antibody or a portion thereof according to claim
1, wherein said monoclonal antibody comprises a property
substantially equivalent to that of a monoclonal antibody produced
by a hybridoma identified by an international deposit accession No.
FERM BP-6208.
6. The monoclonal antibody or a portion thereof according to claim
1, wherein said monoclonal antibody is produced by a hybridoma
identified by an international deposit accession No. FERM
BP-6209.
7. The monoclonal antibody or a portion thereof according to claim
1, wherein said monoclonal antibody comprises a property
substantially equivalent to that of a monoclonal antibody produced
by a hybridoma identified by an international deposit accession No.
FERM BP-6209.
8. A human monoclonal antibody or a portion thereof, reactive to
any human, mouse or rat CTGF.
9. The human monoclonal antibody or a portion thereof according to
claim 8, wherein said human monoclonal antibody is reactive to
human CTGF.
10. A human monoclonal antibody or a portion thereof, reactive to
human CTGF and comprises a property in any of (a) to (d) below: (a)
inhibiting binding of human CTGF to human kidney-derived fibroblast
cell line 293-T (ATCC CRL1573); (b) inhibiting binding of human
CTGF to any of rat kidney-derived fibroblast cell line NRK-49F
(ATCC CRL-1570), human osteosarcoma-derived cell line MG-63 (ATCC
CRL-1427), or human lung-derived fibroblasts; (c) inhibiting the
cell proliferation of rat kidney-derived fibroblast cell line
NRK-49F (ATCC CRL-1570) induced by a stimulus with human or mouse
CTGF; or, (d) inhibiting an increase of hydroxyproline in kidney,
wherein said hydroxyproline level tends to be elevated.
11. The human monoclonal antibody or a portion thereof according to
any one of claims 8 to 10, wherein said human monoclonal antibody
is derived from a non-human transgenic mammal which is capable of
producing a human antibody.
12. The human monoclonal antibody or a portion thereof according to
claim 11, wherein said human monoclonal antibody is obtainable by
immunizing a non-human transgenic mammal which is capable of
producing a human antibody, with human CTGF.
13. The human monoclonal antibody or a portion thereof according to
any one of claims 8 to 12, wherein said non-human transgenic mammal
is a transgenic mouse.
14. The human monoclonal antibody or a portion thereof according to
any one of claims 8 to 13, wherein a V-region DNA encoding a heavy
chain variable region of said human monoclonal antibody is derived
from a gene segment selected from the group consisting of DP-5,
DP-38, DP-65 and DP-75.
15. The human monoclonal antibody or a portion thereof according to
any one of claims 8 to 13, wherein a V-region DNA encoding a light
chain variable region of said human monoclonal antibody is derived
from a gene segment selected from the group consisting of DPK1,
DPK9, DPK12 and DPK24.
16. The human monoclonal antibody or a portion thereof according to
any one of claims 8 to 15, wherein a V-region DNA encoding a heavy
chain variable region of said human monoclonal antibody is derived
from a gene segment selected from the group consisting of DP-5,
DP-38, DP-65 and DP-75, and wherein a V-region DNA encoding a light
chain variable region of said human monoclonal antibody is derived
from a gene segment selected from the group consisting of DPK1,
DPK9, DPK12 and DPK24.
17. The human monoclonal antibody or a portion thereof according to
claim 9, wherein an amino acid sequence of a heavy chain variable
region of said human monoclonal antibody comprises an amino acid
sequence defined below in any of (a) to (j) below: (a) the amino
acid positions 21 to 120 of the amino acid sequence of SEQ ID NO:
6; (b) the amino acid positions 21 to 120 of the amino acid
sequence of SEQ ID NO: 6, wherein one or more amino acids are
deleted, substituted, inserted or added; (c) the amino acid
positions 21 to 118 of the amino acid sequence of SEQ ID NO: 8; (d)
the amino acid positions 21 to 118 of the amino acid sequence of
SEQ ID NO: 8, wherein one or more amino acids are deleted,
substituted, inserted or added; (e) the amino acid positions 21 to
116 of the amino acid sequence of SEQ ID NO: 10; (f) the amino acid
positions 21 to 116 of the amino acid sequence of SEQ ID NO: 10,
wherein one or more amino acids are deleted, substituted, inserted
or added; (g) the amino acid positions 21 to 116 of the amino acid
sequence of SEQ ID NO: 12; (h) the amino acid positions 21 to 116
of the amino acid sequence of SEQ ID NO: 12, wherein one or more
amino acids are deleted, substituted, inserted or added; (i) the
amino acid positions 21 to 117 of the amino acid sequence of SEQ ID
NO: 14; or, (j) the amino acid positions 21 to 117 of the amino
acid sequence of SEQ ID NO: 14, wherein one or more amino acids are
deleted, substituted, inserted or added.
18. The human monoclonal antibody or a portion thereof according to
claim 9, wherein an amino acid sequence of a light chain variable
region of said human monoclonal antibody comprises an amino acid
sequence in any of (a) to (j) below: (a) the amino acid positions
21 to 120 of the amino acid sequence of SEQ ID NO: 16; (b) the
amino acid positions 21 to 120 of the amino acid sequence of SEQ ID
NO: 16, wherein one or more amino acids are deleted, substituted,
inserted or added; (c) the amino acid positions 21 to 121 of the
amino acid sequence of SEQ ID NO: 18; (d) the amino acid positions
21 to 121 of the amino acid sequence of SEQ ID NO: 18, wherein one
or more amino acids are deleted, substituted, inserted or added;
(e) the amino acid positions 23 to 117 of the amino acid sequence
of SEQ ID NO: 20; (f) the amino acid positions 23 to 117 of the
amino acid sequence of SEQ ID NO: 20, wherein one or more amino
acids are deleted, substituted, inserted or added; (g) the amino
acid positions 17 to 111 of the amino acid sequence of SEQ ID NO:
22; (h) the amino acid positions 17 to 111 of the amino acid
sequence of SEQ ID NO: 22, wherein one or more amino acids are
deleted, substituted, inserted or added; (i) the amino acid
positions 23 to 118 of the amino acid sequence of SEQ ID NO: 24;
or, (j) the amino acid positions 23 to 118 of the amino acid
sequence of SEQ ID NO: 24, wherein one or more amino acids are
deleted, substituted, inserted or added.
19. A monoclonal antibody or a portion thereof, reactive to human
CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6535.
20. A monoclonal antibody or a portion thereof, reactive to human
CTGF and comprises a property substantially equivalent to that of a
monoclonal antibody produced by a hybridoma identified by an
international deposit accession No. FERM BP-6535.
21. A monoclonal antibody or a portion thereof, reactive to human
CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6598.
22. A monoclonal antibody or a portion thereof, reactive to human
CTGF and comprises a property substantially equivalent to that of a
monoclonal antibody produced by a hybridoma identified by an
international deposit accession No. FERM BP-6598.
23. A monoclonal antibody or a portion thereof, reactive to human
CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6599.
24. A monoclonal antibody or a portion thereof, reactive to human
CTGF and comprises a property substantially equivalent to that of a
monoclonal antibody produced by a hybridoma identified by an
international deposit accession No. FERM BP-6599.
25. A monoclonal antibody or a portion thereof, reactive to human
CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6600.
26. A monoclonal antibody or a portion thereof, reactive to human
CTGF and comprises a property substantially equivalent to that of a
monoclonal antibody produced by a hybridoma identified by an
international deposit accession No. FERM BP-6600.
27. A monoclonal antibody or a portion thereof, reactive to human
CTGF, and which is non-reactive to an antigen-antibody complex of
human CTGF and the monoclonal antibody reactive to human CTGF of
claim 17 or 18.
28. The monoclonal antibody or a portion thereof according to claim
27, wherein said monoclonal antibody is a human monoclonal
antibody.
29. A monoclonal antibody or a portion thereof, reactive to rat
CTGF.
30. A recombinant chimeric monoclonal antibody, reactive to human
CTGF, and of which a variable region is derived from a variable
region of the monoclonal antibody according to any one of claims 2
to 7, 27 or 29 and of which a constant region is derived from a
constant region of a human immunoglobulin.
31. A recombinant humanized monoclonal antibody, reactive to human
CTGF, of which a whole or portion of the
complementarity-determining regions of a hyper-variable region is
derived from complementarity-determining regions of the monoclonal
antibody of any one of claims 2 to 7, 27 or 29, of which framework
regions of a hyper-variable region are derived from the framework
regions of a human immunoglobulin and of which a constant region is
derived from a constant region of a human immunoglobulin.
32. A cell producing the monoclonal antibody according to any one
of claims 1 to 29.
33. A cell producing the recombinant monoclonal antibody according
to claim 30 or 31.
34. The cell according to claim 32, wherein said cell is a
hybridoma obtainable by fusing a mammalian myeloma cell with a
mammalian B cell which is capable of producing the monoclonal
antibody.
35. The cell according to claim 32 or 33, wherein said cell is a
genetically engineered cell transformed by either one or both of
the DNAs encoding a heavy chain and light chain of the monoclonal
antibody.
36. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6535.
37. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6598.
38. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6599.
39. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6600.
40. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6208.
41. The hybridoma according to claim 34, wherein said hybridoma is
identified by an international deposit accession No. FERM
BP-6209.
42. An antibody-immobilized insoluble carrier on which the
monoclonal antibody according to any one of claims 1 to 31 is
immobilized.
43. The antibody-immobilized insoluble carrier according to 42,
wherein said insoluble carrier is selected from the group
consisting of plates, test tubes, tubes, beads, balls, filters and
membranes.
44. The antibody-immobilized insoluble carrier according to 42,
wherein said insoluble carrier is a filter or membrane, or that
used for affinity column chromatography.
45. A labeled antibody which is prepared by labeling the monoclonal
antibody of any one of claims 1 to 31 with a labeling agent capable
of providing a detectable signal by itself or together with other
substances.
46. The labeled antibody according to claim 45, wherein said
labeling agent is an enzyme, fluorescent substance,
chemiluminescent substance, biotin, avidin, or radioisotope.
47. A kit for detecting or assaying mammalian CTGF, comprising at
least one monoclonal antibody, an antibody-immobilized insoluble
carrier, and a labeled antibody, which is selected from the group
consisting of the monoclonal antibody according to any one of
claims 1 to 31, the antibody-immobilized insoluble carrier
according to claim 42 or 43, and the labeled antibody according to
claim 45 or 46.
48. The kit for detecting or assaying mammalian CTGF according to
claim 47, comprising the antibody-immobilized insoluble carrier
according to claim 42 or 43 and the labeled antibody according to
claim 45 or 46.
49. A method for detecting or assaying mammalian CTGF by an
immunoassay using at least one monoclonal antibody, an
antibody-immobilized insoluble carrier, and a labeled antibody,
which is selected from the group consisting of the monoclonal
antibody according to any one of claims 1 to 31, the
antibody-immobilized insoluble carrier according to claim 42 or 43,
and the labeled antibody according to claim 45 or 46.
50. The method for detecting or assaying mammalian CTGF by an
immunoassay according to claim 49, comprising at least the
following steps of (a) and (b): (a) reacting a sample with the
antibody-immobilized insoluble carrier according to claim 42 or 43;
and, (b) reacting the labeled antibody according to claim 45 or 46
with an antigen-antibody complex formed by binding mammalian CTGF
in said sample to the antibody-immobilized insoluble carrier.
51. The method for detecting or assaying mammalian CTGF by an
immunoassay according to claim 49, comprising at least the
following steps of (a) and (b): (a) reacting a sample with the
labeled antibody according to claim 45 or 46; and, (b) reacting the
antibody-immobilized insoluble carrier according to claim 42 or 43
with the antigen-antibody complex formed by binding said labeled
antibody and mammalian CTGF in said sample.
52. The method for detecting or assaying mammalian CTGF by an
immunoassay according to claim 49, comprising at least the
following step of (a): (a) reacting a mixture comprising the
antibody-immobilized insoluble carrier according to claim 42 or 43,
the labeled antibody according to claim 45 or 46, and a sample.
53. The method for detecting or assaying mammalian CTGF by an
immunoassay according to claim 49, comprising at least the
following step of (a): (a) reacting a sample and a mammalian CTGF
standard labeled with a labeling agent capable of providing a
detectable signal by itself or together with other substances, with
the antibody-immobilized insoluble carrier according to claim 42 or
43.
54. The method for detecting or assaying mammalian CTGFs by an
immunoassay according to claim 49, comprising at least the
following steps of (a) and (b): (a) reacting the monoclonal
antibody according to any one of claims 1 to 31 with a mixture
comprising a sample and a mammalian CTGF standard labeled with a
labeling agent capable of proving a detectable signal by itself or
together with other substances; and, (b) reacting a mammalian
antiserum reactive to said monoclonal antibody with the
antigen-antibody complex formed by binding mammalian CTGF in said
sample or said labeled mammalian CTGF standard and said monoclonal
antibody.
55. The method for detecting or assaying mammalian CTGFs by an
immunoassay according to claim 49, comprising at least the
following steps of any of (a) to (c): (a) reacting the monoclonal
antibody according to any of one claims 1 to 31 with a sample; (b)
reacting a mammalian CTGF standard labeled with a labeling agent
capable of providing a detectable signal by itself or together with
other substances with a reaction product resulted from the reaction
in step (a); and, (c) reacting a mammalian antiserum reactive to
said monoclonal antibody with the antigen-antibody complex formed
by binding mammalian CTGF in said sample or said labeled mammalian
CTGF standard, and said monoclonal antibody.
56. A kit for separating or purifying mammalian CTGF, comprising
the antibody-immobilized insoluble carrier according to claim 42 or
44.
57. A method for separating or purifying mammalian CTGF, comprising
using affinity chromatography with the antibody-immobilized
insoluble carrier according to claim 42 or 44.
58. The purification method for mammalian CTGF according to claim
57, wherein said affinity chromatography is affinity column
chromatography.
59. A transgenic mouse in which DNA encoding human CTGF is
integrated into an endogenous gene locus.
60. A rat CTGF comprising an amino acid sequence of, or
substantially equivalent to an amino acid sequence of SEQ ID NO:
2.
61. A DNA encoding a rat CTGF comprising the amino acid sequence of
SEQ ID NO: 2.
62. The DNA according to claim 61, comprising nucleotide sequence
in the positions of 213 to 1256 of SEQ ID NO: 1.
63. A pharmaceutical composition comprising the monoclonal antibody
or a portion thereof according to any one of claims 2 to 31 and a
pharmaceutically acceptable carrier.
64. A pharmaceutical composition comprising the human monoclonal
antibody or a portion thereof according to any one of claims 9 to
18 or 28 and a pharmaceutically acceptable carrier.
65. A pharmaceutical composition comprising the human monoclonal
antibody or a portion thereof according to any one of claims 14 to
18 and 28.
66. The pharmaceutical composition according to any one of claims
63 to 65, for inhibiting proliferation of cells capable of
proliferating by a stimulus with CTGF.
67. The pharmaceutical composition according to any one of claims
63 to 65, for treating or preventing a disease accompanied by
proliferation of cells capable of proliferating by a stimulus with
CTGF;
68. The pharmaceutical composition according to claim 66 or 67,
wherein said proliferation is cell proliferation in a tissue
selected from the group consisting of brain, neck, lung, heart,
liver, pancreas, kidney, stomach, large intestine, small intestine,
duodenum, bone marrow, uterus, ovary, testis, prostate gland, skin,
mouth, tongue and blood vessels.
69. The pharmaceutical composition according to claim 68, wherein
said tissue is lung, liver, kidney or skin.
70. The pharmaceutical composition according to claim 69, wherein
said tissue is the kidney.
71. The pharmaceutical composition according to claim 67, wherein
said disease is further accompanied by tissue fibrosis.
72. The pharmaceutical composition according to claim 71, wherein
said tissue fibrosis is tissue fibrosis in lung, liver, kidney or
skin.
73. The pharmaceutical composition according to claim 72, wherein
said tissue fibrosis is kidney fibrosis.
74. A pharmaceutical composition for treating or preventing a
kidney disease, comprising a CTGF inhibitor or an agent for
inhibiting CTGF production, and a pharmaceutically acceptable
carrier.
75. The pharmaceutical composition according to claim 74, wherein
said inhibitor is a monoclonal antibody reactive to CTGF.
76. The pharmaceutical composition according to claim 74, wherein
said inhibitor is the monoclonal antibody of any one of claims 9 to
31.
77. The pharmaceutical composition according to 76, wherein said
inhibitor is the human monoclonal antibody according to any one of
claims 14 to 18 and 28.
78. The pharmaceutical composition according to any one of claims
74 to 77, wherein said disease is accompanied by tissue
fibrosis.
79. A pharmaceutical composition for inhibiting proliferation of
cells in kidney which are capable of proliferating by a stimulus
with CTGF, comprising a substance having an activity of inhibiting
proliferation of said cells and a pharmaceutically acceptable
carrier.
80. The pharmaceutical composition according to claim 79, wherein
said substance is a monoclonal antibody reactive to CTGF.
81. The pharmaceutical composition according to claim 79, wherein
said inhibitor is the monoclonal antibody according to any one of
claims 9 to 31.
82. The pharmaceutical composition according to claim 81, wherein
said inhibitor is the human monoclonal antibody according to any
one of claims 14 to 18 and 28.
Description
TECHNICAL FIELD
[0001] The present invention relates to monoclonal antibodies
reactive to mammalian connective tissue growth factor (CTGF) or a
portion thereof; cells producing the monoclonal antibodies;
antibody-immobilized insoluble carriers on which the monoclonal
antibodies or a portion thereof are immobilized; labeled antibodies
obtained by labeling the monoclonal antibodies with labeling
agents; kits for detecting, separating, assaying or purifying
mammalian CTGF; methods for detecting, separating, assaying or
purifying mammalian CTGF; pharmaceutical compositions comprising
the monoclonal antibodies; transgenic mice to which the human CTGF
gene is introduced; a polypeptide of rat CTGF; a DNA encoding rat
CTGF; and antibodies reactive to rat CTGF.
BACKGROUND ART
[0002] Injured tissues are regenerated by the following process:
removal of useless tissue fragments and cell fragments or bacteria
and so on by phagocytes such as macrophages that migrate to the
injured site; recovery of vessels; and the subsequent tissue
renewal. Transforming growth factor .beta. (TGF-.beta.) produced by
macrophages and neutrophils, which appear during the process of the
tissue regeneration and recovery, has been revealed to serve as the
first regulatory factor in the regeneration-recovery process.
[0003] TGF-.beta. has multiple functions. The factor is known to
regulate the production of the extracellular matrix (ECM) from
connective tissue cells as well as to induce the proliferation of
mesenchymal cells and to inhibit the proliferation of vascular
endothelial cells and epithelial cells.
[0004] Increased production of platelet-derived growth factor
(PDGF) and connective tissue growth factor (CTGF; also called
Hcs24) is found in the culture supernatant of the above-mentioned
mesenchymal cells of which proliferation is induced by a stimulus
with TGF-.beta.. Because of this, it is presumed that the cell
proliferation is not directly but indirectly induced by TGF-.beta.
with the help of other regulatory factors.
[0005] Human and mouse CTGFs have been identified previously (so
far, there is no report on the identification of rat CTGF), and
their physicochemical and biological properties have been analyzed
(<human CTGF>: J. Cell Biology, vol. 114, No. 6, p.1285-1294,
1991; Int. J. Biochem. Cell Biol., Vol. 29, No. 1, p. 153-161,
1997; Circulation, vol. 95, No. 4, p.831-839, 1997; Cell Growth
Differ., Vol. 7, No. 4, p. 469-480, 1996; J. Invest. Dermatol.,
Vol. 106, No. 4, p. 729-733, 1996; J. Invest. Dermatol., Vol. 105,
No. 2, p. 280-284, 1995; J. Invest. Dermatol. Vol. 105, No. 1, p.
128-132, 1995; WO96/38172; <mouse CTGF (Fisp12)>: Unexamined
Published Japanese Patent Application (JP-A) No. Hei 5-255397; Cell
Growth Differ., vol. 2, No. 5, p. 225-233, 1991; FEBS Letters, Vol.
327, No. 2, p. 125-130, 1993; DNA Cell Biol., Vol. 10, No. 4, p.
293-308, 1991).
[0006] CTGF is a cysteine-rich secretory glycoprotein with a
molecular weight of about 38 kDa. It has been revealed that the
biosynthesis and secretion of the protein are induced by
TGF-.beta.. CTGF has similar properties with PDGF in the light of
that: their productions are induced by TGF-.beta.; they bind to the
PDGF receptor and induce the proliferation of mesenchymal cells;
and they are produced by fibroblasts and epithelial cells. However,
they exhibit no homology at the amino acid level and thus the two
molecules are distinct to each other (The Journal of Cell Biology,
vol. 114, No. 6, p. 1287-1294, 1991; Molecular Biology of the Cell,
Vol. 4, p.637-645, 1993).
[0007] In recent studies, low molecular weight species of CTGF have
been found in the culture supernatant of human and mouse fibroblast
cells as well as in the secreting fluid derived from the porcine
uterus. They are biologically active but are presumed to be
degradation products of 38 kDa CTGF molecules, since their
molecular weights are about 10-12 kDa (Growth Factors, vol. 15, No.
3, p. 199-213, 1998; J. Biol. Chen., vol. 272, No. 32, p.
20275-20282, 1997).
[0008] Details of the relationship between physiological functions
of CTGF and diseases have yet to be fully clarified. However, it
has been found that: CTGF production is induced by TGF-.beta.; the
expression level of CTGF mRNA is significantly high in tissues and
cells derived from patients affected with various diseases (Int. J.
Biochem. Cell. Biol., Vol. 29, No. 1, p. 153-161, 1997;
Circulation, Vol. 95, No. 4, p.831-839, 1997; J. Invest. Dermatol,,
Vol. 106, No. 4, p.729-733, 1996; J. Invest. Dermatol., Vol. 105,
No. 2, p. 128-132, 1995; J. Cell Physiol., Vol. 165, No. 3, p.
556-565, 1995; Kidney Int., Vol. 48, No. 2, p. 5001-5009, 1995);
and CTGF enhances the chemotaxis and proliferation of the vascular
endothelial cells (J. Cell. Biol., Vol. 114, No. 6, p. 1285-1294,
1991; Exp. Cell Res., Vol. 233, p. 63-77, 1997; Journal of Japanese
Association for Oral Biology, Vol. 38, extra number, p. 463,
PD0187, 1996; the 69th meeting of the Japanese Biochemical Society,
proceedings, p. 683, 1P0535, 1996). These findings suggest the
possibility that CTGF is associated with the onset and/or
advancement of a variety of diseases.
[0009] Identification of the specific diseases awaits further
findings and advancement in research. Nonetheless, CTGF has been
presumed to be involved in the onset and/or advancement of a wide
variety of diseases including, for example, cancers,
arteriosclerosis, and skin diseases (for example, psoriasis,
scleroderma, atopy, and keloid), kidney diseases, arthritis (for
example, rheumatoid arthritis), various fibrotic diseases (fibrotic
diseases in tissues as observed in arteriosclerosis, cirrhosis,
arthritis, scleroderma, keloid, kidney fibrosis and pulmonary
fibrosis, etc.).
[0010] To elucidate the association of CTGF with such various
diseases, it is generally effective to detect and assay CTGF and/or
the protein fragments thereof in the body fluids (serum, etc.) from
patients and mammals affected with the diseases; the values
determined are compared with normal values (obtained from mammals
including normal persons, normal mice, normal rats and normal
rabbits, etc.).
[0011] The detection and assay of secretory proteins such as CTGF
are carried out by immunological assays based on antigen-antibody
interaction by using the antibody (preferably used are monoclonal
antibodies) which is reactive to the secretory protein to be
detected; specifically, immunoassays such as radioimmunoassay (RIA)
and enzyme immunoassay (EIA, ELISA) are widely used as the most
convenient and useful methods for the purpose.
[0012] In this context, for the purpose of assaying CTGF, it is
necessary to develop detection and assay methods using such
immunoassay systems and also to prepare monoclonal antibodies
against CTGF required for the establishment of assay methods. There
are some reports on the preparation of antiserum reactive to CTGF
(Exp. Cell Res., Vol. 233, p. 63-77, 1997; Cell Growth Differ.,
Vol. 8, No. 1, p. 61-68, 1997; the 69th meeting of Japanese
Biochemical Society, proceedings, p. 683, 1P0534, 1996) but no
report on the preparation of functional anti-CTGF monoclonal
antibody which has particularly high affinity for CTGF and/or the
capability of neutralizing the CTGF activity; no immunoassay
systems for CTGF have so far been provided at all.
[0013] Such monoclonal antibodies having the capability of
neutralizing the CTGF activity described hereinabove are useful not
only as components in an immunoassay system but also as
pharmaceutical antibody preparations for the treatment and/or
prevention of the above-mentioned diseases caused by CTGF
secretion. However, there have not been any report on such
monoclonal antibodies yet.
DISCLOSURE OF THE INVENTION
[0014] Thus, the development of monoclonal antibodies reactive to
CTGFs from various mammals such as humans, mice, rats and rabbits,
has been desirably awaited. Such monoclonal antibodies are useful
for the understanding of biological functions of CTGF associated
with the onset and/or advancement of the above-mentioned various
diseases as well as for the understanding of cause-effect relations
between CTGF and the various diseases. Such monoclonal antibodies
are also usable as active ingredients of pharmaceutical products
for treating and preventing the diseases caused by CTGF. In
particular, development of monoclonal antibodies having
sufficiently high affinities for CTGF, the capability of
neutralizing the CTGF activity, and/or the sufficient
crossreactivity to CTGFs from a variety of mammalian species, is
demanded when the antibodies are used as components in immunoassay
systems for detecting CTGF to elucidate the functions of CTGF as
well as the relationship between CTGF and various diseases.
[0015] In addition, it is necessary to develop monoclonal
antibodies with reduced antigenicity or without antigenicity as
well as with the neutralizing activity described above, when the
antibodies are used for the treatment and/or prevention of various
diseases in patients.
[0016] In order to fulfill the social needs, the present inventors
extensively studied the monoclonal antibodies against CTGFs from a
variety of mammals, and using CTGFs from various mammals as
immunogens, succeeded in preparing various monoclonal antibodies
against CTGFs from a variety of mammals; the antibodies are
different in properties such as antigenic specificity, affinity for
the antigens, the neutralizing activity and the
crossreactivity.
[0017] The present inventors also succeeded, for the first time in
the world, in preparing various human monoclonal antibodies against
human CTGF, by immunizing, with human CTGF as an immunogen,
transgenic mice created to produce human antibodies by using
recombinant technology. Furthermore, the present inventors found
that intact CTGFs in body fluids (serum, etc.) from a variety of
mammals (human, mouse, rat, and rabbit) could be highly sensitively
assayed by using various immunoassay systems constructed with the
various monoclonal antibodies described above. Thus the present
inventions were achieved.
[0018] The present invention was also achieved by the findings that
the latter, i.e., the human antibodies, has not only the capability
of significantly neutralizing the human CTGF activity, but also
therapeutic effects on, for example, fibrotic diseases in tissues
(kidney fibrosis, etc.) as well. The fact that these human
antibodies are non-antigenic in humans, dramatically elevates the
utility value of antibody as a pharmaceutical, because antigenicity
is a major therapeutic problem (side effect) in medical treatment
with antibody pharmaceuticals comprising antibodies derived from
non-human mammals such as mice.
[0019] In particular, the present invention provides, for the first
time in the field to which the present invention pertains,
monoclonal antibodies that are reactive to various mammalian CTGFs
and possess various useful properties as pharmaceuticals to treat
and prevent diseases in patients and as components in immunoassay
systems to detect and assay CTGF in body fluids from various
mammals such as humans, mice, and rats.
[0020] In addition to this, the present invention provides methods
and kits of immunoassay for CTGF using such various monoclonal
antibodies against CTGF for the first time.
[0021] The inventive anti-human CTGF monoclonal antibodies are
extremely useful as pharmaceuticals for the treatment and
prevention of various diseases caused by CTGF because the
antibodies are nonantigetic in humans.
[0022] By using an immunoassay with the monoclonal antibodies of
the present invention, it is possible to conveniently and highly
sensitively detect and assay intact CTGF in the body fluids from
healthy and diseased mammals (humans, mice, rats and rabbits).
[0023] Specifically, the present inventions are defined as
follows:
[0024] (1) a monoclonal antibody or a portion thereof, comprising a
property in any of (a) to (g) below:
[0025] (a) reactive to human, mouse and rat connective tissue
growth factors (CTGFs);
[0026] (b) reactive to both human and mouse CTGFs but not reactive
to rat CTGF;
[0027] (c) reactive to both mouse and rat CTGFs but not reactive to
human CTGF;
[0028] (d) inhibiting binding of human CTGF to human kidney-derived
fibroblast cell line 293-T (ATCC CRL1573), or the binding of mouse
CTGF to said cell line 293-T;
[0029] (e) inhibiting binding of human CTGF to any cells of rat
kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570), human
osteosarcoma-derived cell line MG-63 (ATCC CRL-1427), or human
lung-derived fibroblasts;
[0030] (f) inhibiting cell proliferation of rat kidney-derived
fibroblast cell line NRK-49F (ATCC CRL-1570)induced by a stimulus
with human or mouse CTGF; or,
[0031] (g) inhibiting an increase of hydroxyproline in the kidney,
wherein said hydroxyproline level tends to be elevated;
[0032] (2) the monoclonal antibody or a portion thereof according
to (1), comprising a property in any of (a) to (c) below:
[0033] (a) obtainable by immunizing a mouse with human CTGF or a
portion thereof, and reactive to human, mouse and rat CTGFs;
[0034] (b) obtainable by immunizing a hamster with mouse CTGF or a
portion thereof, and reactive to human, mouse and rat CTGFs;
or,
[0035] (c) obtainable by immunizing a rat with mouse CTGF or a
portion thereof, and reactive to human, mouse and rat CTGFs;
[0036] (3) the monoclonal antibody or a portion thereof according
to (1), comprising a property in any of (a) to (c) below:
[0037] (a) obtainable by immunizing a mouse with human CTGF or a
portion thereof, reactive to human, mouse and rat CTGFs and
inhibiting binding of human CTGF to human kidney-derived fibroblast
cell line 293-T (ATCC CRL1573);
[0038] (b) obtainable by immunizing a rat with mouse CTGF or a
portion thereof, reactive to human, mouse and rat CTGFs and
inhibiting binding of mouse CTGF to human kidney-derived fibroblast
cell line 293-T (ATCC CRL1573); or,
[0039] (c) obtainable by immunizing a hamster with mouse CTGF or a
portion thereof, and reactive to human, mouse and rat CTGFs and
inhibiting binding of mouse CTGF to human kidney-derived fibroblast
cell line 293-T (ATCC CRL1573);
[0040] (4) the monoclonal antibody or a portion thereof according
to (1), wherein said monoclonal antibody is produced by a hybridoma
identified by an international deposit accession No. FERM
BP-6208;
[0041] (5) the monoclonal antibody or a portion thereof according
to (1), wherein said monoclonal antibody comprises a property
substantially equivalent to that of a monoclonal antibody produced
by a hybridoma identified by an international deposit accession No.
FERM BP-6208;
[0042] (6) the monoclonal antibody or a portion thereof according
to (1), wherein said monoclonal antibody is produced by a hybridoma
identified by an international deposit accession No. FERM
BP-6209;
[0043] (7) the monoclonal antibody or a portion thereof according
to (1), wherein said monoclonal antibody comprises a property
substantially equivalent to that of a monoclonal antibody produced
by a hybridoma identified by an international deposit accession No.
FERM BP-6209;
[0044] (8) a human monoclonal antibody or a portion thereof,
reactive to any human, mouse or rat CTGF;
[0045] (9) the human monoclonal antibody or a portion thereof
according to (8), wherein said human monoclonal antibody is
reactive to human CTGF;
[0046] (10) a human monoclonal antibody or a portion thereof,
reactive to human CTGF and comprises a property in any of (a) to
(d) below:
[0047] (a) inhibiting binding of human CTGF to human kidney-derived
fibroblast cell line 293-T (ATCC CRL1573);
[0048] (b) inhibiting binding of human CTGF to any of rat
kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570), human
osteosarcoma-derived cell line MG-63 (ATCC CRL-1427), or human
lung-derived fibroblasts;
[0049] (c) inhibiting the cell proliferation of rat kidney-derived
fibroblast cell line NRK-49F (ATCC CRL-1570)induced by a stimulus
with human or mouse CTGF; or,
[0050] (d) inhibiting an increase of hydroxyproline in kidney,
wherein said hydroxyproline level tends to be elevated;
[0051] (11) the human monoclonal antibody or a portion thereof
according to any one of (8) to (10), wherein said human monoclonal
antibody is derived from a non-human transgenic mammal which is
capable of producing a human antibody;
[0052] (12) the human monoclonal antibody or a portion thereof
according to (11), wherein said human monoclonal antibody is
obtainable by immunizing a non-human transgenic mammal which is
capable of producing a human antibody, with human CTGF;
[0053] (13) the human monoclonal antibody or a portion thereof
according to any one of (8) to (12), wherein said non-human
transgenic mammal is a transgenic mouse;
[0054] (14) the human monoclonal antibody or a portion thereof
according to any one of (8) to (13), wherein a V-region DNA
encoding a heavy chain variable region of said human monoclonal
antibody is derived from a gene segment selected from the group
consisting of DP-5, DP-38, DP-65 and DP-75;
[0055] (15) the human monoclonal antibody or a portion thereof
according to any one of (8) to (13), wherein a V-region DNA
encoding a light chain variable region of said human monoclonal
antibody is derived from a gene segment selected from the group
consisting of DPK1, DPK9, DPK12 and DPK24;
[0056] (16) the human monoclonal antibody or a portion thereof
according to any one of (8) to (15), wherein a V-region DNA
encoding a heavy chain variable region of said human monoclonal
antibody is derived from a gene segment selected from the group
consisting of DP-5, DP-38, DP-65 and DP-75, and wherein a V-region
DNA encoding a light chain variable region of said human monoclonal
antibody is derived from a gene segment selected from the group
consisting of DPK1, DPK9, DPK12 and DPK24;
[0057] (17) the human monoclonal antibody or a portion thereof
according to (9), wherein an amino acid sequence of a heavy chain
variable region of said human monoclonal antibody comprises an
amino acid sequence defined below in any of (a) to (j) below:
[0058] (a) the amino acid positions 21 to 120 of the amino acid
sequence of SEQ ID NO: 6;
[0059] (b) the amino acid positions 21 to 120 of the amino acid
sequence of SEQ ID NO: 6, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0060] (c) the amino acid positions 21 to 118 of the amino acid
sequence of SEQ ID NO: 8;
[0061] (d) the amino acid positions 21 to 118 of the amino acid
sequence of SEQ ID NO: 8, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0062] (e) the amino acid positions 21 to 116 of the amino acid
sequence of SEQ ID NO: 10;
[0063] (f) the amino acid positions 21 to 116 of the amino acid
sequence of SEQ ID NO: 10, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0064] (g) the amino acid positions 21 to 116 of the amino acid
sequence of SEQ ID NO: 12;
[0065] (h) the amino acid positions 21 to 116 of the amino acid
sequence of SEQ ID NO: 12, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0066] (i) the amino acid positions 21 to 117 of the amino acid
sequence of SEQ ID NO: 14; or,
[0067] (j) the amino acid positions 21 to 117 of the amino acid
sequence of SEQ ID NO: 14, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0068] (18) the human monoclonal antibody or a portion thereof
according to (9), wherein an amino acid sequence of a light chain
variable region of said human monoclonal antibody comprises an
amino acid sequence in any of (a) to (j) below:
[0069] (a) the amino acid positions 21 to 120 of the amino acid
sequence of SEQ ID NO: 16;
[0070] (b) the amino acid positions 21 to 120 of the amino acid
sequence of SEQ ID NO: 16, wherein one -or more amino acids are
deleted, substituted, inserted or added;
[0071] (c) the amino acid positions 21 to 121 of the amino acid
sequence of SEQ ID NO: 18;
[0072] (d) the amino acid positions 21 to 121 of the amino acid
sequence of SEQ ID NO: 18, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0073] (e) the amino acid positions 23 to 117 of the amino acid
sequence of SEQ ID NO: 20;
[0074] (f) the amino acid positions 23 to 117 of the amino acid
sequence of SEQ ID NO: 20, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0075] (g) the amino acid positions 17 to 111 of the amino acid
sequence of SEQ ID NO: 22;
[0076] (h) the amino acid positions 17 to 111 of the amino acid
sequence of SEQ ID NO: 22, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0077] (i) the amino acid positions 23 to 118 of the amino acid
sequence of SEQ ID NO: 24; or,
[0078] (j) the amino acid positions 23 to 118 of the amino acid
sequence of SEQ ID NO: 24, wherein one or more amino acids are
deleted, substituted, inserted or added;
[0079] (19) a monoclonal antibody or a portion thereof, reactive to
human CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6535;
[0080] (20) a monoclonal antibody or a portion thereof, reactive to
human CTGF and comprises a property substantially equivalent to
that of a monoclonal antibody produced by a hybridoma identified by
an international deposit accession No. FERM BP-6535;
[0081] (21) a monoclonal antibody or a portion thereof, reactive to
human CTGF, and which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6598;
[0082] (22) a monoclonal antibody or a portion thereof, reactive to
human CTGF and comprises a property substantially equivalent to
that of a monoclonal antibody produced by a hybridoma identified by
an international deposit accession No. FERM BP-6598;
[0083] (23) a monoclonal antibody or a portion thereof, reactive to
human CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6599;
[0084] (24) a monoclonal antibody or a portion thereof, reactive to
human CTGF and comprises a property substantially equivalent to
that of a monoclonal antibody produced by a hybridoma identified by
an international deposit accession No. FERM BP-6599;
[0085] (25) a monoclonal antibody or a portion thereof, reactive to
human CTGF, which is produced by a hybridoma identified by an
international deposit accession No. FERM BP-6600;
[0086] (26) a monoclonal antibody or a portion thereof, reactive to
human CTGF and comprises a property substantially equivalent to
that of a monoclonal antibody produced by a hybridoma identified by
an international deposit accession No. FERM BP-6600;
[0087] (27) a monoclonal antibody or a portion thereof, reactive to
human CTGF, and which is non-reactive-to a antigen-antibody complex
of human CTGF and the monoclonal antibody reactive to human CTGF of
(17) or (18);
[0088] (28) the monoclonal antibody or a portion thereof according
to (27), wherein said monoclonal antibody is a human monoclonal
antibody;
[0089] (29) a monoclonal antibody or a portion thereof, reactive to
rat CTGF;
[0090] (30) a recombinant chimeric monoclonal antibody, reactive to
human CTGF, and of which a variable region is derived from a
variable region of the monoclonal antibody according to any one of
(2) to (7), (27) or (29) and of which a constant region is derived
from a constant region of a human immunoglobulin;
[0091] (31) a recombinant humanized monoclonal antibody, reactive
to human CTGF, of which a whole or portion of the
complementarity-determining regions of a hyper-variable region is
derived from complementarity-determining regions of the monoclonal
antibody of any one of (2) to (7), (27) or (29), of which framework
regions of a hyper-variable region are derived from the framework
regions of a human immunoglobulin and of which a constant region is
derived from a constant region of a human immunoglobulin;
[0092] (32) a cell producing the monoclonal antibody according to
any one of (1) to (29);
[0093] (33) a cell producing the recombinant monoclonal antibody
according to (30) or (31);
[0094] (34) the cell according to (32), wherein said cell is a
hybridoma obtainable by fusing a mammalian myeloma cell with a
mammalian B cell which is capable of producing the monoclonal
antibody;
[0095] (35) the cell according to (32) or (33), wherein said cell
is a genetically engineered cell transformed by either one or both
of the DNAs encoding a heavy chain and light chain of the
monoclonal antibody;
[0096] (36) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6535;
[0097] (37) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6598;
[0098] (38) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6599;
[0099] (39) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6600;
[0100] (40) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6208;
[0101] (41) the hybridoma according to (34), wherein said hybridoma
is identified by an international deposit accession No. FERM
BP-6209;
[0102] (42) an antibody-immobilized insoluble carrier on which the
monoclonal antibody according to any one of (1) to (31) is
immobilized;
[0103] (43) the antibody-immobilized insoluble carrier according to
(42), wherein said insoluble carrier is selected from the group
consisting of plates, test tubes, tubes, beads, balls, filters and
membranes;
[0104] (44) the antibody-immobilized insoluble carrier according to
(42), wherein said insoluble carrier is a filter or membrane, or
that used for affinity column chromatography;
[0105] (45) a labeled antibody which is prepared by labeling the
monoclonal antibody of any one of (1) to (31) with a labeling agent
capable of providing a detectable signal by itself or together with
other substances;
[0106] (46) the labeled antibody according to (45), wherein said
labeling agent is an enzyme, fluorescent substance,
chemiluminescent substance, biotin, avidin, or radioisotope;
[0107] (47) a kit for detecting or assaying mammalian CTGF,
comprising at least one monoclonal antibody, an
antibody-immobilized insoluble carrier, and a labeled antibody,
which is selected from the group consisting of the monoclonal
antibody according to any one of (1) to (31), the
antibody-immobilized insoluble carrier according to (42) or (43),
and the labeled antibody according to (45) or (46);
[0108] (48) the kit for detecting or assaying mammalian CTGF
according to (47), comprising the antibody-immobilized insoluble
carrier according to (42) or (43) and the labeled antibody
according to (45) or (46);
[0109] (49) a method for detecting or assaying mammalian CTGF by an
immunoassay using at least one monoclonal antibody, an
antibody-immobilized insoluble carrier, and a labeled antibody,
which is selected from the group consisting of the monoclonal
antibody according to any one of (1) to (31), the
antibody-immobilized insoluble carrier according to (42) or (43),
and the labeled antibody according to (45) or (46);
[0110] (50) the method for detecting or assaying mammalian CTGF by
an immunoassay according to (49), comprising at least the following
steps of (a) and (b):
[0111] (a) reacting a sample with the antibody-immobilized
insoluble carrier according to (42) or (43); and,
[0112] (b) reacting the labeled antibody according to (45) or (46)
with an antigen-antibody complex formed by binding mammalian CTGF
in said sample to the antibody-immobilized insoluble carrier;
[0113] (51) the method for detecting or assaying mammalian CTGF by
an immunoassay according to (49), comprising at least the following
steps of (a) and (b):
[0114] (a) reacting a sample with the labeled antibody according to
(45) or (46); and,
[0115] (b) reacting the antibody-immobilized insoluble carrier
according to (42) or (43) with the antigen-antibody complex formed
by binding said labeled antibody and mammalian CTGF in said
sample;
[0116] (52) the method for detecting or assaying mammalian CTGF by
an immunoassay according to (49), comprising at least the following
step of (a):
[0117] (a) reacting a mixture comprising the antibody-immobilized
insoluble carrier according to (42) or (43), the labeled antibody
according to (45) or (46), and a sample;
[0118] (53) the method for detecting or assaying mammalian CTGF by
an immunoassay according to (49), comprising at least the following
step of (a):
[0119] (a) reacting a sample and a mammalian CTGF standard labeled
with a labeling agent capable of providing a detectable signal by
itself or together with other substances, with the
antibody-immobilized insoluble carrier according to (42) or
(43);
[0120] (54) the method for detecting or assaying mammalian CTGFs by
an immunoassay according to (49), comprising at least the following
steps of (a) and (b):
[0121] (a) reacting the monoclonal antibody according to any one of
(1) to (31) with a mixture comprising a sample and a mammalian CTGF
standard labeled with a labeling agent capable of providing a
detectable signal by itself or together with other substances;
and,
[0122] (b) reacting a mammalian antiserum reactive to said
monoclonal antibody with the antigen-antibody complex formed by
binding mammalian CTGF in said sample or said labeled mammalian
CTGF standard and said monoclonal antibody;
[0123] (55) the method for detecting or assaying mammalian CTGFs by
an immunoassay according to (49), comprising at least the following
steps of any of (a) to (c):
[0124] (a) reacting the monoclonal antibody according to any of one
s (1) to (31) with a sample;
[0125] (b) reacting a mammalian CTGF standard labeled with a
labeling agent capable of providing a detectable signal by itself
or together with other substances with a reaction product resulted
from the reaction in step (a); and,
[0126] (c) reacting a mammalian antiserum reactive to said
monoclonal antibody with the antigen-antibody complex formed by
binding mammalian CTGF in said sample or said labeled mammalian
CTGF standard, and said monoclonal antibody;
[0127] (56) a kit for separating or purifying mammalian CTGF,
comprising the antibody-immobilized insoluble carrier according to
(42) or (44);
[0128] (57) a method for separating or purifying mammalian CTGF,
comprising using affinity chromatography with the
antibody-immobilized insoluble carrier according to (42) or
(44);
[0129] (58) the purification method for mammalian CTGF according to
(57), wherein said affinity chromatography is affinity column
chromatography;
[0130] (59) a transgenic mouse in which DNA encoding human CTGF is
integrated into an endogenous gene locus;
[0131] (60) a rat CTGF comprising an amino acid sequence of, or
substantially equivalent to the amino acid sequence of SEQ ID NO:
2;
[0132] (61) a DNA encoding a rat CTGF comprising the amino acid
sequence of SEQ ID NO: 2;
[0133] (62) the DNA according to (61), comprising nucleotide
sequence in the positions of 213 to 1256 of SEQ ID NO: 1;
[0134] (63) a pharmaceutical composition comprising the monoclonal
antibody or a portion thereof according to any one of (2) to (31)
and a pharmaceutically acceptable carrier;
[0135] (64) a pharmaceutical composition comprising the human
monoclonal antibody or a portion thereof according to any one of
(9) to (18) or (28) and a pharmaceutically acceptable carrier;
[0136] (65) a pharmaceutical composition comprising the human
monoclonal antibody or a portion thereof according to any one of
(14) to (18) and (28);
[0137] (66) the pharmaceutical composition according to any one of
(63) to (65), for inhibiting proliferation of cells capable of
proliferating by a stimulus with CTGF;
[0138] (67) the pharmaceutical composition according to any one of
(63) to (65), for treating or preventing a disease accompanied by
proliferation of cells capable of proliferating by a stimulus with
CTGF;
[0139] (68) the pharmaceutical composition according to (66) or
(67), wherein said proliferation is cell proliferation in a tissue
selected from the group consisting of brain, neck, lung, heart,
liver, pancreas, kidney, stomach, large intestine, small intestine,
duodenum, bone marrow, uterus, ovary, testis, prostate gland, skin,
mouth, tongue and blood vessels;
[0140] (69) the pharmaceutical composition according to (68),
wherein said tissue is the lung, liver, kidney or skin;
[0141] (70) the pharmaceutical composition according to (69),
wherein said tissue is the kidney;
[0142] (71) the pharmaceutical composition according to (67),
wherein said disease is further accompanied by tissue fibrosis;
[0143] (72) the pharmaceutical composition according to (71),
wherein said tissue fibrosis is tissue fibrosis in lung, liver,
kidney or skin;
[0144] (73) the pharmaceutical composition according to (72),
wherein said tissue fibrosis is kidney fibrosis;
[0145] (74) a pharmaceutical composition for treating or preventing
a kidney disease, comprising a CTGF inhibitor or an agent for
inhibiting CTGF production, and a pharmaceutically acceptable
carrier;
[0146] (75) the pharmaceutical composition according to (74),
wherein said inhibitor is a monoclonal antibody reactive to
CTGF;
[0147] (76) the pharmaceutical composition according to (74),
wherein said inhibitor is the monoclonal antibody of any one of (9)
to (31);
[0148] (77) the pharmaceutical composition according to (76),
wherein said inhibitor is the human monoclonal antibody according
to any one of (14) to (18) and (28);
[0149] (78) the pharmaceutical composition according to any one of
(74) to (77), wherein said disease is accompanied by tissue
fibrosis;
[0150] (79) a pharmaceutical composition for inhibiting
proliferation of cells in kidney which are capable of proliferating
by a stimulus with CTGF, comprising a substance having an activity
of inhibiting proliferation of said cells and a pharmaceutically
acceptable carrier;
[0151] (80) the pharmaceutical composition according to (79),
wherein said substance is a monoclonal antibody reactive to
CTGF;
[0152] (81) the pharmaceutical composition according to (79),
wherein said inhibitor is the monoclonal antibody according to any
one of (9) to (31);
[0153] (82) the pharmaceutical composition according to (81),
wherein said inhibitor is the human monoclonal antibody according
to any one of (14) to (18) and (28).
[0154] The present inventions are described in detail herein below
by defining terminologies used herein.
[0155] Herein, "mammals" mean humans, bovine, goats, rabbits, mice,
rats, hamsters and guinea pigs; preferred are humans, rabbits,
rats, hamsters or mice, and particularly preferred are humans,
rats, hamsters or mice.
[0156] The terminologies "mammals except human" and "non-human
mammals" in the present invention have the same meaning, and both
indicate all the above-defined mammals except humans.
[0157] "Amino acids" used in the present invention mean any amino
acid existing in nature and preferably the following amino acids
presented by alphabetical triplets or single letter codes used to
represent amino acids. (Gly/G) glycine, (Ala/A) alanine, (Val/V)
valine, (Leu/L) leucine, (Ile/I) isoleucine, (Ser/S) serine,
(Thr/T) threonine, (Asp/D) aspartic acid, (Glu/E) glutamic acid,
(Asn/N) asparagine, (Gln/Q) glutamine, (Lys/K) lysine, (Arg/R)
arginine, (Cys/C) cysteine, (Met/M) methionine, (Phe/F)
phenylalanine, (Tyr/Y) tyrosine, (Trp/W) tryptophane, (His/H)
histidine, (Pro/P) proline.
[0158] The term "connective tissue growth factor (CTGF)" as
referred to in the present invention means CTGF derived from the
above-mentioned mammals, and includes, for example, human and mouse
CTGFs having the above-described structure and function as reported
in previous reports (for example: The Journal of Cell Biology Vol.
114, No. 6, p. 1287-1294, 1991; Molecular Biology of the Cell, Vol.
4, p. 637-645, 1993; Biochem. Biophys. Res. Comm. Vol. 234, p.
206-210, 1997, etc.). As a matter of course, the "connective tissue
growth factor" also includes rat CTGF that is included within the
scope of the present invention.
[0159] Moreover, "connective tissue growth factor" as referred to
in the present invention includes not only the CTGF (for example,
human CTGF) with a molecular weight of about 38 kDa as documented-
in reports but also a low-molecular-weight CTGF protein, with a
molecular weight ranging from about 10 to about 12 kDa. The
low-molecular-weight protein is assumed to be a degradation product
of the full-length CTGF (for example, human CTGF) with a molecular
weight of about 38 kDa (Growth Factors, Vol. 15, No. 3, p. 199-213,
1998; J. Biol. Chem., Vol. 272, No. 32, p. 20275-20282, 1997).
Although the structure of this low-molecular-weight CTGF remains
to, be clarified, there is a possibility that, in the case of human
CTGF, the low-molecular-weight-CTGF corresponds to a C-terminal
protein (molecular weight: about 11,800 Da) consisting of 103 amino
acid residues resulted from the cleavage of the full-length human
CTGF consisting of 349 amino acids between leucine at amino acid
position 246 (Leu246) and glutamic acid at amino acid position 247
(Glu247) or another C-terminal protein (molecular weight: about
11,671 Da) consisting of 102 amino acid residues resulted from the
cleavage of the full-length human CTGF between glutamic acid at
amino acid position 247 (Glu247) and glutamic acid at amino acid
position 248 (Glu248).
[0160] In addition, CTGF as referred to in the present invention
includes CTGFs having substantially the same amino acid sequence as
that of the natural CTGF (in particular, human CTGF) having the
native primary structure (amino acid sequence) or a portion
thereof, as long as the "monoclonal antibody" of the present
invention, which is described hereinafter, is reactive to the
natural CTGF or a portion thereof.
[0161] Here, "having substantially the same amino acid sequence"
means to include a protein having an amino acid sequence where
multiple amino acids, preferably 1 to 10 amino acids, particularly
preferably 1 to 5 amino acids, in the amino acid sequence of the
natural CTGF protein, are substituted, deleted and/or modified, and
a protein having an amino acid sequence where multiple amino acids,
preferably 1 to 10 amino acids, particularly preferably 1 to 5
amino acids, are added to the amino acid sequence, as long as the
protein has substantially the same biological properties as the
natural CTGF protein. Furthermore, a combination of two or more of
the above alterations including a substitution, deletion,
modification and addition is also included.
[0162] The CTGF of the present invention can be produced by
suitably using a method known in the technical field, such as
recombinant technology, chemical synthesis or cell culture, or by
using a modified method thereof.
[0163] The CTGF of the present invention also includes "a portion"
of the CTGF. The terminology "a portion of CTGF" here refers to a
polypeptide comprising any arbitrary partial amino acid sequence
derived from the above-defined CTGF (including the above-mentioned
low-molecular-weight CTGF of about 10 to 12 kDa). Specifically, the
polypeptide includes CTGF peptide fragments with 5 to 100 amino
acid residues (for example, the peptides in the C-terminus), more
specifically, includes CTGF peptide fragments with 5 to 50 amino
acid residues, and even more specifically the peptide fragments
with 5 to 30 amino acid residues. Preferably, the polypeptide has a
partial structure of CTGF comprising a domain that binds or
interacts with the receptor thereof (receptor binding site, etc.)
or comprising a domain necessary to the biological function of CTGF
(active site, etc.).
[0164] These polypeptides (partial structures or fragments) can be
produced according to a method known in the technical field, or a
modified method thereof, by using recombinant technology or
chemical synthesis. The polypeptides can also be produced by
appropriately digesting the CTGF isolated by the cell culture
method with proteases and such.
[0165] "Monoclonal antibody" as referred to in the present
invention is a monoclonal antibody reactive to mammalian connective
tissue growth factor (CTGF) or a portion thereof. Specifically, the
"monoclonal antibody" is a monoclonal antibody having a property
described above in any of the inventions (1) to (31). More
specifically, "monoclonal antibody" means the various monoclonal
antibodies with a variety of properties and industrial utilities
described below in the examples and as indicated in the
drawings.
[0166] As a preferable embodiment, the monoclonal antibody of the
present invention is exemplified by the following monoclonal
antibodies described in (i) to (iv):
[0167] (i) the monoclonal antibody according to (1), wherein the
monoclonal antibody comprises a property described in any of (d) to
(g);
[0168] (ii) the monoclonal antibody according to (2);
[0169] (iii) the monoclonal antibody according to any one of (4) to
(7);
[0170] (iv) the monoclonal antibody according to any one of (9) to
(31).
[0171] In this embodiment, for the purpose of usage as a
pharmaceutical for treating or preventing various diseases,
preferable monoclonal antibody is a human monoclonal antibody
included by the antibodies described above in (i) to (iv).
[0172] In this embodiment, any of the monoclonal antibodies
described above in (i) to (iv) are usable for the detection, assay,
separation or purification of mammalian CTGFs, which is another
subject matter of the present invention.
[0173] As a more preferable embodiment, the monoclonal antibody of
the present invention is exemplified by the following monoclonal
antibodies described in (v) and (vi):
[0174] (v) the monoclonal antibody according to (1), wherein the
monoclonal antibody comprises a property described in any of (d) to
(g);
[0175] (vi) the monoclonal antibody according to any of (4) to (7),
(10), and (14) to (28).
[0176] In this embodiment, for the purpose of usage as a
pharmaceutical for treating or preventing various diseases,
preferable monoclonal antibody is a human monoclonal antibody
included in the antibodies described above in (v) and (vi).
[0177] Furthermore, in this embodiment, any of the monoclonal
antibodies described above in (v) and (vi) are usable for the
detection, assay, separation or purification of mammalian CTGFs
which is another subject matter of the present invention.
[0178] As a particularly preferable embodiment, the monoclonal
antibody of the present invention is exemplified by the following
monoclonal antibodies described in (vii) and (viii):
[0179] (vii) the monoclonal antibodies described above in any of
(iv) to (vii);
[0180] (viii) the monoclonal antibody according to any of (14) to
(26) and (28).
[0181] In this embodiment, for the purpose of usage as a
pharmaceutical for treating or preventing various diseases,
preferable monoclonal antibody is the human monoclonal antibody
described above in (viii).
[0182] In this embodiment, any of the monoclonal antibodies
described above in (vii) and (viii)are usable for the detection,
assay, separation or purification of mammalian CTGFs which is
another subject matter of this invention.
[0183] As a more particularly preferable embodiment, the monoclonal
antibody of the present invention is exemplified by the following
monoclonal antibodies described in (ix) to (xiv):
[0184] (ix) the monoclonal antibody according to (4) or (6);
[0185] (x) the monoclonal antibody according to any of (14) to
(16);
[0186] (xi) the monoclonal antibody according to (17), wherein the
monoclonal antibody comprises a property described in any of (a),
(c), (e), (g) and (i);
[0187] (xii) the monoclonal antibody according to (18), wherein the
monoclonal antibody comprises a property described in any of (a),
(c), (e), (g) and (i);
[0188] (xiii) the monoclonal antibody according to any of (19),
(21), (23) and (25);
[0189] (xiv) the monoclonal antibody according to (28).
[0190] In this embodiment, for the purpose of usage as a
pharmaceutical for treating or preventing various diseases,
preferable monoclonal antibody is the monoclonal antibody described
above in any of (x) to (xiv).
[0191] In this embodiment, any of the monoclonal antibodies
described above in (ix) to (xiv) are usable for the detection,
assay, separation or purification of mammalian CTGFs, which is
another subject matter of the present invention. However, the
monoclonal antibody described above in (ix) is particularly
preferable for the purpose.
[0192] The "monoclonal antibody" of the present invention also
includes a natural monoclonal antibody prepared by immunizing
mammals such as mice, rats, hamsters, guinea pigs or rabbits with
the above-defined connective tissue growth factor (including
natural, recombinant, and chemically synthesized protein and cell
culture supernatant) or a portion thereof as an antigen
(immunogen); a chimeric antibody and a humanized antibody
(CDR-grafted antibody) produced by recombinant technology; and a
human monoclonal antibody, for example, obtained by using human
antibody-producing transgenic animals.
[0193] The "monoclonal antibody" of the present invention further
includes a recombinant monoclonal antibody produced by the "cells
producing recombinant monoclonal antibody" described
hereinafter.
[0194] The monoclonal antibody includes those having any one of the
isotypes of IgG, IgM, IgA (IgA1 and IgA2), IgD, or IgE. IgG (IgG1,
IgG2, IgG3, and IgG4, preferably IgG2 or IgG4) or IgM is
preferable. IgG is most preferred.
[0195] The polyclonal antibody (antisera) or monoclonal antibody of
the present invention can be produced by known methods. Namely,
mammals (including transgenic animals generated so as to produce an
antibody derived from another animal species, such as the human
antibody producing transgenic mice described below), preferably,
mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs,
goats, horses, or bovine, or more preferably, mice, rats, hamsters,
guinea pigs, or rabbits are immunized, for example, with an antigen
mentioned above with Freund's adjuvant, if necessary. The
polyclonal antibody can be obtained from the serum obtained from
the animal so immunized. The monoclonal antibodies are produced as
follows. Hybridomas are produced by fusing the antibody-producing
cells obtained from the animal so immunized and myeloma cells
incapable of producing autoantibodies. Then the hybridomas are
cloned, and clones producing the monoclonal antibodies showing the
specific affinity to the antigen used for immunizing the mammal are
screened.
[0196] The antibodies can also be produced using "recombinant
monoclonal antibody producing cells" of the present invention
described below.
[0197] Specifically, the monoclonal antibody can be produced as
follows. Immunizations are done by injecting or implanting once or
several times the CTGF (including natural, recombinant, and
synthetic proteins, and cell culture supernatant) or its fragment
as mentioned above as an immunogen, if necessary, with Freund's
adjuvant, subcutaneously, intramuscularly, intravenously, through
the footpad, or intraperitoneally into non-human mammals, such as
mice, rats, hamsters, guinea pigs, or rabbits, preferably mice,
rats or hamsters (including transgenic animals generated so as to
produce antibodies derived from another animal such as the
transgenic mouse producing human antibody described below).
Usually, immunizations are performed once to four times every one
to fourteen days after the first immunization. Antibody-producing
cells are obtained from the mammal so immunized in about one to
five days after the last immunization. The times and interval of
the immunizations can be adequately altered according to the
properties of the immunogen used.
[0198] Hybridomas that secrete a monoclonal antibody can be
prepared by the method of Kohler and Milstein (Nature, Vol.256,
pp.495-497 (1975)) and by its modified method. Namely, hybridomas
are prepared by fusing antibody-producing cells contained in a
spleen, lymph node, bone marrow, or tonsil obtained from the
non-human mammal immunized as mentioned above, preferably a spleen,
with myelomas without autoantibody-producing ability, which are
derived from, preferably, a mammal such as mice, rats, guinea pigs,
hamsters, rabbits, or humans, or more preferably, mice, rats, or
humans.
[0199] For example, mouse-derived myeloma P3/X63-AG8.653 (653, ATCC
No. CRL1580), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1),
SP2/0-Ag14 (Sp2/0, Sp2), PAI, F0, or BW5147; rat-derived myeloma
210RCY3-Ag.2.3.; or human-derived myeloma U-266AR1 GM1500-6TG-A1-2,
UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used as a myeloma used
for the cell fusion.
[0200] Monoclonal antibody producing cells (e.g., hybridoma) can be
screened by cultivating the cells, for example, in microtiter
plates and by measuring the reactivity of the culture supernatant
in the well in which hybridoma growth is observed, to the immunogen
used for the immunization mentioned above, for example, by an
enzyme immunoassay such as RIA and ELISA.
[0201] The monoclonal antibodies can be produced from hybridomas by
cultivating the hybridomas in vitro or in vivo such as in the
ascites of mice, rats, guinea pigs, hamsters, or rabbits,
preferably mice or rats, more preferably mice and isolating the
antibodies from the resulting the culture supernatant or ascites
fluid of a mammal.
[0202] Furthermore, monoclonal antibodies can be obtained in a
large quantity by cloning a gene encoding a monoclonal antibody
from a hybridoma or "recombinant monoclonal antibody producing
cells" of the present invention described below, generating
transgenic animals such as bovine, goats, sheep, or pigs in which
the gene encoding the monoclonal antibody is integrated in its
endogenous gene using transgenic animal generating technique, and
recovering the monoclonal antibody derived from the antibody gene
from milk of the transgenic animals (Nikkei Science, No.4, pp.78-84
(1997)).
[0203] Cultivating the cells in vitro can be performed depending on
the property of cells to be cultured, on the object of a test
study, and on various culture, by using known nutrient media or any
nutrient media derived from known basal media for growing,
maintaining, and storing the hybridomas to produce monoclonal
antibodies in the culture supernatant.
[0204] Examples of basal media are low calcium concentration media
such as Ham'F12 medium, MCDB153 medium, or low calcium
concentration MEM medium, and high calcium concentration media such
as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium,
ASF104 medium, or RD medium. The basal media can contain, for
example, sera, hormones, cytokines, and/or various inorganic or
organic substances depending on the objective.
[0205] Monoclonal antibodies can be isolated and purified from the
culture supernatant or ascites mentioned above by saturated
ammonium sulfate precipitation, euglobulin precipitation method,
caproic acid method, caprylic acid method, ion exchange
chromatography (DEAE or DE52), affinity chromatography using
anti-immunoglobulin column or protein A column.
[0206] The monoclonal antibody of the present invention also
includes a monoclonal antibody comprising the heavy chain and/or
the light chain in which either or both of the chains have
deletions, substitutions or additions of one or several amino acids
in the sequences thereof; "several amino acids" as referred to here
means multiple amino acid residues, specifically means one to ten
amino acid residues, preferably one to five amino acid
residues.
[0207] The partial modification of amino acid sequence (deletion,
substitution, insertion, and addition) described above, can be
introduced into the monoclonal antibody of the present invention by
partially modifying the nucleotide sequence encoding the amino acid
sequence. The partial modification of the nucleotide sequence can
be performed by the usual method of site-specific mutagenesis
(Proc. Natl. Acad. Sci. USA, Vol. 81, p. 5662-5666, 1984).
[0208] "Human monoclonal antibody" as referred to in this invention
is a human monoclonal antibody reactive to the above-defined
mammalian CTGFs (preferably human CTGF). The human monoclonal
antibody is exemplified by the various human monoclonal antibodies
with a variety of properties described below in the examples and as
indicated in the drawings.
[0209] Specifically, the monoclonal antibody is a human
immunoglobulin which is encoded by the human immunoglobulin gene
segments in the entire region thereof including the variable region
of the heavy chain (H chain), the constant region of the H chain,
the variable region of the light chain (L chain) and the constant
region of the L chain. The L chain is exemplified by a human
.kappa. chain and a human .lambda. chain.
[0210] The human monoclonal antibody of the present invention can
be produced, for example, by immunizing, with the above-defined
mammalian CTGFs, "non-human transgenic mammals which are capable of
producing human antibodies" such as "transgenic mice which are
capable of producing human antibodies" which can be produced by
previously reported methods. By using the above-mentioned usual
methods, it is possible to immunize non-human mammals, to prepare
and screen hybridomas producing the antibodies, and to prepare the
human monoclonal antibody in large quantities (Nature Genetics,
Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997;
Published Japanese Translation of PCT International Publication No.
Hei 4-504365; Published Japanese Translation of PCT International
Publication No. Hei 7-509137; Nikkei Science, June edition, p.
40-50, 1995; WO94/25585; Nature, Vol. 368, p. 856-859, 1994;
Published Japanese Translation of PCT International Publication No.
Hei 6-500233, etc.).
[0211] The human antibody-producing transgenic mice can be
produced, specifically, for example, via the following processes;
other human antibody-producing non-human transgenic mammals can be
produced in the same manner.
[0212] (1) A process for preparing knockout mice in which
endogenous immunoglobulin heavy chain gene has been functionally
inactivated and the inactivation is done by substituting at least a
portion of the endogenous gene locus of the mouse immunoglobulin
heavy chain for a drug-resistance gene (the neomycin resistance
gene, etc.) through homologous recombination;
[0213] (2) A process for preparing knockout mice in which
endogenous gene of immunoglobulin light chain (a .kappa. chain gene
in particular) has been functionally inactivated and the
inactivation is done by substituting at least a portion of the
endogenous gene locus of the mouse immunoglobulin light chain for a
drug-resistance gene (the neomycin resistance gene, etc.) through
homologous recombination;
[0214] (3) A process for preparing transgenic mice in which a
desired portion of the human immunoglobulin heavy chain gene locus
has been integrated into a mouse chromosome, by using a vector,
such as yeast artificial chromosome (YAC) vector, capable of
transporting mega base genes;
[0215] (4) A process for preparing transgenic mice in which a
desired portion of the human immunoglobulin light chain (a .kappa.
gene in particular) gene locus has been integrated into a mouse
chromosome, by using a vector, such as YAC vector, capable of
transporting mega base genes;
[0216] (5) A process for preparing transgenic mice in which both
the mouse endogenous heavy chain and light chain gene loci have
been functionally inactivated and both desired portions of the
human immunoglobulin heavy chain and light chain genes loci have
been integrated in a chromosome, of which preparation is achieved
by crossbreeding, in arbitrary order, the knockout mice and the
transgenic mice described above in (1) to (4).
[0217] The knockout mice mentioned above can be prepared by
substituting any suitable region in the mouse endogenous
immunoglobulin gene locus for a foreign marker gene (neomycin
resistance gene, etc.) through homologous recombination so that the
immunoglobulin gene locus can be inactivated so as not to cause a
rearrangement of the gene locus.
[0218] For example, the method designated as positive-negative
selection (PNS) can be used for the inactivation with homologous
recombination (Nikkei Science, May edition, p. 52-62, 1994).
[0219] The functional inactivation of the immunoglobulin heavy
chain locus can be achieved, for example, by introducing a lesion
into a portion of the J region or a portion of the C region (the
C.mu. region, for example). The functional inactivation of the
immunoglobulin light chain locus can also be achieved, for example,
by introducing a lesion into a portion of the J region, a portion
of the C region, or a region extending from the J region to the C
region.
[0220] The transgenic mouse can be prepared according to the method
as usually used for producing a transgenic animal (for example, see
"Newest Manual of Animal Cell Experiment", LIC press, Chapter 7,
pp.361-408, (1990)). Specifically, for example, a transgenic mouse
can be produced as follows. Hypoxanthine-guanine phosphoribosyl
transferase (HPRT)-negative embryonic stem cells (ES cells)
obtained from a normal mouse blastocyst is fused with a yeast cell
containing an YAC vector, in which the gene encoding human
immunoglobulin heavy chain locus or light chain locus, or its
fragment and a HPRT gene have been inserted, by spheroplast fusion
method. ES cells in which the foreign gene has been integrated into
the mouse endogenous gene are screened by the HAT selection method.
Then, the ES cells screened are microinjected into a fertilized egg
(blastocyst) obtained from another normal mouse (Proc. Natl. Acad.
Sci. USA, Vol.77, No.12, pp.7380-7384 (1980); U.S. Pat. No.
4,873,191). The blastocyst is transplanted into the uterus of
another normal mouse as the foster mother. Then, chimeric
transgenic mice are born from the foster mother mouse. By mating
the chimeric transgenic mice with normal mice, heterozygous
transgenic mice are obtained. By mating the heterozygous transgenic
mice with each other, homozygous transgenic mice are obtained
according to Mendel's laws.
[0221] The "chimeric monoclonal antibody" of the present invention
is a monoclonal antibody prepared by genetic engineering, whose
variable region is non-human mammal (e.g. mice, rats, hamsters, and
so forth) immunoglobulin-derived variable region and whose constant
region is human immunoglobulin-derived constant region and is
exemplified by mouse/human chimeric antibody.
[0222] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG (IgG1,
IgG2,IgG3 and IgG4), IgM, IgA, IgD, and IgE. The constant region of
the recombinant chimeric monoclonal antibody of the present
invention can be that of human immunoglobulin belonging to any
isotype. Preferably, it is the constant region of human IgG.
[0223] The chimeric monoclonal antibody of the present invention
can be produced, for example, as follows. Needless to say, the
production method is not limited thereto.
[0224] For example, mouse/human chimeric monoclonal antibody can be
prepared, by referring to Experimental Medicine: SUPPLEMENT,
Vol.1.6, No.10 (1988); and Examined Published Japanese Patent
Application (JP-B), No. Hei 3-73280. Namely, it can be prepared by
ligating C.sub.H gene (C gene encoding the constant region of H
chain) obtained from the DNA encoding human immunoglobulin to the
downstream of active V.sub.H genes (rearranged VDJ gene encoding
the variable region of H chain) obtained from the DNA encoding
mouse monoclonal antibody isolated from the hybridoma producing the
mouse monoclonal antibody, and by ligating the C.sub.L gene (C gene
encoding the constant region of L chain) obtained from the DNA
encoding human immunoglobulin to the downstream of active V.sub.L
genes (rearranged VJ gene encoding the variable region of L chain)
obtained from the DNA encoding the mouse monoclonal antibody
isolated from the hybridoma, and operably inserting those into the
same or different vectors in an expressible manner, followed by
transformation of host cells with the expression vector, and
cultivation of the transformants.
[0225] Specifically, DNAs are first extracted from mouse monoclonal
antibody-producing hybridoma by the usual method, digested with
appropriate restriction enzymes (for example, EcoRI and HindIII),
electrophoresed (using, for example, 0.7% agarose gel), and
analyzed by Southern blotting. After the electrophoresed gel is
stained, for example, with ethidium bromide and photographed, the
gel is given marker positions, washed twice with water, and soaked
in 0.25 M HCl for 15 minutes. Then, the gel is soaked in 0.4 N NaOH
solution for 10 minutes with gentle stirring. The DNAs are
transferred to a filter for 4 hours following the usual method. The
filter is recovered and washed twice with 2.times.SSC. After the
filter is sufficiently dried, it is baked at 75.degree. C. for 3
hours, treated with 0.1.times.SSC/0.1% SDS at 65.degree. C. for 30
minutes, and then soaked in 3.times.SSC/0.1% SDS. The filter
obtained is treated with prehybridization solution in a plastic bag
at 65.degree. C. for 3 to 4 hours.
[0226] Next, .sup.32P-labeled probe DNA and hybridization solution
are added to the bag and reacted at 65.degree. C. about 12 hours.
After hybridization, the filter is washed under an appropriate salt
concentration, reaction temperature, and time (for example,
2.times.SSC-0.1% SDS, room temperature, 10 minutes). The filter is
put into a plastic bag with a little 2.times.SSC, and subjected to
autoradiography after the bag is sealed.
[0227] Rearranged VDJ gene and VJ gene encoding H chain and L chain
of mouse monoclonal antibody respectively are identified by
Southern blotting mentioned above. The region comprising the
identified DNA fragment is fractionated by sucrose density gradient
centrifugation and inserted into a phage vector (for example,
Charon 4A, Charon 28, .lambda.EMBL3, .lambda.EMBL4, etc.). E. coli
(for example, LE392, NM539, etc.) are transformed with the phage
vector to generate a genomic library. The genomic library is
screened by plaque hybridization such as the Benton-Davis method
(Science, Vol.196, pp.180-182 (1977)) using appropriate probes (H
chain j gene, L chain (.kappa.) J gene, etc.) to obtain positive
clones comprising rearranged VDJ gene or VJ gene respectively. By
making the restriction map and determining the nucleotide sequence
of the clones obtained, it is confirmed that genes comprising the
desired, rearranged V.sub.H (VDJ) gene or V.sub.L (VJ) gene have
been obtained.
[0228] Separately, human C.sub.H gene and human C.sub.L gene used
for chimerization are isolated. For example, when a chimeric
antibody with human IgG1 is produced, C.gamma..sub.1 gene is
isolated as a C.sub.H gene, and C.kappa. gene is also isolated as a
C.sub.L gene, are isolated. These genes can be isolated from human
genomic library with mouse C.gamma..sub.1 gene and mouse C.kappa.
gene, corresponding to human C.gamma..sub.1 gene and human C.kappa.
gene, respectively, as probes, taking advantage of the high
homology between the nucleotide sequences of mouse immunoglobulin
gene and that of human immunoglobulin gene.
[0229] Specifically, DNA fragments comprising human C.kappa. gene
and an enhancer region are isolated from human .lambda. Charon 4A
HaeIII-AluI genomic library (Cell, Vol.15, pp.1157-1174 (1978)),
for example, using a 3 kb HindIII-BamHI fragment from clone Ig146
(Proc. Natl. Acad. Sci. USA, Vol.75, pp.4709-4713 (1978)) and a 6.8
kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA,
Vol.78, pp.474-478 (1981)) as probes. In addition, for example,
after human fetal hepatocyte DNA is digested with HindIII and
fractioned by agarose gel electrophoresis, a 5.9 kb fragment is
inserted into .lambda.788 and then human C.gamma..sub.1 gene is
isolated with the probes mentioned above.
[0230] Using mouse V.sub.H gene, mouse V.sub.L gene, human C.sub.H
gene, and human C.sub.L gene so obtained, and taking promoter
region and enhancer region into consideration, human C.sub.H gene
is inserted downstream of mouse V.sub.H gene and human C.sub.L gene
is inserted downstream of mouse V.sub.L gene in an expression
vector such as pSV2gpt or pSV2neo with appropriate restriction
enzymes and DNA ligase following the usual method. In this case,
chimeric genes of mouse V.sub.H gene/human C.sub.H gene and mouse
V.sub.L gene/human C.sub.L gene can be respectively inserted into a
same or different expression vector.
[0231] Chimeric gene-inserted expression vector(s) thus prepared
are introduced into myelomas (e.g., P3X63.Ag8.653 cells or SP210
cells) that do not produce antibodies by the protoplast fusion
method, DEAE-dextran method, calcium phosphate method, or
electroporation method. The transformants are screened by
cultivating in a medium containing a drug corresponding to the drug
resistance gene inserted into the expression vector and, then,
cells producing desired chimeric monoclonal antibodies are
obtained.
[0232] Desired chimeric monoclonal antibodies are obtained from the
culture supernatant of antibody-producing cells thus screened.
[0233] The "humanized monolonal antibody (CDR-grafted antibody)" of
the present invention is a monoclonal antibody prepared by genetic
engineering and specifically means a humanized monoclonal antibody
wherein a portion or the whole of the complementarity determining
regions of the hyper-variable region are derived from the those of
the hyper-variable region from non-human mammal (mouse, rat,
hamster, etc.) monoclonal antibody, the framework regions of the
variable region are derived from those of the variable region from
human immunoglobulin, and the constant region is derived from that
from human-immunoglobulin.
[0234] The complementarity determining regions of the
hyper-variable region exists in the hyper-variable region in the
variable region of an antibody and means three regions which
directly binds, in a complementary manner, to an antigen
(complementarity-determining residues, CDR1, CDR2, and CDR3). The
framework regions of the variable region mean four comparatively
conserved regions intervening upstream, downstream or between the
three complementarity-determining regions (framework region, FR1,
FR2, FR3, and FR4).
[0235] In other words, a humanized monoclonal antibody means that
in which the whole region except a portion, or the whole region, of
the complementarity determining regions of the hyper-variable
region of a nonhuman mammal-derived monoclonal antibody have been
replaced with their corresponding regions derived from human
immunoglobulin.
[0236] The constant region derived from human immunoglobulin has
the amino acid sequence inherent in each isotype such as IgG (IgG1,
IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The constant region of a
humanized monoclonal antibody in the present invention can be that
from human immunoglobulin belonging to any isotype. Preferably, it
is the constant region of human IgG. The framework regions of the
constant region derived from human immunoglobulin are not
particularly limited.
[0237] The humanized monoclonal antibody of the present invention
can be produced, for example, as follows. Needless to say, the
production method is not limited thereto.
[0238] For example, a recombinant humanized monoclonal antibody
derived from mouse monoclonal antibody can be prepared by genetic
engineering, referring to Published Japanese Translations of PCT
International Publication No. Hei 4-506458 and Unexamined Published
Japanese Patent Application (JP-A) No. Sho 62-296890. Namely, at
least one mouse H chain CDR gene and at least one mouse L chain CDR
gene corresponding to the mouse H chain CDR gene are isolated from
hybridomas producing mouse monoclonal antibody, and human H chain
gene encoding the whole region except human H chain CDR
corresponding to mouse H chain CDR mentioned above and human L
chain gene encoding the whole region except human L chain CDR
corresponding to mouse L chain CDR mentioned above are isolated
from human immunoglobulin genes.
[0239] The mouse H chain CDR gene(s) and the human H chain gene(s)
so isolated are inserted, in an expressible manner, into an
appropriate vector so that they can be expressed. Similarly, the
mouse L chain CDR gene(s) and the human L chain gene(s) are
inserted, in an expressible manner, into another appropriate vector
so that they can be expressed. Alternatively, the mouse H chain CDR
gene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human L
chain gene(s) can be inserted, in an expressible manner, into the
same expression vector so that they can be expressed. Host cells
are transformed with the expression vector thus prepared to obtain
transformants producing humanized monoclonal antibody. By
cultivating the transformants, desired humanized monoclonal
antibody is obtained from culture supernatant.
[0240] The "monoclonal antibody" of the invention includes "a
portion" of the monoclonal antibody as well. The "portion of an
antibody" used in the present invention means a partial region of
the antibody, preferably monoclonal antibody of the present
invention as mentioned above, and specifically, means F(ab').sub.2,
Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulfide
stabilized Fv), or dAb (single domain antibody) (Exp. Opin. Ther.
Patents, Vol.6, No.5, pp.441-456 (1996)).
[0241] "F(ab').sub.2" and "Fab'" can be produced by treating
immunoglobulin (monoclonal antibody) with a protease such as pepsin
and papain, and means an antibody fragment generated by digesting
immunoglobulin near the disulfide bonds existing between the hinge
regions in each of the two H chains. For example, papain cleaves
IgG upstream of the disulfide bonds existing between the hinge
regions in each of the two H chains to generate two homologous
antibody fragments in which an L chain composed of V.sub.L (L chain
variable region) and C.sub.L (L chain constant region), and an H
chain fragment composed of V.sub.H (H chain variable region) and
C.sub.H.gamma.1 (.gamma.1 region in the constant region of H chain)
are connected at their C terminal regions through a disulfide bond.
Each of these two homologous antibody fragments is called Fab'.
Pepsin also cleaves IgG downstream of the disulfide bonds existing
between the hinge regions in each of the two H chains to generate
an antibody fragment slightly larger than the fragment in which the
two above-mentioned Fab' are connected at the hinge region. This
antibody fragment is called F(ab').sub.2.
[0242] The "monoclonal antibody producing cells" or "recombinant
monoclonal antibody producing cells" of this invention mean any
cells producing the above-described monoclonal antibody of this
invention. Specific examples include the cells described in (1) to
(3) below.
[0243] (1) monoclonal antibody-producing B cells that are
obtainable from the above-described non-human mammal or human
antibody producing transgenic mouse (or other transgenic non-human
mammals) that produces a monoclonal antibody reactive with CTGF,
which animal can be produced by immunizing the animal with the
above-defined mammalian CTGF (preferably human CTGF) or a portion
thereof or cells secreting the CTGF, etc.;
[0244] (2) the above-described hybridomas prepared by fusing
antibody producing B cells obtained described above with myelomas
derived from mammals; and
[0245] (3) monoclonal antibody producing transformants (recombinant
cells) obtained by transforming other cells than the monoclonal
antibody producing B cells and hybridomas (e.g. Chinese hamster
ovarian (CHO) cells, Baby hamster kidney (BHK) cells, etc.) with
genes (either the heavy chain-encoding gene or the light chain
encoding gene, or both) encoding the monoclonal antibody isolated
from the monoclonal antibody producing B cells or hybridomas.
[0246] The monoclonal antibody producing transformants (recombinant
cells) of (3) mean recombinant cells producing a recombinant
product of the monoclonal antibody produced by B cells of (1) or
hybridomas of (2). These antibody producing transformants can be
produced using known recombinant technology as used for the
above-described chimeric monoclonal antibody and humanized
monoclonal antibody.
[0247] The term "monoclonal antibody comprising a property
substantially equivalent to" as referred to in the present
invention indicates that, when biological properties of two
monoclonal antibodies are compared with each other, one monoclonal
antibody is not significantly different from the other in at least
the following biological properties:
[0248] (1) the reactivity to CTGF derived from a particular animal,
which is used as an immunogen for immunizing a non-human mammal to
prepare the monoclonal antibody;
[0249] (2) the reactivity to any CTGF derived from animals other
than the particular animal (namely, crossreactivity);
[0250] (3) the properties measured by a variety of experiments
described below in the examples.
[0251] The term "mammalian antiserum" as referred to in the present
invention indicates a serum containing antibody reactive to the
monoclonal antibody of the present invention or a portion thereof.
The antiserum can be produced, according to the above-described
method described in the production of monoclonal antibody, by
immunizing mammals such as mice, rats, guinea pigs, rabbits, goats,
pigs or bovine, preferably rats, guinea pigs, rabbits or goats,
with the above-mentioned monoclonal antibody or a portion thereof
as an immunogen.
[0252] The term "insoluble carrier" as referred to in the present
invention indicates a supporting material thereon used for
immobilizing the monoclonal antibody or a portion thereof (antibody
fragment) of the present invention, or CTGF in samples (for
example, body fluids such as plasma, culture supernatant,
supernatant fluids obtained by centrifugation, etc.) by physical
adsorption or chemical bonding.
[0253] The insoluble carrier is exemplified below in (A) and
(B):
[0254] (A) plates, containers having internal spaces such as test
tubes or tubes, beads (microbeads in particular), balls, filters or
membranes, made of water-insoluble materials, for example, glass or
plastics such as polystyrene resin, polycarbonate resin, silicone
resin or nylon resin;
[0255] (B) insoluble carriers, used for affinity chromatography,
such as cellulose carriers, agarose carriers, polyacrylamide
carriers, dextran carriers, polystyrene carriers, polyvinyl alcohol
carriers, poly(amino acid) carriers or porous silica carriers.
[0256] The term "antibody-immobilized insoluble carrier" as
referred to in the present invention indicates the above-defined
insoluble carrier on which the monoclonal antibody (or a portion of
the antibody, namely an antibody fragment) of this invention is
immobilized by physical adsorption or chemical bonding. These
insoluble carriers with immobilized antibodies are usable for the
detection, assay, separation or purification of CTGF in samples
(for example, body fluids such as serum and plasma; culture
supernatant; the supernatant fluids obtained by centrifugation,
etc.).
[0257] The insoluble carriers shown above in (A) can be used for
the detection and the assay; from the standpoint of the simplicity
of operation and the simultaneous processing of many samples, in
particular, the multi-well microtiter plates, which are made of
plastics and have many wells, such as 96-well microtiter plates or
48-well microtiter plates, are used preferably as the insoluble
carrier in the assay for assaying CTGF. The filters or membranes
shown above in (A), or the insoluble carriers shown above in (B),
are usable for the separation or the purification.
[0258] A "labeling agent capable of providing a detectable signal
through the reaction with the labeling agent alone or together with
other substances" as referred to in this invention means a
substance used for converting the monoclonal antibody or a portion
thereof (antibody fragment) described above, or a CTGF standard
into detectable forms; the conversion can be performed by the
physical binding or chemical bonding between the labeling agent and
the monoclonal antibody or a portion thereof, or between the
labeling agent and the CTGF standard material.
[0259] Specifically, the labeling agent includes enzymes,
fluorescent materials, chemiluminescent materials, biotin, avidin
or radioisotopes, etc., more specifically, enzymes such as
peroxidase (for example, horseradish peroxidase), alkaline
phosphatase, .beta.-D-galactosidase, glucose oxidase,
glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate
dehydrogenase, penicillinase, catalase, apo-glucose oxidase,
urease, luciferase or acetylcholinesterase; fluorescent materials
such as fluorescein isothiocyanate, phycobiliprotein, chelating
compounds of rare-earth metals, dansyl chloride or
tetramethylrhodamine isothiocyanate; radioisotopes such as .sup.3H,
.sup.14C, .sup.125I or .sup.131I; biotin; avidin; or
chemiluminescent materials.
[0260] Radioisotopes and fluorescent materials, even when used
alone, give a detectable signal. On the other hand, enzymes,
chemiluminescent materials, biotin, and avidin give no detectable
signals, when used alone. In these cases, one or more substances
are needed with the substances in order to give a detectable
signal. For example, when the substance is an enzyme, at least a
substrate for the enzyme is necessary to give a detectable signal.
Various types of substrates are selectable depending on the methods
for measuring the enzyme activity (colorimetry, immunofluorescence
method, bioluminescence method or chemiluminescence method, etc.).
For example, hydrogen peroxide is used as a substrate for
peroxidase. When biotin is selected, avidin or enzyme-conjugated
avidin is used for the reaction with biotin generally but not
always. According to needs, various coloring agents are further
used for the reaction depending on the type of the substrate.
[0261] The terminologies, "labeled antibody" and "labeled mammalian
CTGF standard" as referred to in the present invention indicate,
respectively, monoclonal antibody (or antibody fragment) and CTGF
labeled with the above-mentioned various labeling agents. The
labeled antibody and labeled standard can be used to detect, assay,
separate or purify CTGFs in samples (for example, body fluid
samples such as serum and plasma; culture supernatants; or the
supernatant fluids obtained by centrifugation, etc.). In the
present invention, any of the above-mentioned labeling agents are
usable. However, biotin or enzymes such as peroxidase are used
favorably for the labeling from the standpoint of the high
detection sensitivity or high assay sensitivity and the simplicity
of operation.
[0262] Being different from a CTGF of an unknown concentration
(amount) in a sample, the "CTGF standard" is a CTGF isolated
previously and the standard adjustable to any desired concentration
thereof to suit the purpose of each assay. For example, the
standard substance can be used for the preparation of calibration
curves.
[0263] The term "immunoassay" as referred to in the present
invention means the method of detecting or assaying the antigens in
samples (for example, body fluid samples such as plasma; culture
supernatants; or the supernatant fluids obtained by centrifugation)
based on the principle of antigen-antibody reaction. In the present
invention, for the immunoassay, one or more monoclonal antibodies
(or antibody fragment(s)) to be used as the antibody in the
antigen-antibody reaction are selected from the above-mentioned
monoclonal antibodies (or antibody fragment) reactive to the
mammalian CTGF of the present invention, the above-mentioned
antibody-immobilized insoluble carrier (or antibody
fragment-immobilized insoluble carrier) and the above-mentioned
labeled antibody (or labeled antibody fragment) as well as the
antigen is the mammalian CTGF, but otherwise previously known
immunoassay methods are applicable in the assay.
[0264] Specifically, the immunoassay is exemplified by single
antibody solid phase method, two-antibodies liquid phase method,
two-antibodies solid phase method, sandwich method, enzyme
multiplied immunoassay technique (EMIT method), enzyme channeling
immunoassay, enzyme modulator mediated enzyme immunoassay (EMMIA),
enzyme inhibitor immunoassay, immuno enzymometric assay, enzyme
enhanced immunoassay or proximal linkage immunoassay all of which
are described in "Enzyme Immunoassay (3rd Ed., eds., Eiji Ishikawa
et al., Igakushoin, 1987); or the one-pot method which is described
in JP-B Hei 2-39747.
[0265] In this invention, any of these immunoassays can be selected
appropriately to suit each assay purpose. However, from the
standpoint of the simplicity of operation and/or the economical
advantage, and especially when considering the clinical
applicability, the sandwich method, the one pot method, the single
antibody solid phase method and the two-antibodies solid phase
method are preferably used in this invention; more preferable are
the sandwich method and the one pot method. Particularly preferable
is the sandwich method using a labeled antibody prepared by
labeling the monoclonal antibody of the present invention with an
enzyme or biotin as well as using an antibody-immobilized insoluble
carrier prepared by immobilizing the monoclonal antibody on a
multi-well microplate having many wells thereon, such as a 96-well
microplate; another particularly preferable method is the one-pot
method using a labeled antibody prepared by labeling the monoclonal
antibody of the present invention with an enzyme or biotin as well
as using an antibody-immobilized insoluble carrier prepared by
immobilizing the monoclonal antibody on beads, such as microbeads,
or small balls.
[0266] A specific example of a particularly preferable embodiment
is the sandwich method or the one-pot method using a labeled
antibody prepared by labeling the monoclonal antibody "8-86-2," as
indicated in FIG. 1, with an enzyme or biotin, as well as using an
antibody-immobilized insoluble carrier prepared by immobilizing the
monoclonal antibody "8-64-6" or "13-51-2," as indicated in FIG. 1,
on the microplate or the microbeads.
[0267] Human and mouse CTGFs can be detected or quantified in high
sensitivity by immunoassays using the monoclonal antibody
"8-64-6"-immobilized insoluble carrier, in combination with the
monoclonal antibody "8-86-2" labeled with an enzyme or biotin. Rat
CTGF (first disclosed in the present application) and mouse CTGF
can be detected or assayed in high sensitivity by the immunoassay
using the monoclonal antibody "13-51-2"-immobilized insoluble
carrier, in combination with the monoclonal antibody "8-86-2"
labeled with an enzyme or biotin.
[0268] The sandwich method, the one-pot method, the single antibody
solid phase method, and the two-antibodies liquid phase method are
described in detail herein below.
[0269] The sandwich method corresponds to the method described
above in (50) of the present invention, and specifically, is an
immunoassay that comprises at least the following steps (a) and
(b):
[0270] (a) reacting a sample with the antibody-immobilized
insoluble carrier of the present invention; and
[0271] (b) reacting a labeled antibody of the present invention
with the antigen-antibody complex formed by the binding between the
antibody-immobilized insoluble carrier and mammalian CTGF in the
sample.
[0272] According to the present invention, a specific example of
the method of assaying human or mouse CTGF is indicated below, in
which the "antibody-immobilized insoluble carrier" is an
"antibody-immobilized microplate" prepared by immobilizing the
monoclonal antibody "8-64-6" as indicated in FIG. 1 on a
microplate, and the "labeled antibody" is the monoclonal antibody
"8-86-2", as indicated in FIG. 1, labeled with biotin or an enzyme
such as peroxidase; the method comprises, for example, the steps
described below, but the method is not to be construed as being
restricted thereto.
[0273] Not only mouse CTGF but also rat CTGF (first disclosed in
the present application) can be assayed by the same procedures as
indicated below, when the "antibody-immobilized insoluble carrier"
is an "antibody-immobilized microplate" prepared by immobilizing
the monoclonal antibody "13-51-2" as indicated in FIG. 1 on a
microplate and the "labeled antibody" is the monoclonal antibody
"8-86-2", as indicated in FIG. 1, labeled with biotin or an enzyme
such as peroxidase.
[0274] (Step 1) preparing an antibody-immobilized microplate by
immobilizing the monoclonal antibody "8-64-6" of the present
invention on a microplate;
[0275] (Step 2) reacting a sample such as a human or mouse serum
with the monoclonal antibody immobilized on the
antibody-immobilized microplate by adding the sample to the
microplate;
[0276] (Step 3) washing the microplate to remove the unreacted
sample from the microplate;
[0277] (Step 4) preparing a labeled antibody by labeling the
monoclonal antibody "8-86-2" of the present invention with biotin
or an enzyme such as peroxidase;
[0278] (Step 5) reacting the labeled antibody with the
antigen-antibody complex formed through the reaction between human
or mouse CTGF in the sample and the monoclonal antibody immobilized
on the microplate, by adding the labeled antibody to the microplate
washed in Step 3;
[0279] (Step 6) washing out the unreacted labeled antibody from the
microplate;
[0280] (Step 7) reacting the labeling agent moiety of the labeled
antibody with a substrate selected depending on the type of the
enzyme used (when the labeled antibody used in Step 5 is labeled
with an enzyme such as peroxidase), avidin or enzyme-conjugated
avidin avidin (when the labeled antibody used in Step 5 is labeled
with biotin), by adding, if necessary together with a coloring
agent, the substrate, or avidin or enzyme-conjugated avidin to the
microplate;
[0281] (Step 8) reacting a substrate for the enzyme selected
depending on the type of the enzyme conjugated with avidin, with
the enzyme conjugated with avidin, by adding the substrate, when
enzyme-conjugated avidin is used in Step 7;
[0282] (Step 9) stopping the enzyme reaction and the coloring
reaction by adding a reaction stop solution into the reaction
mixture of step 7 or 8; and
[0283] (Step 10) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0284] The one-pot method corresponds to each of the methods
described above in (50), (51) and (52) of the present
invention.
[0285] Specifically, the first is the immunoassay method,
comprising at least the following steps (a) and (b);
[0286] (a) reacting a sample with an antibody-immobilized insoluble
carrier of the present invention; and
[0287] (b) reacting a labeled antibody of the present invention
with the antigen-antibody complex formed by the binding between the
antibody-immobilized insoluble carrier and mammalian CTGF in the
sample.
[0288] The second is the immunoassay method comprising at least the
following steps (a) and (b);
[0289] (a) reacting a sample with a labeled antibody of the present
invention; and
[0290] (b) reacting an antibody-immobilized insoluble carrier of
the present invention with the antigen-antibody complex formed by
the binding between the labeled antibody and mammalian CTGF in the
sample.
[0291] The third is the immunoassay method comprising at least the
following step (a);
[0292] (a) reacting a mixture of an antibody-immobilized insoluble
carrier of the present invention, a labeled antibody of the present
invention and a sample.
[0293] According to the present invention, a specific example of
the method of assaying human or mouse CTGF is indicated below, in
which the "antibody-immobilized insoluble carrier" is
"antibody-immobilized microbeads" prepared by immobilizing the
monoclonal antibody "8-64-6" as indicated in FIG. 1 on microbeads,
and the "labeled antibody" is the monoclonal antibody "8-86-2", as
indicated in FIG. 1, labeled with biotin or an enzyme such as
peroxidase; the method comprises, for example, the steps described
below, but the method is not to be construed as being restricted
thereto.
[0294] Not only mouse CTGF but also rat CTGF (first disclosed in
the application) can be assayed by the same procedures as indicated
below, when the "antibody-immobilized insoluble carrier" is
"antibody-immobilized microbeads" prepared by immobilizing the
monoclonal antibody "13-51-2" as indicated in FIG. 1 on microbeads
and the "labeled antibody" is the monoclonal antibody "8-86-2", as
indicated in FIG. 1, labeled with biotin or an enzyme such as
peroxidase.
[0295] The first method comprises the following steps:
[0296] (Step 1) preparing antibody-immobilized microbeads by
immobilizing the monoclonal antibody "8-64-6" of the present
invention on microbeads;
[0297] (Step 2) reacting a sample such as a human or mouse serum
with the monoclonal antibody immobilized on the microbeads by
adding the sample and the antibody-immobilized microbeads together
with a buffer solution into a container having internal spaces such
as a test tube, microplate, or tube;
[0298] (Step 3) washing the beads to remove the liquid content from
the container;
[0299] (Step 4) preparing a labeled antibody by labeling the
monoclonal antibody "8-86-2" with biotin or an enzyme such as
peroxidase;
[0300] (Step 5) reacting the labeled antibody with the
antigen-antibody complex formed through the reaction between the
human or mouse CTGF in the sample and the monoclonal antibody
immobilized on the beads by adding the labeled antibody into the
container containing the beads washed in Step 3;
[0301] (Step 6) removing the unreacted labeled antibody by removing
the liquid content from the container and washing the beads;
[0302] (Step 7) reacting the labeling agent moiety of the labeled
antibody with a substrate selected depending on the type of the
enzyme used (when the labeled antibody used in Step 5 is labeled
with an enzyme such as peroxidase), avidin or the enzyme-conjugated
avidin (when the labeled antibody used in Step 5 is labeled with
biotin) by adding, if necessary together with a coloring agent, the
substrate, or avidin or enzyme-conjugated avidin into the container
containing the beads washed in Step 6;
[0303] (Step 8) reacting a substrate for the enzyme selected
depending on the type of the enzyme conjugated with avidin, with
the enzyme conjugated with avidin by adding the substrate, when
enzyme-conjugated avidin is used in Step 7;
[0304] (Step 9) stopping the enzyme reaction and the coloring
reaction by adding a reaction stop solution into the reaction
mixture of Step 7 or Step 8; and
[0305] (Step 10) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0306] The second method comprises the following steps:
[0307] (Step 1) preparing a labeled antibody by labeling the
monoclonal antibody "8-86-2" of the present invention with biotin
or an enzyme such as peroxidase;
[0308] (Step 2) reacting a sample such as a human or mouse serum
with the labeled antibody by adding the sample and the labeled
antibody together with a buffer solution into a container having
internal spaces such as a test tube, microplate or tube;
[0309] (Step 3) preparing antibody-immobilized microbeads by
immobilizing the monoclonal antibody "8-64-6" of the present
invention on microbeads;
[0310] (Step 4) reacting the monoclonal antibody immobilized on the
beads with the antigen-antibody complex formed through the reaction
between the labeled antibody and human CTGF or mouse CTGF in the
sample by adding the beads into the reaction system in Step 3;
[0311] (Step 5) removing the unreacted labeled antibody by removing
the liquid content from the container and washing the beads;
[0312] (Step 6) reacting the labeling agent moiety of the labeled
antibody with a substrate selected depending on the type of the
enzyme used (when the labeled antibody used in Step 2 is labeled
with an enzyme such as peroxidase), avidin or the enzyme-conjugated
avidin (when the labeled antibody used in Step 2 is labeled with
biotin) by adding, if necessary together with a coloring agent, the
substrate, or avidin or enzyme-conjugated avidin into the container
containing the beads washed in Step 5;
[0313] (Step 7) reacting a substrate selected depending on the type
of the enzyme conjugated with avidin, with the enzyme conjugated
with avidin by adding the substrate, when enzyme-conjugated avidin
is used in Step 6;
[0314] (Step 8) stopping the enzyme reaction and the coloring
reaction by adding a stop solution to the reaction system in Step 6
or 7; and
[0315] (Step 9) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0316] The third method comprises the following steps:
[0317] (Step 1) preparing antibody-immobilized microbeads by
immobilizing the monoclonal antibody "8-64-6" of the present
invention on the microbeads;
[0318] (Step 2) preparing a labeled antibody by labeling the
monoclonal antibody "8-86-2" of the present invention with biotin
or an enzyme such as peroxidase;
[0319] (Step 3) reacting the labeled antibody and a sample such as
a human or mouse serum simultaneously with the monoclonal antibody
immobilized on microbeads by adding the sample and the
antibody-immobilized microbeads prepared in Step 1 and the labeled
antibody prepared in Step 2 together with a buffer solution into a
container having internal spaces such as a test tube, plate, or
tube.
[0320] (Step 4) removing the unreacted labeled antibody by removing
the liquid content from the container and washing the beads;
[0321] (Step 5) reacting the labeling agent moiety of the labeled
antibody with a substrate selected depending on the type of the
enzyme used (when the labeled antibody used in Step 3 is labeled
with an enzyme such as peroxidase), avidin or the enzyme-conjugated
avidin (when the labeled antibody used in Step 3 is labeled with
biotin) by adding, if necessary together with a coloring agent, the
substrate, or avidin or enzyme-conjugated avidin into the container
containing the beads washed in Step 4;
[0322] (Step 6) reacting a substrate selected depending on the type
of the enzyme conjugated with avidin, with the enzyme conjugated
with avidin by adding the substrate, when enzyme-conjugated avidin
is used in Step 5;
[0323] (Step 7) stopping the enzyme reaction and the coloring
reaction by adding a stop solution to the reaction system in Step 5
or 6; and
[0324] (Step 8) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0325] The single antibody solid phase method corresponds to the
method described above in (53) of the present invention, and
specifically, the immunoassay method that comprises at least the
following step (a):
[0326] (a) reacting a sample and mammalian CTGF standard labeled
with a labeling agent capable of providing a detectable signal by
itself or by reacting with other substances, with an
antibody-immobilized insoluble carrier of the present
invention.
[0327] A specific example of the method of assaying human or mouse
CTGF according to the present invention is indicated below, in
which the "antibody-immobilized insoluble carrier" is an
"antibody-immobilized microplate" prepared by immobilizing the
monoclonal antibody "8-64-6" as indicated in FIG. 1 on a microplate
and widely used biotin or enzyme such as peroxidase is used here as
a "labeling agent"; the method comprises, for example, the steps
described below, but the method is not to be construed as being
restricted thereto.
[0328] Not only mouse CTGF but also rat CTGF (first disclosed in
the application) can be assayed by the same procedures as indicated
below, when the "antibody-immobilized insoluble carrier" is an
"antibody-immobilized microplate" prepared by immobilizing the
monoclonal antibody "13-51-2" as indicated in FIG. 1 on a
microplate and widely used biotin or an enzyme such as peroxidase
is use as the "labeling agent."
[0329] (Step 1) preparing an antibody-immobilized microplate by
immobilizing the monoclonal antibody "8-64-6" on a microplate;
[0330] (Step 2) preparing a labeled CTGF standard by labeling the
standard with biotin or an enzyme such as peroxidase;
[0331] (Step 3) reacting a sample such as a human or mouse serum
and the labeled CTGF standard competitively with the monoclonal
antibody immobilized on the microplate by adding the sample and the
labeled standard to the microplate;
[0332] (Step 4) washing out the unreacted labeled standard from the
microplate;
[0333] (Step 5) reacting the labeling agent moiety of the labeled
standard with a substrate selected from depending on the type of
the enzyme used (when the labeled standard used in Step 3 is
labeled with an enzyme such as peroxidase), avidin or
enzyme-conjugated avidin (when the labeled standard in Step 3 is
labeled with biotin) by adding, if necessary together with a
coloring agent, the substrate, or avidin or enzyme-conjugated
avidin to the microplate washed in Step 4;
[0334] (Step 6) reacting a substrate selected depending on the type
of the enzyme conjugated with avidin, with the enzyme conjugated
with avidin by adding the substrate, when enzyme-conjugated avidin
is used in Step 5;
[0335] (Step 7) stopping the enzyme reaction and the coloring
reaction by adding a stop solution to the microplate; and
[0336] (Step 8) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0337] The two antibodies solid phase method corresponds to the
methods described above in (54) and (55) of the present
invention.
[0338] Specifically, the first is the immunoassay method comprising
at least the following steps (a) and (b):
[0339] (a) reacting the monoclonal antibody of the present
invention with a mixture comprising a sample and a mammalian CTGF
standard labeled with a labeling agent capable of providing a
detectable signal by itself or by reacting with other substances;
and,
[0340] (b) reacting a mammalian antiserum reactive to the
monoclonal antibody with the antigen-antibody complex formed
through binding of the monoclonal antibody and the mammalian CTGF
in the sample or the labeled mammalian CTGF standard.
[0341] The second is the immunoassay method comprising at least the
following steps (a) to (c):
[0342] (a) reacting a monoclonal antibody of the present invention
with a sample;
[0343] (b) reacting a mammalian CTGF standard labeled with a
labeling agent capable of providing a detectable signal by itself
or by reacting other substances, with the reaction mixture in Step
(a); and,
[0344] (c) reacting a mammalian antiserum reactive to the
monoclonal antibody with the antigen-antibody complex formed
through the binding of the monoclonal antibody and the mammalian
CTGF in the sample or the labeled mammalian CTGF standard.
[0345] A specific example of the method of assaying human or mouse
CTGF according to the present invention is indicated below, in
which the "monoclonal antibody" is the monoclonal antibody "8-64-6"
or the monoclonal antibody "8-86-2" as indicated in FIG. 1 and
widely used biotin or an enzyme such as peroxidase is used as the
"labeling agent"; the method comprises, for example, the steps
described below, but the method is not to be construed as being
restricted thereto.
[0346] Not only mouse CTGF but also rat CTGF (first disclosed in
the application) can be assayed by the same procedures as indicated
below, when the "monoclonal antibody" is the monoclonal antibody
"13-51-2" as indicated in FIG. 1 and widely used biotin or an
enzyme such as peroxidase is used as the "labeling agent."
[0347] The first method comprises the following steps:
[0348] (Step 1) preparing the labeled CTGF standard by labeling a
human or mouse CTGF standard with biotin or an enzyme such as
peroxidase;
[0349] (Step 2) reacting a sample such as a human or mouse serum
and the labeled CTGF standard prepared in Step 1 competitively with
the monoclonal antibody "8-64-6" or "8-86-2" of the present
invention by adding a mixture comprising the sample and the labeled
CTGF standard into a container having internal spaces such as a
test tube, plate or tube and by subsequently adding thereto the
monoclonal antibody;
[0350] (Step 3) reacting an antiserum, derived from mammals except
mice, reactive to the mouse monoclonal antibody, such as a goat
anti-mouse .gamma.-globulin antiserum, with the antigen-antibody
complex, formed in Step 2, consisting of the monoclonal antibody
and the mammalian CTGF in the sample or the labeled mammalian CTGF
standard, to give the resulting precipitated immune-complex;
[0351] (Step 4) separating the precipitated complex by the
centrifugation of the reaction mixture of Step 3;
[0352] (Step 5) reacting the labeling agent moiety of the labeled
standard with a substrate selected depending on the type of the
enzyme used (when the labeled standard used in Step 2 is labeled
with an enzyme such as peroxidase), avidin or the enzyme-conjugated
avidin (when the labeled standard used in Step 2 is labeled with
the biotin) by adding, if necessary together with a coloring agent,
the substrate, or avidin or enzyme-conjugated avidin to the
precipitated complex separated in Step 4;
[0353] (Step 6) reacting a substrate selected depending on the type
of the enzyme conjugated with avidin, with the enzyme conjugated
with avidin by adding the substrate, when enzyme-conjugated avidin
is used in Step 5;
[0354] (Step 7) stopping the enzyme reaction and the coloring
reaction by adding a stop solution to the reaction system in Step 5
or 6; and,
[0355] (Step 8) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0356] The second method comprises the following steps:
[0357] (Step 1) preparing a labeled CTGF standard by labeling a
human or mouse CTGF standard with biotin or an enzyme such as
peroxidase;
[0358] (Step 2) reacting a sample such as a human or mouse serum
with the monoclonal antibody "8-64-6" or "8-86-2" of the present
invention by adding the sample into a container having internal
spaces such as a test tube, plate or tube and by subsequently
adding thereto the monoclonal antibody;
[0359] (Step 3) reacting the labeled CTGF standard prepared in Step
1 with the remaining unreacted monoclonal antibody, by adding the
labeled CTGF standard to the reaction mixture in Step 2;
[0360] (Step 4) reacting an antiserum, derived from mammals except
mice, reactive to the mouse monoclonal antibody, such as a goat
anti-mouse .gamma.-globulin antiserum, with the antigen-antibody
complex, formed in Step 2, consisting of the monoclonal antibody
and the mammalian CTGF in the sample and/or the antigen-antibody
complex, formed in Step 3, consisting of the monoclonal antibody
and the labeled mammalian CTGF standard, to give the precipitated
immune-complex consisting of the antiserum and the antigen-antibody
complex;
[0361] (Step 5) separating the precipitated complex by the
centrifugation of the reaction mixture of Step 4;
[0362] (Step 6) reacting the labeling agent moiety of the labeled
standard with a substrate selected depending on the type of the
enzyme used (when the labeled standard used in Step 3 is labeled
with an enzyme such as peroxidase), avidin or the enzyme-conjugated
avidin (when the labeled standard used in Step 3 is labeled with
biotin) by adding, if necessary together with a coloring agent, the
substrate, or avidin or enzyme-conjugated avidin to the
precipitated complex separated in Step 5;
[0363] (Step 7) reacting a substrate selected depending on the type
of the enzyme conjugated with avidin, with the enzyme conjugated
with avidin by adding the substrate, when enzyme-conjugated avidin
is used in Step 6;
[0364] (Step 8) stopping the enzyme reaction and the coloring
reaction are stopped by adding a stop solution to the reaction
system in Step 6 or 7, and;
[0365] (Step 9) measuring the colorimetric intensity, fluorescence
intensity or luminescence intensity.
[0366] The "affinity chromatography" as referred to in the present
invention indicates the method of separating or purifying the
materials of interest in samples (for example, the body fluid
samples such as a serum and plasma; culture supernatants; or the
supernatant fluids obtained by centrifugation, etc.) by utilizing
the interaction (affinity) between a pair of materials, for
example, antigen and antibody, enzyme and substrate, or receptor
and ligand.
[0367] The method of the present invention relates to the method
for separating or purifying the mammalian CTGFs in samples (for
example, the body fluid samples such as a serum and plasma; culture
supernatants; or the supernatant fluids obtained by centrifugation,
etc.) by utilizing the antigen-antibody interaction, specifically,
the affinity of the monoclonal antibody of the present invention
for mammalian CTGFs as antigens; specifically includes,
[0368] (1) a method for separating CTGF in samples by immobilizing
the monoclonal antibody (or antibody fragment) reactive to mammlian
CTGF on the above-defined insoluble carriers, such as a filter or a
membrane and contacting the sample with the filter or membrane;
and
[0369] (2) a method for separating or purifying CTGF in the
samples, by immobilizing, in a usual manner (immobilization by
physical adsorption, cross-linking to the carrier polymer, trapping
in the carrier matrix or non-covalent bonding, etc.), the inventive
monoclonal antibody (or the antibody fragment) reactive to
mammalian CTGF on insoluble carriers such as cellulose carriers,
agarose carriers, polyacrylamide carriers, dextran carriers, and
polystyrene carriers, polyvinyl alcohol carriers, poly(amino acid)
carriers or porous silica carriers; by filling a column made of
glass, plastics, or stainless, with the insoluble carriers; and by
loading and eluting samples (for example, the body fluid samples
such as a serum and plasma; culture supernatants; or the
supernatant fluids obtained by centrifugation, etc.) through the
column (for example, the cylindrical column). The method described
above in (2) is in particular designated as affinity column
chromatography.
[0370] Any of the insoluble carriers are usable as insoluble
carriers for affinity column chromatography, as long as the
monoclonal antibody (or antibody fragment) of the present invention
can be immobilized on the carriers. Such carriers include, for
example, commercially available carriers such as SEPHAROSE 2B,
SEPHAROSE 4B, SEPHAROSE 6B, CNBR-ACTIVATED SEPHAROSE 4B,
AH-SEPHAROSE 4B, CH-SEPHAROSE 4B, ACTIVATED CH-SEPHAROSE 4B,
EPOXY-ACTIVATED SEPHAROSE 6B, ACTIVATED THIOL-SEPHAROSE 4B,
SEPHADEX, CM-SEPHADEX, ECH-SEPHAROSE 4B, EAH-SEPHAROSE 4B,
NHS-ACTIVATED SEPHAROSE or THIOPROPYL SEPHAROSE 6B, etc., all of
which are supplied by Pharmacia; BIO-GEL A, CELLEX, CELLEX AE,
CELLEX-CM, CELLEX PAB, BIO-GEL P, HYDRAZIDE BIO-GEL P, AMINOETHYL
BIO-GEL P, BIO-GEL CM, AFFI-GEL 10, AFFI-GEL 15, AFFI-PREP10,
AFFI-GEL HZ, AFFI-PREP HZ, AFFI-GEL 102, CM BIO-GEL A, AFFI-GEL
HEPARIN, AFFI-GEL 501 OR AFFI-GEL 601, etc., all of which are
supplied by Bio-Rad; CHROMAGEL A, CHROMAGEL P, ENZAFIX P-HZ,
ENZAFIX P-SH OR ENZAFIX P-AB, etc., all of which are supplied by
Wako Pure Chemical Industries Ltd.; AE-CELLUROSE, CM-CELLUROSE or
PAB CELLUROSE etc., all of which are supplied by Serva.
[0371] The term "pharmaceutical composition" as referred to in the
present invention means a composition useful as a pharmaceutical
comprising as an active ingredient the monoclonal antibody of the
present invention or a portion thereof, or any of the
after-mentioned "CTGF inhibitor", "CTGF production inhibitor" and
"substance with the activity to inhibit the CTGF-stimulated
proliferation of cells having the capability of proliferating by
CTGF stimulation", as well as comprising a "pharmaceutically
acceptable carrier."
[0372] The "pharmaceutically acceptable carrier" includes an
excipient, a diluent, an expander, a disintegrating agent, a
stabilizer, a preservative, a buffer, an emulsifier, an aromatic, a
colorant, a sweetener, a viscosity increasing agent, a flavor, a
dissolving agent, or other additives.
[0373] Using one or more of such carriers, a pharmaceutical
composition can be formulated into tablets, pills, powders,
granules, injections, solutions, capsules, troches, elixirs,
suspensions, emulsions, or syrups.
[0374] The pharmaceutical composition can be administered orally or
parenterally. Other forms for parenteral administration include a
solution for external application, suppository for rectal
administration, and pessary, prescribed by the usual method, which
comprises one or more active ingredient.
[0375] The dosage can vary depending on the age, sex, weight, and
symptoms of a patient, effect of treatment, administration route,
period of treatment, or the kind of active ingredient (protein or
antibody mentioned above) contained in the pharmaceutical
composition. Usually, the pharmaceutical composition can be
administered to an adult in a dose of 10 .mu.g to 1000 mg (or 10
.mu.g to 500 mg) per one administration. Depending on various
conditions, the lower dosage may be sufficient in some cases, and a
higher dosage may be necessary in other cases.
[0376] In particular, the injection can be produced by dissolving
or suspending the antibody in a non-toxic, pharmaceutically
acceptable carrier such as physiological saline or commercially
available distilled water for injections by adjusting the
concentration to 0.1 .mu.g antibody/ml carrier to 10 mg antibody/ml
carrier.
[0377] The injection thus produced can be administered to a human
patient in need of treatment in a dose of 1 .mu.g to 100 mg/kg body
weight, preferably 50 .mu.g to 50 mg/kg body weight, once or more
times a day. Examples of administration routes are medically
appropriate administration routes such as intravenous injection,
subcutaneous injection, intradermal injection, intramuscular
injection, or intraperitoneal injection, preferably intravenous
injection.
[0378] The injection can also be prepared into a non-aqueous
diluent (for example, propylene glycol, polyethylene glycol,
vegetable oil such as olive oil, and alcohols such as ethanol),
suspension, or emulsion.
[0379] The injection can be sterilized by filtration with a
bacteria-non-penetratable filter, by mixing bacteriocide, or by
irradiation. The injection can be prepared at the time of use.
Namely, it is freeze-dried to make a sterile solid composition, and
can be dissolved in sterile distilled water for injection or
another solvent before use.
[0380] The pharmaceutical composition of the present invention is
useful for inhibiting the proliferation of various cells having the
capability of proliferating (for example, various fibroblast cells,
various vascular endothelial cells, and others, etc.) in response
to the stimulation of CTGFs from a variety of tissues. Examples of
the tissues are, the brain, neck, lung, heart, liver, pancreas,
kidney, stomach, large intestine, small intestine, duodenum, bone
marrow, uterus, ovary, testis, prostate, skin, mouth, tongue, and
blood vessels, and preferably, the lung, liver, kidney or skin.
[0381] As described hereinabove, the pharmaceutical composition of
the present invention can inhibit the proliferation of cells having
the capability of proliferating in response to the stimulation of
CTGF. Therefore, the pharmaceutical composition of the present
invention is also useful as a pharmaceutical for treating or
preventing a variety of diseases associated with the cell
proliferation in various tissues mentioned above. Examples of such
tissues are the brain, neck, lung, heart, liver, pancreas, kidney,
stomach, large intestine, small intestine, duodenum, bone marrow,
uterus, ovary, testis, prostate, skin, mouth, tongue, and blood
vessels, and preferably, the lung, liver, kidney or skin.
[0382] The diseases, to which the pharmaceutical composition of the
present invention is applicable for the treatment or prevention,
are, for example, fibrotic diseases in various tissues (kidney
fibrosis, pulmonary fibrosis, hepatic fibrosis, fibrosis in the
skin, etc.), kidney diseases (for example, kidney fibrosis,
nephritis, renal failure, etc.), lung diseases (for example,
pulmonary fibrosis, pneumonia, etc.), skin diseases (for example,
psoriasis, scleroderma, atopy, keloid, etc.), liver diseases (for
example, hepatic fibrosis, hepatitis, cirrhosis, etc.), arthritis
(for example, rheumatoid arthritis), various cancers, or
arteriosclerosis.
[0383] Preferable examples of the diseases are kidney diseases (for
example, kidney fibrosis, nephritis, renal failure, etc.), lung
diseases (for example, pulmonary fibrosis, pneumonia, etc.), skin
diseases (for example, psoriasis, scleroderma, atopy, keloid,
etc.), liver diseases (for example, hepatic fibrosis, hepatitis,
cirrhosis, etc.).
[0384] More preferable are kidney diseases (for example, kidney
fibrosis, nephritis, renal failure, etc.).
[0385] The pharmaceutical composition of the present invention
includes the pharmaceutical composition comprising a "CTGF
inhibitor," a "CTGF production inhibitor" or a "substance with the
activity to inhibit the CTGF-stimulated proliferation of the cells
having the capability of proliferating by CTGF stimulation."
[0386] Each of the "CTGF inhibitor," the "CTGF production
inhibitor," and the "substance" means a substance having the
activity of suppressing or inhibiting the biological function of
CTGF, or a substance having the activity of suppressing or
inhibiting the production of CTGF in a variety of cells. Such
substances are exemplified by a substance having any of the
following activities:
[0387] (1) the activity of suppressing or inhibiting the binding of
human kidney-derived fibroblast cells (for example, cell line 293-T
(ATCC CRL1573)) to human CTGF, or the binding of the cells to mouse
CTGF;
[0388] (2) the activity of suppressing or inhibiting the binding of
human CTGF with rat kidney-derived fibroblast cells (for example,
cell line NRK-49F (ATCC CRL-1570)), human osteosarcoma cell line
MG-63 (ATCC CRL-1427), or human lung-derived fibroblast cells;
[0389] (3) the activity of suppressing or inhibiting the
proliferation of rat kidney-derived fibroblast cells (for example,
cell line NRK-49F (ATCC CRL-1570)) in response to the stimulation
of human CTGF or mouse CTGF;
[0390] (4) the activity of suppressing or inhibiting an increase of
hydroxyproline in the kidney where the synthesis of hydroxyproline
level tends to be increased.
[0391] Specifically, the above-mentioned "substance" is exemplified
by the following substances:
[0392] (a) the above-mentioned monoclonal antibody of the present
invention (which is not restricted to the wild-type antibody and
the recombinant antibody) or a portion thereof;
[0393] (b) antisense DNA;
[0394] (c) antisense RNA;
[0395] (d) low molecular weight chemical substances (chemically
synthesized compounds or naturally-occurring substances) other than
the substances indicated in (a) to (c).
[0396] The antisense DNA as referred to in the present invention
includes a DNA comprising a partial nucleotide sequence of a DNA
encoding the mammalian (preferably human) CTGF protein or a DNA
corresponding to the above DNA that is chemically modified in part,
or a DNA comprising a complementary sequence to the partial
nucleotide sequence, or a DNA corresponding to this DNA that is
chemically modified in part.
[0397] The "partial nucleotide sequence" as referred to here
indicates a partial nucleotide sequence comprising an arbitrary
number of nucleotides contained in an arbitrary region of the DNA
sequence encoding the mammalian (preferably human) CTGF
protein.
[0398] The DNA, hybridizing to a DNA or an RNA encoding the CTGF
protein, can inhibit the CTGF production by suppressing
transcription of the DNA to mRNA or suppressing the translation of
the mRNA into the protein.
[0399] The partial nucleotide sequence consists of 5 to 100
consecutive nucleotides, preferably 5 to 70 consecutive
nucleotides, more preferably 5 to 50 consecutive nucleotides, and
still more preferably 5 to 30 consecutive nucleotides.
[0400] When the DNA is used as an antisense DNA pharmaceutical, the
DNA sequence can be modified chemically in part for extending the
half-life (stability) of the blood concentration of the DNA
administered to patients, for increasing the
intracytoplasmic-membrane permeability of the DNA, or for
increasing the degradation resistance or the absorption of the
orally administered DNA in the digestive organs. The chemical
modification includes, for example, the modification of the
phosphate bonds, the riboses, the nucleotide bases, the sugar
moiety, the 3' end and/or the 5' end in the structure of the
oligonucleotide DNA.
[0401] The modification of phosphate bond includes, for example,
the conversion of one or more of the bonds to phosphodiester bonds
(D-oligo), phosphorothioate bonds, phosphorodithioate bonds
(S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds,
non-phosphate bonds or methyl phosphonothioate bonds, or
combinations thereof. The modification of the ribose includes, for
example, the conversion to 2'-fluororibose or 2'-O-methylribose.
The modification of the nucleotide base includes, for example, the
conversion to 5-propynyluracil or 2-aminoadenine.
[0402] The antisense RNA as referred to in the present invention
includes an RNA comprising a partial nucleotide sequence of an RNA
encoding mammalian (preferably human) CTGF protein or an RNA
corresponding to the RNA which is chemically modified in part, or
an RNA comprising a complementary sequence to the partial
nucleotide sequence or an RNA corresponding to this RNA which is
chemically modified in part.
[0403] The "partial nucleotide sequence" as referred to here
indicates a partial nucleotide sequence comprising an arbitrary
number of nucleotides contained in an arbitrary region of the RNA
sequence encoding mammalian (preferably human) CTGF protein.
[0404] The RNA, hybridizing to a DNA or an RNA encoding the CTGF
protein, can inhibit the CTGF production by inhibiting the
transcription of the DNA to mRNA or inhibiting the translation of
the mRNA into the protein.
[0405] The partial nucleotide sequence consists of 5 to 100
consecutive nucleotides, preferably 5 to 70 consecutive
nucleotides, more preferably 5 to 50 consecutive nucleotides, and
still more preferably 5 to 30 consecutive nucleotides.
[0406] When the RNA is used as an antisense RNA pharmaceutical, the
RNA sequence can be modified chemically in part for extending the
half-life (stability) of the blood concentration of the RNA
administered to patients, for increasing the
intracytoplasmic-membrane permeability of the RNA, or for
increasing the degradation resistance or the absorption of the
orally administered RNA in the digestive organ. The chemical
modification includes, for example, the modification of the
phosphate bonds, the riboses, the nucleotide bases, the sugar
moiety, the 3' end and/or the 5' end in the structure of the
oligonucleotide RNA.
[0407] The modification of phosphate bonds includes, for example,
the conversion of one or more of the bonds to phosphodiester bonds
(D-oligo), phosphorothioate bond, phosphorodithioate bonds
(S-oligo), methyl phosphonate (MP-oligo), phosphoroamidate bonds,
non-phosphate bonds or methyl phosphonothioate bonds, or
combinations thereof. The modification of the ribose includes, for
example, the conversion to 2'-fluororibose or 2'-O-methylribose.
The modification of the nucleotide base includes, for example, the
conversion to 5-propynyluracil or 2-aminoadenine.
[0408] The therapeutic effects of the pharmaceutical composition of
the present invention on various diseases can be examined and
evaluated according to a usual method by administering the
composition to known animals as disease models.
[0409] For example, evaluation of the therapeutic effect on kidney
fibrosis, which is a tissue fibrosis as well as a kidney disease,
can be performed by a method using a renal failure model mouse
(unilateral ureteral obstruction (UUO) model), in which unilateral
ureteral ligation obstructs renal blood filtration in the kidney
and results in renal failure in the mouse. After administration of
the inventive pharmaceutical composition to the mouse, the
examination is achieved by measuring the degree of inhibition of an
increase of hydroxyproline production , which is an index of the
onset of nephritis and kidney fibrosis induced by the renal
failure. A decrease in the hydroxyproline concentration indicates
the efficacy of the pharmaceutical composition for the treatment of
the kidney disease.
[0410] By using the model animals described in detail in a previous
report ("Preparation of animals as disease models; Testing and
experimental methods for the development of new drugs" p. 34-46,
1993, Technological Information Society), the evaluation can be
performed for kidney diseases including, for example, minimal
change glomerular disease (for example, minimal change nephrotic
syndrome (MCNS)), focal glomerular sclerosis (FGS), membraneous
glomerulonephritis (membranous nephropathy (MN)), IgA nephropathy,
mesangial proliferative glomerulonephritis, acute
post-streptococcal glomerulonephritis (APSGN, crescentic
(extracapillary) glomerulonephritis, interstitial nephritis, or
acute renal failure.
[0411] By using the model animals described in detail in the
previous report ("Preparation of animals as disease models: Testing
and experimental methods for the development of new drugs" p.
229-235, 1993, Technological Information Society), the evaluation
can be performed for skin diseases including, for example,
injuries, keloid, atopy, dermatitis, scleroderma or psoriasis.
[0412] By using the model animals described in detail in the
previous report ("Preparation of animals as disease models: Testing
and experimental methods for the development of new drugs" p.
349-358, 1993, Technological Information Society), the evaluation
can be performed for liver diseases including, for example,
hepatitis (for example, viral hepatitis (type A, type B, type C,
type E, etc.)), cirrhosis or drug induced hepatic injuries.
[0413] For example, the effect on arteriosclerosis and restenosis
can be evaluated by using a restenosis model rat, in which the
pseudo-restenosis is causeed by percutaneous transluminal coronary
angioplasty (PTCA) with balloon catheter inserted in the aorta.
[0414] For example, the effect on tumor growth and metastasis can
be confirmed by using mice as cancer metastasis models. The model
mice are prepared by transplanting cancer cells into the
subcutaneous tissue, caudal vein, spleen, tissues under the
renicapsule, peritoneal cavity or cecum wall tissue, of
commercially available mice including normal mice such as Balb/c
mouse, or model mice such as nude mouse and SCID mouse.
[0415] The "rat CTGF" of the present invention (specifically,
having an amino acid sequence of, or substantially equivalent to
that of SEQ ID NO: 2) and the "DNA encoding rat CTGF"
(specifically, comprising the nucleotide sequence spanning from
nucleotide position 213 to 1256 of the nucleotide sequence of SEQ
ID NO: 1) are defined below, and can be prepared according to a
usual method as shown below.
[0416] Here, the terminology "substantially equivalent" has the
meaning defined above.
[0417] The "rat CTGF" of the present invention can be produced by
suitably using a method known in this technical field, such as
chemical synthesis and cell culture as well as the recombinant
technique described below, or by using a modified method
thereof.
[0418] The "DNA" of the present invention indicates the DNA
encoding rat CTGF, and includes any nucleotide sequences as long as
the nucleotide sequence encodes rat CTGF of the present invention.
Specifically, the DNA includes any DNAs encoding the polypeptide
with the amino acid sequence of SEQ ID NO:2. In a preferred
embodiment, the DNA comprises the nucleotide sequence spanning from
nucleotide position 213 to 1256 in the nucleotide sequence of SEQ
ID NO: 1 (for example, the DNA having the nucleotide sequence of
SEQ ID NO: 1).
[0419] The DNA of the present invention includes both cDNA and
genomic DNA encoding rat CTGF.
[0420] The DNA of the present invention also includes the DNAs
consisting of any codons as long as the codons encodeidentical
amino acids.
[0421] The DNA of the present invention can be a DNA obtained by
any method. For example, the DNA includes complementary DNA (cDNA)
prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by
chemical synthesis, DNA obtained by PCR amplification with RNA or
DNA as a template, and DNA constructed by appropriately combining
these methods.
[0422] The DNA encoding the rat CTGF of the present invention can
be prepared by the usual methods: cloning cDNA from mRNA encoding
rat CTGF, isolating genomic DNA and splicing it, PCR using the cDNA
or mRNA sequence as a template, chemical synthesis, and so on.
[0423] The DNA encoding the rat CTGF can be prepared by cleaving
(digesting) each DNA encoding the rat CTGF as prepared above with
an appropriate restriction enzyme, and linking the obtained DNA
fragments, in combination with linker DNA or Tag if necessary,
using an appropriate DNA polymerase and such.
[0424] cDNA encoding rat CTGF (hereinafter referred to as the
desired protein) can be cloned from mRNA by, for example, the
method described below.
[0425] First, the mRNA encoding the desired protein is prepared
from tissues or cells (for example, rat fibroblasts, etc.)
expressing and producing the desired protein. mRNA can be prepared
by isolating total RNA by a known method such as
quanidine-thiocyanate method (Chirgwin et al., Biochemistry,
Vol.18, p5294, 1979), hot phenol method, or AGPC method, and
subjecting it to affinity chromatography using oligo-dT cellulose
or poly-U Sepharose.
[0426] Then, with the mRNA obtained as a template, cDNA is
synthesized, for example, by a well-known method using reverse
transcriptase, such as the method of Okayama et al (Mol. Cell.
Biol. Vol.2, p.161 (1982); ibid. Vol.3, p.280 (1983)) or the method
of Hoffman et al. (Gene Vol.25, p.263 (1983)), and converted into
double-stranded cDNA. A cDNA library is prepared by transforming E.
coli with plasmid vectors, phage vectors, or cosmid vectors having
this cDNA or by transfecting E. coli after in vitro packaging.
[0427] The plasmid vectors used in this invention are not limited
as long as they are replicated and maintained in hosts. Any phage
vector that can be replicated in hosts can also be used. Examples
of usually used cloning vectors are pUC19, .lambda.gt10,
.lambda.gt11, and so on. When the vector is applied to
immunological screening as mentioned below, a vector having a
promoter that can express a gene encoding the desired protein in a
host is preferably used.
[0428] cDNA can be inserted into a plasmid by, for example, the
method of Maniatis et al. (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p.1.53, 1989). cDNA
can be inserted into a phage vector by, for example, the method of
Hyunh et al. (DNA cloning, a practical approach, Vol.1, p.49
(1985)). These methods can be simply performed by using a
commercially available cloning kit (for example, a product from
Takara Shuzo). The recombinant plasmid or phage vector thus
obtained is introduced into an appropriate host cell such as a
prokaryote (for example, E. coli: HB101, DH5.alpha., Y1090, DH10B,
MC1061/P3, etc).
[0429] Examples of a method for introducing a plasmid into a host
are, calcium chloride method, calcium chloride/rubidium chloride
method and electroporation method, described in Molecular Cloning,
A Laboratory Manual (second edition, Cold Spring Harbor Laboratory,
p.1.74 (1989)). Phage vectors can be introduced into host cells by,
for example, a method in which the phage DNAs are introduced into
grown hosts after in vitro packaging. In vitro packaging can be
easily performed with a commercially available in vitro packaging
kit (for example, a product from Stratagene or Amersham).
[0430] The cDNA encoding the desired protein can be isolated from
the cDNA library so prepared according to the method mentioned
above by combining general cDNA screening methods.
[0431] For example, a clone comprising the desired cDNA can be
screened by a known colony hybridization method (Crunstein et al.
Proc. Natl. Acad. Sci. USA, Vol.72, p.3961 (1975)) or plaque
hybridization method (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p.2.108 (1989))
using .sup.32P-labeled chemically synthesized oligonucleotides as
probes, which correspond to the amino acid sequence of the desired
protein. Alternatively, a clone having a DNA fragment encoding a
specific region within the desired protein can be screened by
amplifying the region by PCR with synthetic PCR primers.
[0432] When a cDNA library prepared using a cDNA expression vector
(for example, .lambda.gt11 phage vector) is used, the desired clone
can be screened by the antigen-antibody reaction using an antibody
against the desired protein. A screening method using PCR method is
preferably used when many clones are subjected to screening.
[0433] The nucleotide sequence of the DNA thus obtained can be
determined by Maxam-Gilbert method (Maxam et al. Proc. Natl. Acad.
Sci. USA, Vol.74, p.560 (1977)) or the dideoxynucleotide synthetic
chain termination method using phage M13 (Sanger et al. Proc. Natl.
Acad. Sci. USA, Vol.74, pp.5463-5467 (1977)). The whole or a part
of the gene encoding the desired protein can be obtained by
excising the clone obtained as mentioned above with restriction
enzymes and so on.
[0434] Also, the DNA encoding the desired protein can be isolated
from the genomic DNA derived from the cells expressing the desired
protein as mentioned above by the following methods.
[0435] Such cells are solubilized preferably by SDS or proteinase
K, and the DNAs are deproteinized by repeating phenol extraction.
DNAs are digested preferably with ribonuclease. The DNAs obtained
are partially digested with appropriate restriction enzymes, and
the DNA fragments obtained are amplified with appropriate phage or
cosmid to generate a library. Then, clones having the desired
sequence are detected, for example, by using radioactively labeled
DNA probes, and the whole or a portion of the gene encoding the
desired protein is obtained from the clones by excision with
restriction enzymes etc.
[0436] A DNA encoding a desired protein can be prepared by
following standard methods using known mRNA or cDNA of the desired
protein as a template by means of PCR (Gene Amplification PCR
method, Basics and Novel Development, Kyoritsu Publishers, 1992,
etc).
[0437] A DNA encoding a desired protein can also be produced by
chemical synthesis according to a usual method based on the
nucleotide sequence encoding the protein.
[0438] The rat CTGF of the present invention can be prepared as a
recombinant protein according to the frequently used recombinant
technology by using DNA obtained by digesting the rat CTGF-encoding
DNA (the cDNA or the genomic DNA comprising introns) prepared by
the method indicated above with appropriate restriction enzymes;
linking the resulting DNA fragment encoding the rat CTGF, according
to need, with a linker DNA or Tag by using an appropriate DNA
polymerase or other enzymes.
[0439] Specifically, the preparation of the protein is illustrated
as follows: the DNA construct as prepared above is inserted into a
vector, described below in detail, to obtain an expression vector;
a host cell, which will be described hereinafter, is transformed
with the expression vector to obtain a transformant; the resulting
transformant cells are cultured for the production and accumulation
of the desired protein in the culture supernatant; the protein
accumulated in the culture supernatant can be purified easily by
using column chromatography, etc.
[0440] The present invention also relates to an expression vector
comprising the DNA encoding the rat CTGF of the present invention.
As an expression vector of the present invention, any vector can be
used as long as it is capable of retaining replication or
self-multiplication in each host cell of prokaryotic and/or
eukaryotic cells, including plasmid vectors and phage vectors
(Cloning Vectors: A laboratory Manual, Elsevier, New York,
1985).
[0441] The recombinant vector can easily be prepared by ligating
the DNA encoding rat CTGF of the present invention with a vector
for recombination available in the art (plasmid DNA and
bacteriophage DNA) by the usual method. Specific examples of the
vectors for recombination used are E. coli-derived plasmids such as
pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids
such as pSH19 and pSH15, and Bacillus subtilis-derived plasmids
such as pUB110, pTP5, and pC194. Examples of phages are a
bacteriophages such as .lambda. phage, and an animal or insect
virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus,
and nuclear polyhedrosis virus.
[0442] A plasmid vector is useful for expressing the DNA encoding
rat CTGF and for producing rat CTGF. The plasmid vector is not
limited as long as it expresses the gene encoding the rat CTGF in
various prokaryotic and/or eukaryotic host cells and produces this
polypeptide. Examples thereof are pMAL C2, pcDNA3.1(-), pEF-BOS
(Nucleic Acids Res. Vol.18, p.5322 (1990) and so on), pME18S
(Experimental Medicine: SUPPLEMENT, "Handbook of Genetic
Engineering" (1992) and so on), etc.
[0443] When bacteria, particularly E. coli are used as host cells,
an expression vector is generally comprised of, at least, a
promoter/operator region, an initiation codon, the DNA encoding the
protein of the present invention, termination codon, terminator
region, and replicon.
[0444] When yeast, animal cells, or insect cells are used as hosts,
an expression vector is preferably comprised of, at least, a
promoter, an initiation codon, the DNA encoding the rat CTGF of
,the present invention, and a termination codon. It may also
comprise the DNA encoding a signal peptide, enhancer sequence, 5'-
and 3'-untranslated region of the gene encoding the rat CTGF of the
present invention, splicing junctions, polyadenylation site,
selectable marker region, and replicon. The expression vector may
also contain, if required, a gene for gene amplification (marker)
that is usually used.
[0445] A promoter/operator region to express the fusion polypeptide
of the present invention in bacteria comprises a promoter, an
operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG).
For example, when the host is Escherichia, it preferably comprises
Trp promoter, lac promoter, recA promoter, .lambda.PL promoter, lpp
promoter, tac promoter, or-the like.
[0446] Examples of a promoter to express the rat CTGF of the
present invention in yeast are PH05 promoter, PGK promoter, GAP
promoter, ADH promoter, and so on. When the host is Bacillus,
examples thereof are SL01 promoter, SP02 promoter, penP promoter
and so on.
[0447] When the host is a eukaryotic cell such as a mammalian cell,
examples thereof are SV40-derived promoter, retrovirus promoter,
heat shock promoter, and soon, and preferably SV-40 and
retrovirus-derived one. As a matter of course, the promoter is not
limited to the above examples. In addition, using an enhancer is
effective for expression.
[0448] A preferable initiation codon is, for example, a methionine
codon (ATG).
[0449] A commonly used termination codon (for example, TAG, TAA,
TGA) is exemplified as a termination codon.
[0450] Usually, used natural or synthetic terminators are used as a
terminator region.
[0451] A replicon means a DNA capable of replicating the whole DNA
sequence in host cells, and includes a natural plasmid, an
artificially modified plasmid (DNA fragment prepared from a natural
plasmid), a synthetic plasmid, and so on. Examples of preferable
plasmids are pBR322 or its artificial derivatives (DNA fragment
obtained by treating pBR322 with appropriate restriction enzymes)
for E. coli, yeast 2.mu. plasmid or yeast chromosomal DNA for
yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo
ATCC 37224, pSV2neo ATCC 37149, pSV2bsr, and such for mammalian
cells.
[0452] An enhancer sequence, polyadenylation site, and splicing
junction that are usually used in the art, such as those derived
from SV40 can also be used.
[0453] A selectable marker usually employed can be used according
to the usual method. Examples thereof are resistance genes for
antibiotics, such as tetracycline, ampicillin, or kanamycin.
[0454] Examples of genes for gene amplification are dihydrofolate
reductase (DHFR) gene, thymidine kinase gene, neomycin resistance
gene, glutamate synthase gene, adenosine deaminase gene, ornithine
decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate
transcarbamylase gene, etc.
[0455] The expression vector of the present invention can be
prepared by continuously and circularly linking at least the
above-mentioned promoter, initiation codon, DNA encoding the
protein of the present invention, termination codon, and terminator
region, to an appropriate replicon. If desired, appropriate DNA
fragments (for example, linkers, restriction sites generated with
other restriction enzyme), can be used by the usual method such as
digestion with a restriction enzyme or ligation using T4 DNA
ligase.
[0456] Transformants of the present invention can be prepared by
introducing the expression vector mentioned above into host
cells.
[0457] Host cells used in the present invention are not limited as
long as they are compatible with an expression vector mentioned
above and can be transformed. Examples thereof are various cells
such as wild-type cells or artificially established recombinant
cells usually used in technical field of the present invention (for
example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces,
Pichia, and such), animal cells, or insect cells).
[0458] E. coli or animal cells are preferably used. Specific
examples are E. coli (DH5 .alpha., DH10B, TB1, HB101, XL-2Blue, and
such), mouse-derived cells (COP, L, C127, Sp2/0, NS-1, NIH 3T3, and
such), rat-derived cells, hamster-derived cells (BHK, CHO, and
such), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, and
such), and human-derived cells (Hela, diploid fibroblast-derived
cells, myeloma, Namalwa, and such).
[0459] An expression vector can be introduced (transformed
(transduced)) into host cells by known methods.
[0460] Transformation can be performed, for example, according to
the method of Cohen et al. (Proc. Natl. Acad. Sci. USA, Vol.69,
p.2110 (1972)), protoplast method (Mol. Gen. Genet., Vol.168, p.111
(1979)), or competent method (J. Mol. Biol., Vol.56, p.209 (1971))
when the hosts are bacteria (E. coli, Bacillus subtilis, and such),
the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA, Vol.75,
p.1927 (1978)), or lithium method (J. Bacteriol., Vol.153, p.163
(1983)) when the host is Saccharomyces cerevisiae, the method of
Graham (Virology, Vol.52, p.456 (1973)) when the hosts are animal
cells, and the method of Summers et al. (Mol. Cell. Biol., Vol.3,
pp.2156-2165 (1983)) when the hosts are insect cells.
[0461] Rat CTGF of the present invention can be produced by
cultivating transformants (in the following this term includes
transductants) comprising an expression vector prepared as
mentioned above in nutrient media.
[0462] The nutrient media preferably comprise carbon source,
inorganic nitrogen source, or organic nitrogen source necessary for
the growth of host cells (transformants). Examples of the carbon
source are glucose, dextran, soluble starch, and sucrose, and
examples of the inorganic or organic nitrogen source are ammonium
salts, nitrates, amino acids, corn steep liquor, peptone, casein,
meet extract, soy bean cake, and potato extract. If desired, they
may comprise other nutrients (for example, an inorganic salt (for
example, calcium chloride, sodium dihydrogenphosphate, and
magnesium chloride), vitamins, antibiotics (for example,
tetracycline, neomycin, ampicillin, kanamycin, and so on).
[0463] Cultivation is performed by a method known in the art.
Cultivation conditions such as temperature, pH of the media, and
cultivation time are selected appropriately so that the protein of
the present invention is produced in large quantities.
[0464] Specific media and cultivation conditions used depending on
host cells are illustrated below, but are not limited thereto.
[0465] When the hosts are bacteria, actinomycetes, yeasts,
filamentous fungi, liquid media comprising the nutrient source
mentioned above are appropriate. The media with pH 5 to 8 are
preferably used.
[0466] When the host is E. coli, examples of preferable media are
LB media, M9 media (Miller et al. Exp. Mol. Genet., Cold Spring
Harbor Laboratory, p.431 (1972)), YT medium, and so on. Using these
media, cultivation can be performed usually at 14 to 43.degree. C.
for about 3 to 24 hours with aeration and stirring, if
necessary.
[0467] When the host is Bacillus, cultivation can be performed
usually at 30 to 40.degree. C. for about 16 to 96 hours with
aeration and stirring, if necessary.
[0468] When the host is yeast, an example of media is Burkholder
minimal media (Bostian, Proc. Natl. Acad. Sci. USA, Vol.77, p.4505
(1980)). The pH of the media is preferably 5 to 8. Cultivation can
be performed usually at 20 to 35.degree. C. for about 14 to 144
hours with aeration and stirring, if necessary.
[0469] When the host is an animal cell, examples of media are MEM
media containing about 5 to 20% fetal bovine serum (Science,
Vol.122, p.501 (1952)), DMEM media (Virology, Vol.8, p.396 (1959)),
RPMI1640 media (J. Am. Med. Assoc., Vol.199, p.519 (1967)), 199
media (Proc. Soc. Exp. Biol. Med., Vol.73, p.1 (1950)), HamF12
media, and so on. The pH of the media is preferably about 6 to 8.
Cultivation can be performed usually at about 30 to 40.degree. C.
for about 15 to 72 hours with aeration and stirring, if
necessary.
[0470] When the host is an insect cell, an example of media is
Grace's media containing fetal bovine serum (Proc. Natl. Acad. Sci.
USA, Vol.82, p.8404 (1985)). The pH thereof is preferably about 5
to 8. Cultivation can be performed usually at about 20 to
40.degree. C. for 15 to 100 hours with aeration and stirring, if
necessary.
[0471] Rat CTGF of the present invention can be produced by
cultivating transformants as mentioned above (in particular animal
cells or E. coli) and allowing them to secrete the protein into the
culture supernatant. Namely, a culture filtrate (supernatant) is
obtained by a method such as filtration or centrifugation of the
obtained culture, and the rat CTGF of the present invention is
purified and isolated from the culture filtrate by methods commonly
used in order to purify and isolate a natural or synthetic
protein.
[0472] Examples of the isolation and purification method are a
method utilizing affinity, such as affinity column chromatography;
a method utilizing solubility, such as salting out and solvent
precipitation method; a method utilizing the difference in
molecular weight, such as dialysis, ultrafiltration, gel
filtration, and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis; a method utilizing charges, such as ion exchange
chromatography and hydroxylapatite chromatography; a method
utilizing the difference in hydrophobicity, such as reverse phase
high performance liquid chromatography; and a method utilizing the
difference in isoelectric point, such as isoelectric focusing.
[0473] When the rat CTGF of the present invention exists in the
periplasm or cytoplasm of cultured transformants, first, the cells
are harvested by a usual method such as filtration or
centrifugation and suspended in appropriate buffer. After the cell
wall and/or cell membrane of the cells and such are disrupted by
the method such as lysis with sonication, lysozyme, and
freeze-thawing, the membrane fraction comprising the rat CTGF of
the present invention is obtained by the method such as
centrifugation or filtration. The membrane fraction is solubilized
with a detergent such as Triton-X100 to obtain the crude extract.
Finally, the protein is isolated and purified from the crude
extract by the usual method as illustrated above.
[0474] The "transgenic mouse" of the present is a transgenic mouse
in which the above human CTGF encoding DNA (cDNA or genomic DNA)
prepared by the method mentioned above has been integrated into the
endogenous gene locus of the mouse. This transgenic mouse expresses
and secretes the human CTGF in vivo.
[0475] The transgenic mouse can be prepared according to the method
usually used for producing a transgenic animal (for example, see
"Newest Manual of Animal Cell Experiment", LIC press, Chapter 7,
pp.361-408, (1990)). Specifically, for example, a transgenic mouse
can be produced as follows. Embryonic stem cells (ES cells)
obtained from normal mouse blastocysts are transformed with an
expression vector in which the gene encoding the human CTGF has
been inserted in an expressible manner. ES cells in which the gene
encoding the human CTGF has been integrated into the endogenous
gene are screened by a usual method. Then, the ES cells screened
are microinjected into a fertilized egg (blastocyst) obtained from
another normal mouse (Proc. Natl. Acad. Sci. USA, Vol.77, No.12,
pp.7380-7384 (1980); U.S. Pat. No. 4,873,191). The blastocyst is
transplanted into the uterus of another normal mouse as the foster
mother and chimeric transgenic mice are born. By mating the
chimeric transgenic mice with normal mice, heterozygous transgenic
mice are obtained. By mating the heterozygous transgenic mice with
each other, homozygous transgenic mice are obtained according to
Mendel's laws.
BRIEF DESCRIPTION OF THE DRAWINGS
[0476] FIG. 1 shows the properties of various monoclonal antibodies
prepared by immunizing a variety of mammals with human CTGF or
mouse CTGF.
[0477] FIG. 2 shows the properties of various human monoclonal
antibodies prepared by immunizing human antibody-producing
transgenic mice with human CTGF.
[0478] FIG. 3 is an SDS-polyacrylamide gel electrophoretic pattern
of human, mouse, and rat recombinant CTGFs. The proteins were
affinity purified by using a column coupled with the monoclonal
antibody "8-86-2" reactive to all of human, mouse and rat
CTGFs.
[0479] FIG. 4 shows the growth-promoting activities of human,
mouse, and rat CTGFs for the rat kidney-derived fibroblast cell
line NRK-49F. The proteins were affinity purified by using a column
coupled with the monoclonal antibody "8-86-2" reactive to all of
human, mouse, and rat CTGFs.
[0480] The ordinate indicates the [.sup.3H]-thymidine uptake of the
cells as an index of the growth promoting activity; the abscissa
indicates the concentrations of the respective recombinant CTGFs
used.
[0481] FIG. 5 shows the reactivity of various monoclonal antibodies
to human CTGF. The monoclonal antibodies were prepared by
immunizing a variety of mammals with human or mouse CTGF.
[0482] The ordinate indicates the fluorescence intensity as an
index of the reactivity of the monoclonal; the abscissa indicates
clone names of the monoclonal antibodies tested by ELISA with
different concentrations of the human CTGF.
[0483] FIG. 6 shows the reactivity of various monoclonal antibodies
to the mouse CTGF. The monoclonal antibodies were prepared by
immunizing a variety of mammals with human or mouse CTGF.
[0484] The ordinate indicates the fluorescence intensity as an
index of the reactivity of the monoclonal antibody; the abscissa
indicates clone names of the monoclonal antibodies tested by ELISA
with different concentrations of mouse CTGF.
[0485] FIG. 7 shows the reactivity of various monoclonal antibodies
to rat CTGF. The monoclonal antibodies were prepared by immunizing
a variety of mammals with human or mouse CTGF.
[0486] The ordinate indicates the fluorescence intensity-as an
index of the reactivity of the monoclonal antibody; the abscissa
indicates clone names of the monoclonal antibodies tested by ELISA
with different concentrations of rat CTGF.
[0487] FIG. 8 shows the reactivity of various human monoclonal
antibodies to human CTGF. The antibodies were prepared by
immunizing the human antibody-producing transgenic mice with human
CTGF.
[0488] The ordinate indicates the fluorescence intensity as an
index of the reactivity of the monoclonal antibody; the abscissa
indicates clone names of the human monoclonal antibodies tested by
ELISA with different concentrations of human CTGF.
[0489] FIG. 9 shows the reactivity of various human monoclonal
antibodies to mouse CTGF. The antibodies were prepared by
immunizing the human antibody-producing transgenic mice with human
CTGF.
[0490] The ordinate indicates the fluorescence intensity as an
index of the reactivity of the monoclonal antibody; the abscissa
indicates clone names of the human monoclonal antibodies tested by
ELISA with different concentrations of mouse CTGF.
[0491] FIG. 10 shows the reactivity of various human monoclonal
antibodies to rat CTGF. The antibodies were prepared by immunizing
the human antibody-producing transgenic mice with human CTGF.
[0492] The ordinate indicates the fluorescence intensity as an
index of the reactivity of the monoclonal antibody; the abscissa
indicates clone names of the human monoclonal antibodies tested by
ELISA with different concentrations of rat CTGF.
[0493] FIG. 11 shows the inhibiting activity of various monoclonal
antibodies for the binding of human or mouse CTGF to the human
kidney-derived fibroblast cell line 293-T. The monoclonal
antibodies were prepared by immunizing a variety of mammals with
human or mouse CTGF.
[0494] The ordinate indicates that fluorescence intensity as an
index of the inhibiting activity; the abscissa indicates clone
names of various monoclonal antibodies tested.
[0495] FIG. 12 shows the inhibiting activity of various human
monoclonal antibodies for the binding of human CTGF to the rat
kidney-derived fibroblast cell line NRK-49F. The antibodies were
prepared by immunizing human antibody-producing transgenic mice
with human CTGF.
[0496] The ordinate indicates the rate of bound cells (%); the
abscissa indicates clone names of the various monoclonal antibodies
tested. The total number of cells added is taken as 100%.
[0497] FIG. 13 shows the inhibiting activity of various human
monoclonal antibodies for the binding of human CTGF to the rat
kidney-derived fibroblast cell line NRK-49F, the human
osteosarcoma-derived cell line MG-63, or human lung-derived
fibroblast cells. The antibodies were prepared by immunizing the
human antibody-producing transgenic mice with human CTGF. The
ordinate indicates the rate of bound cells (%); the abscissa
indicates clone names of the various monoclonal antibodies tested.
The total number of cells added is taken as 100%.
[0498] FIG. 14 shows immunological staining patterns of tissue
sections from arteriosclerotic lesions of a rabbit as an
arteriosclerosis model rabbit. By using various monoclonal
antibodies prepared by immunizing a variety of mammals with human
or mouse CTGF, the sections were stained and the reactivity of the
antibodies to the lesions was assessed.
[0499] The panel (a) shows a control stain; (b) shows the stain
with the monoclonal antibody B35.1; (c) shows the stain with-the
monoclonal antibody B29.6; (d) shows the stain with the monoclonal
antibody 13-51-2; (e) shows the stain with the monoclonal antibody
A4.3; (f) shows the stain with the monoclonal antibody C114.4; (g)
shows the stain with the monoclonal antibody A11.1; (h) shows the
stain with the monoclonal antibody A29.6; and (1) shows the stain
with the monoclonal antibody C26.11.
[0500] FIG. 15 shows the inhibiting activity of various human
monoclonal antibodies towards for the proliferation of the rat
kidney-derived fibroblast cells NRK-49F stimulated by the purified
human CTGF. The antibodies were prepared by immunizing the human
antibody-producing transgenic mice with human CTGF.
[0501] The ordinate indicates the [.sup.3H]-thymidine uptake of the
cells as an index of the growth promoting activity; the abscissa
indicates clone names of the human monoclonal antibodies tested by
using the purified human CTGF of different concentrations.
[0502] FIG. 16 shows the inhibiting activity of various human
monoclonal antibodies towards the proliferation of the rat
kidney-derived fibroblast cells NRK-49F stimulated by the purified
human CTGF. The antibodies were prepared by immunizing the human
antibody-producing transgenic mice with human CTGF.
[0503] The ordinate indicates the [.sup.3H]-thymidine uptake of the
cells as an index of the growth promoting activity; the abscissa
indicates clone names of the human monoclonal antibodies tested by
using purified human CTGF of different concentrations.
[0504] FIG. 17 shows the therapeutic effect of various human
monoclonal antibodies on kidney diseases and fibrotic diseases in
tissues. The antibodies were prepared by immunizing the human
antibody-producing transgenic mice with human CTGF.
[0505] The ordinate indicates the concentration of hydroxyproline
which is an index of advancement of the disease; the abscissa
indicates clone names of the human monoclonal antibodies
administered.
[0506] FIG. 18 schematically shows the procedure for the sequence
determination of the DNAs encoding the heavy chain and the light
chain of the human anti-human CTGF monoclonal antibody.
[0507] FIG. 19 shows the calibration curves of the human CTGF
standard, mouse CTGF standard, and rat CTGF standard. The curves
were obtained by sandwich ELISA using the monoclonal antibodies
8-64-6 and 8-86-2.
[0508] The ordinate indicates the fluorescence intensity; the
abscissa indicates the concentration of the standards of CTGF.
[0509] FIG. 20 shows the calibration curves of the human CTGF
standard, mouse CTGF standard, and rat CTGF standard. The curves
were obtained by sandwich ELISA using the monoclonal antibodies
13-51-2 and -8-86-2.
[0510] The ordinate indicates the fluorescence intensity; the
abscissa indicates the concentration of the standards of CTGF.
[0511] FIG. 21 shows CTGF concentrations in various serum samples
from patients affected with biliary atresia. The concentration was
determined by sandwich ELISA with the monoclonal antibodies 8-64-6
and 8-86-2.
[0512] The ordinate indicates the concentrations (CTGF content)
determined; the abscissa indicates the subject groups tested. The
samples were obtained from three groups; Group 1 (I) consisting of
patients with normal clinical findings; Group 2 (II) consisting of
patients with symptoms progressing; and Group 3 (III) consisting of
patients with severe symptoms in need of liver transplantation.
[0513] FIG. 22 shows CTGF concentrations in various serum samples
from patients affected various diseases. The concentration was
determined by sandwich ELISA with the monoclonal antibodies 8-64-6
and 8-86-2.
[0514] The ordinate indicates the concentrations (CTGF content)
determined; the abscissa indicates the diseases with which the
patients affected.
[0515] FIG. 23 shows CTGF concentrations in the synovial fluid
samples; from patients affected with rheumatoid arthritis or
osteoarthritis. The concentration was determined by sandwich ELISA
with the monoclonal antibodies 8-64-6 and 8-86-2.
[0516] The ordinate indicates the concentrations (CTGF content)
determined; the abscissa indicates the diseases with which the
patients affected.
[0517] FIG. 24 are SDS-polyacrylamide gel patterns showing the
result of Western blotting of human CTGF fractions purified from
human fetal skin-derived fibroblast cells by using a heparin
column.
[0518] Lanes 1, 2, and 3 contain fractions eluted with 0.2 M, 0.6
M, and 2.0 M NaCl, respectively, and the samples were treated with
pre-immune antibody (pre-immune serum from normal rabbit). Lanes 4,
5, and 6 contain the fractions eluted with 0.2 M, 0.6 M, and 2.0 M
NaCl, respectively, and the samples were treated with an anti-human
CTGF polyclonal antibody.
[0519] FIG. 25 shows the growth-promoting activity of human CTGF
for rat kidney-derived fibroblast cells NRK-49F. The human CTGF is
purified from human fetal skin-derived fibroblast cells by using a
heparin column. The fraction eluted with 0.6 M NaCl was used after
diluting 10-, 30-, 100-, or 300 times.
[0520] "PDGF" denotes the PDGF used as a positive control in this
assay; "NC" indicates negative control where the CTGF was omitted
in the assay.
BEST MODE FOR CARRYING OUT THE INVENTION
[0521] The present inventions are further described in detail by
referring to Examples herein below, but are not to be construed as
being restricted thereto.
EXAMPLE 1
Preparation of a Polyclonal Antibody to Human CTGF
[0522] The peptide (Cys-Glu-Ala-Asp-Leu-Glu-Glu-Ash-Ile-Lys)
corresponding to amino acid residues in the positions of 242 to 252
of human CTGF was synthesized with a peptide synthesizer (Applied
Biosystems) according to a usual method. The peptide emulsified
with Freund's complete adjuvant was used as an immunogen. The
peptide (0.32 mg/kg) was given subcutaneously to a New Zealand
white rabbit (NZW; Simunek, Inc.). The dose and the interval were:
0.8 mg of the peptide on day 1; 0.8 mg on day 14; 0.8 mg on day 35;
and 0.8 mg on day 49. The antibody titer in the serum was assayed
from time to time by using the peptide. The serum was then
collected according to a usual method. The polyclonal antibody
(IgG) against human CTGF was purified from the serum by affinity
chromatography using agarose on which the peptide was immobilized.
The reactivity to human CTGF was verified by Enzyme-linked
immunosorbent assay (ELISA ) and Western blotting.
EXAMPLE 2
Preparation of Recombinant Human CTGF
[0523] <2-1> The Transient Expression of Human Recombinant
CTGF in Human Kidney-Derived Fibroblast Cell Line 293
[0524] Complementary DNA encoding human CTGF was prepared according
to a usual method by PCR. Specifically, the cDNA was prepared by
using, as a template, heterogeneous cDNAs derived from a human
chondroma cell line, HCS2/8, and by using primers designed based on
the-human CTGF cDNA sequence (The Journal of Cell Biology, Vol.
114, No. 6, p. 1287-1294, 1991). The human CTGF cDNA so obtained
comprising the coding region was inserted into a plasmid, pcDNA3.1
(-) (Invitrogen Co.) to construct the expression vector. The human
kidney-derived fibroblast cell line 293-T (ATCC CRT1573) was
transformed with the prepared vector by electroporation. The
transformant was cultured in a serum-free medium, ASF104 (Ajinomoto
Co. Inc.), for three days, for the transient expression of the
recombinant human CTGF. The human CTGF expression was confirmed by
Western blotting using the polyclonal antibody prepared in Example
1.
[0525] The collected culture supernatant was subjected to
salting-out with ammonium sulfate, and then to heparin-column
chromatography. The column was washed with 0.3M NaCl/PBS and the
human CTGF fraction was eluted with 0.5M NaCl/PBS. Thus, the
partially purified human CTGF was obtained.
[0526] <2-2> Stable Expression of Human Recombinant CTGF in a
Human Epithelioid Cell Line, Hela Cell
[0527] Complementary DNA encoding human CTGF was prepared according
to a usual method by PCR in the same manner as described in Example
<2-1>. The human CTGF cDNA so obtained comprising the coding
region was inserted into a plasmid, pcDNA3.1 (-) (Invitrogen Co.)
to construct the expression vector. The human epithelioid cell
line, Hela cell (ATCC CCL-2), was transformed with the prepared
vector by electroporation. The transformants were cultured in a
RPMI1640 medium containing GENETICIN (0.8 mg/ml; GIBCO-BRL) and 10%
fetal calf serum for about two weeks, in order to select
GENETICIN-resistant clones of the transformant. The transformant
selected was cultured in the serum-free medium ASF104 (Ajinomoto
Co. Inc.) for the expression of the recombinant human CTGF. The
expression of the human CTGF was verified by Western blotting using
the polyclonal antibody prepared in Example 1.
[0528] The collected culture supernatant was subjected to
salting-out with ammonium sulfate, and then to heparin-column
chromatography. The column was washed with 0.3M NaCl/PBS and the
human CTGF fraction was eluted with 0.5M NaCl/PBS. Thus, the
partially purified human CTGF was obtained.
EXAMPLE 3
Preparation of Recombinant Mouse CTGF
[0529] Partially purified recombinant mouse CTGF was prepared by
the same method described in Example 2, based on the cDNA sequence
of mouse CTGF reported previously (Unexamined Published Japanese
Patent Application (JP-A) No. Hei 5-255397; Cell Growth Differ.,
Vol. 2, No. 5, p. 225-233, 1991; FEBS Letters, Vol. 327, No. 2, p.
125-130, 1993; DNA Cell Biol., Vol. 10, No. 4, p. 293-300,
1991).
EXAMPLE 4
Preparation of Anti-Human CTGF Monoclonal Antibody and Anti-Mouse
CTGF Monoclonal Antibody
[0530] Preparation of the monoclonal antibodies in this example was
performed by the conventional method described in "Experimental
Medicine (supplement): Handbook for Cellular Engineering
Technology, eds., T. Kuroki et al., Yodosha, page 66-74, 1992),"
and "Introductory Manual for Monoclonal Antibody Experiment (T.
Ando et al., Kodansha, 1991)."
[0531] Here in this example, either of the recombinant human CTGF
preparations obtained by either of the two methods described in
Example 2, or a mixture thereof, was used as an immunogen. The
mouse CTGF used was the recombinant mouse CTGF prepared in Example
3.
[0532] The immune animals used were: (1) normal mouse (Balb/c
mouse, female, 4- to 5-week old; Shizuoka experimental animal
center); (2) normal rat (Wistar rat, female, 4- to 5-week old;
Shizuoka experimental animal center); (3) normal hamster (Armenian
hamster, male, 4- to 5-week-old; Oriental Yeast Co., Ltd.); and (4)
the human antibody-producing transgenic mouse created by using the
method described above (refer the following reports: Nature
Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p.
146-156, 1997; Published Japanese Translation of PCT International
Application No. Hei 4-504365; Published Japanese Translation of PCT
International Application No. Hei 7-509137; Nikkei Science, June
edition, page 40-50, 1995).
[0533] Unless otherwise stated, the same method was used for the
preparation of monoclonal antibodies derived from any animal.
Multi-well microplates were used for culturing cells.
[0534] <4-1> Preparation of Hybridomas Producing Anti-Human
CTGF Monoclonal Antibody
[0535] The above-mentioned normal mouse and the human-antibody
producing transgenic mouse were immunized with the partially
purified recombinant human CTGF (1 .mu.g/animal) prepared in
Example 2. The immunogen, together with complete Freund's adjuvant,
was given to the mice by footpad injection for primary immunization
(day 0). The recombinant human CTGF was given to the mice by
footpad injection every week after the primary immunization. The
booster immunization was performed four times or more in total. The
final immunization was carried out by the same procedure two days
before the collection of lymph node cells described
hereinafter.
[0536] The lymph node cells collected from each animal and mouse
myeloma cells were mixed at a ratio of 5:1. Hybridomas were
prepared by cell fusion using polyethylene glycol 4000 or
polyethylene glycol 1500 (GIBCO) as a fusing agent. The lymph node
cells of the normal mouse were fused with mouse myeloma PAI cells
(JCR No. B0113; Res. Disclosure Vol. 217, p. 155, 1982), and the
lymph node cells of human antibody producing-transgenic mouse were
fused with mouse myeloma P3/X63-AG8.653 cells (ATCC No. CRL
1580).
[0537] The resulting hybridomas were selected by culturing the
fused cells in an ASF104 medium (Ajinomoto Co. Inc.) containing HAT
supplemented with 10% fetal calf serum (FCS) and aminopterin.
[0538] The reactivity of the culture supernatant of each hybridoma
clone to the recombinant human CTGF used as the immunogen was
measured by ELISA described hereinafter. Many antibody
producing-hybridomas were thus obtained from each animal
species.
[0539] The clones named 8-64-6, 8-86-2, 8-97-3, 8-149-3, and
15-38-1 (FIG. 1) were obtained from normal mice (mouse anti-human
CTGF monoclonal antibodies).
[0540] The hybridoma clones, 8-86-2 and 8-64-6, both have been
deposited internationally since Dec. 18, 1997 at The National
Institute of Bioscience and Human-Technology, The Agency of
Industrial Science and Technology, The Ministry of International
Trade and Industry (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) (clone
8-86-2: international deposit accession No. FERM BP-6208; clone
8-64-6: international deposit accession No. FERM BP-6209).
[0541] The clones (producing the human anti-human CTGF monoclonal
antibodies) named A4.3, A11.1, A15.1, A29.6, B13.7, B22.2, B29.6,
B35.1, C2.1, C26.11, C59.1, C114.4, M32.2, M33.3, M84.4, M107.2,
M122, M124.6, M194.2, M244, M255, M268.1, M288.5, M291.2, M295.2,
M315, M320.2, N45.2, N50.1, and N60.1 (FIGS. 1 and 2) were obtained
from the human antibody-producing transgenic mice.
[0542] The hybridoma clone A11.1 has been deposited internationally
since Sep. 25, 1998 at The National Institute of Bioscience and
Human-Technology, The Agency of Industrial Science and Technology,
The Ministry of International Trade and Industry (1-1-3 Higashi,
Tsukuba, Ibaraki, Japan) (international deposit accession No. FERM
BP-6535).
[0543] The hybridoma clones, B22.2, M84.4, and M320.2 have been
deposited internationally since Dec. 15, 1998 at The National
Institute of Bioscience and Human-Technology, The Agency of
Industrial Science and Technology, The Ministry of International
Trade and Industry (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) (clone
B22.2: international deposit accession No. FERM BP-6598; clone
M84.4: international deposit accession No. FERM BP-6599; clone
M320.2: international deposit accession No. FERM BP-6600).
[0544] Described above, the hybridoma clones producing the human
monoclonal antibodies of the present invention are indicated by
symbols all through the examples including the present example and
the drawings or the tables showing the experimental results
obtained in each example.
[0545] The alphabet followed by numerals up to the dot mark of each
symbol corresponds to the name of parental clone. The numerals
after the dot mark of the symbol means a subclone obtained from the
parental clone by subcloning.
[0546] The numerals indicating the subclones may occasionally be
abbreviated in any of the examples including the present example
and the drawings or the tables showing the experimental results
obtained in each example. However, it should be noted that the
abbreviated symbols indicate the same clones as those indicated by
the non-abbreviated symbols in FIGS. 1 and 2.
[0547] <4-2> Preparation of Hybridomas Producing Anti-Mouse
CTGF Monoclonal Antibody
[0548] The above-mentioned normal rat and normal hamster were
immunized with partially purified recombinant mouse CTGF (2
.mu.g/animal) prepared in Example 3, together with complete
Freund's adjuvant, by footpad injection for primary immunization
(day 0). The booster immunization was performed by footpad
injection every week after the primary immunization four times or
more in total. The final immunization was carried out by the same
procedure two days before the collection of lymph node cells. The
collection of lymph node cells is described below.
[0549] Popliteal lymph node cells were collected from each
immunized animals according to a usual method by a surgical
operation.
[0550] The lymph node cells collected from each animal and myeloma
cells PAI (JCR No.B0113; Res. Disclosure Vol. 217, p. 155, 1982)
were mixed at a ratio of 5:1. The hybridomas were prepared by cell
fusion using polyethylene glycol 4000 or polyethylene glycol 1500
(GIBCO) as a fusing agent.
[0551] The hybridomas were selected by culturing the fused cells in
an ASF104 medium (Ajinomoto Co. Inc.) containing HAT supplemented
with 10% fetal calf serum (FCS) and aminopterin.
[0552] The reactivity of the culture supernatant of each hybridoma
to the recombinant mouse CTGF used as the immunogen was measured by
ELISA described hereinafter. Many antibody producing-hybridomas
were, thus, obtained from each animal.
[0553] The clones (producing rat anti-mouse CTGF monoclonal
antibodies) named 13-51-2, 17-132, 23-96, 24-53, 24-67, 25-91,
25-101, 25-256, 25-338, 25-410, and 25-463 (FIG. 1) were obtained
from normal rats.
[0554] The clone (producing hamster anti-mouse CTGF monoclonal
antibody) named 2-228-1 (FIG. 1) was obtained from normal
hamster.
[0555] <4-3> Screening of Hybridomas Producing Monoclonal
Antibodies by ELISA
[0556] ELISA performed in Examples <4-1> and <4-2> is
as follows.
[0557] The recombinant human CTGF (0.2 .mu.g/well) prepared in
Example 2 or the recombinant mouse CTGF (0.2 .mu.g/well) prepared
in Example 3 was added into each well of a 96-well ELISA microplate
(Corning Costar Co.). The plate was incubated at room temperature
for 2 hours for the adsorption of the recombinant human CTGF or the
recombinant mouse CTGF onto the microplate. The supernatants were
discarded and then the blocking reagent (200 .mu.l; phosphate
buffer containing 3% BSA) was added into each well. The plate was
incubated at room temperature for 2 hours for the blocking of
CTGF-free sites on the microplate. Each well was washed three times
with 200 .mu.l of phosphate buffer containing 0.1% Tween 20. Thus,
each well of the microplate was coated with the recombinant human
CTGF or recombinant mouse CTGF.
[0558] Culture supernatant (100 .mu.l) of each hybridoma was added
into each well of the plate, and the reaction was allowed to
proceed for 40 minutes. Each well was then washed three times with
200 .mu.l of phosphate buffer containing 0.1% Tween 20.
[0559] In the next step, biotin-labeled sheep anti-mouse
immunoglobulin antibody (50 .mu.l; Amersham) was added to the wells
where the culture supernatant of the monoclonal antibody-producing
hybridoma derived from normal mouse had been placed; biotin-labeled
sheep anti-rat immunoglobulin antibody (50 .mu.l; Amersham). was
added to the wells where the culture supernatant of the monoclonal
antibody-producing hybridoma derived from normal rat had been
placed; biotin-labeled goat anti-hamster immunoglobulin antibody
(50 .mu.l; Cedarlane) was added to the wells where the culture
supernatant of the monoclonal antibody-producing hybridoma derived
from normal hamster had been placed; biotin-labeled goat anti-human
immunoglobulin antibody (50 .mu.l; American Qualex International
Inc.) was added to the wells where the culture supernatant of the
monoclonal antibody-producing hybridoma derived from the human
antibody-producing transgenic mouse had been placed. The plates
were incubated at room temperature for 1 hour.
[0560] The microplate was washed with phosphate buffer containing
0.1% Tween 20. A solution of streptavidin-.beta.-galactosidase (50
.mu.l; Gibco-BRL), diluted 1000 times with a solution (pH7.0)
containing 20 mM HEPES, 0.5M NaCl and bovine serum albumin (BSA, 1
mg/ml), was added into each well. The plate was incubated at room
temperature for 30 minutes.
[0561] Subsequently, the microplate was washed with phosphate
buffer containing 0.1% Tween 20. A solution of 1%
4-Methyl-umbelliferyl-.beta.-D- -galactoside (50 .mu.l; Sigma) in a
phosphate buffer (pH7.0) containing 100 mM NaCl, 1 mM MgCl.sub.2
and 1 mg/ml BSA, was added into each well. The plate was incubated
at room temperature for 10 minutes. 1M Na.sub.2CO.sub.3 (100 .mu.l)
was added into each well to stop the reaction. The fluorescence
intensity was measured in a FLUOROSCAN II MICROPLATE FLUOROMETER
(Flow Laboratories Inc.) at a wavelength of 460 nm (excitation
wavelength: 355 nm).
[0562] <4-4> Large-Scale Preparation of Monoclonal
Antibodies
[0563] Each hybridoma clone (10.sup.6-10.sup.7 cells/0.5 ml/each
mouse) described above was injected intraperitoneally to ICR nude
mice (female, 7- to 8-week old; Charles River). Ten to twenty days
after the injection, the ascitic fluids were collected from the
mice according to a usual method by opening the abdomen under
anesthesia. The monoclonal antibodies were prepared from the
ascitic fluids in a large quantity.
[0564] <4-5> Purification of Monoclonal Antibodies
[0565] Each monoclonal antibody-containing ascitic fluid obtained
in <4-4> was centrifuged, and the resulting supernatant was
diluted 3 times with 0.06M acetate buffer (pH 4.0). The pH of the
dilute was adjusted to 4.8 by adding 1N hydrochloric acid.
Subsequently, 0.033 ml of caprylic acid (Wako Pure Chemical
Industries. Ltd.) was added little by little to every 1 ml of the
ascitic fluid, while stirring the mixture at room temperature. The
mixture was allowed to react for 30 minutes, while being stirred.
The proteins, except the antibody, were removed by centrifugation
(10,000 rpm, for 20 minutes). The supernatant obtained by
centrifugation was filtered by using a filter (Millipore Co.), the
white precipitate was discarded. The filtrate obtained was dialyzed
with phosphate buffer (for 2 hours).
[0566] After the dialysis, ammonium sulfate (26.2 g/100 ml) was
added thereto little by little, while stirring the mixture at room
temperature. The reaction of the mixture was carried out at
4.degree. C. for 120 minutes, while being stirred. The resulting
precipitate was then recovered by centrifugation (10,000 rpm, for
20 minutes). The recovered precipitate was dissolved with phosphate
buffer and dialyzed with phosphate buffer (at 4.degree. C., for 24
hours). Each monoclonal antibody was thus purified.
[0567] <4-6> Determination of the Isotype
[0568] Each isotype of the above-mentioned anti-human CTGF
monoclonal antibody derived from normal mouse (8-64-6, 8-86-2,
8-97-3, 8-149-3, and 15-38-1) was determined by using an
isotype-determining kit for mouse monoclonal antibody (Amersham).
The determination was performed according to the supplier's
protocol attached to the kit. All the antibodies were determined to
be IgG1/.kappa. (FIG. 1).
[0569] Each isotype of the above-mentioned anti-human CTGF
monoclonal antibody derived from the human antibody-producing
transgenic mouse (A4.3, A11.1, A15.1, A29.6, B13.7, B22.2, B29.6,
B35.1, C2.1, C26.11, C59.1, C114.4, M32.2, M33.3, M84.4, M107.2,
M122, M124.6, M194.2, M244, M255, M268.1, M288.5, M291.2, M295.2,
M315, M320.2, N45.2, N50.1, and N60.1) was determined by using an
isotype-determining kit for human monoclonal antibody (American
Qualex International Inc.). The determination was performed
according to the supplier's protocol attached to the kit. All the
antibodies were determined to be IgG2/.kappa. (FIGS. 1 and 2).
[0570] <4-7> Preparation of the Affinity Column
[0571] An affinity column was prepared by using NHS-activated
HiTrap column (HITRAP-NHS-ACTIVATED SEPHAROSE HP; 5 ml; Pharmacia
Biotec.) according to the protocol attached to the product.
Specifically, the preparation was done as follows:
[0572] The monoclonal antibody 8-86-2 (10 mg/ml SEPHAROSE) prepared
in Example <4-5> was dissolved in a 0.2M sodium hydrogen
carbonate solution (pH8.3) containing 0.5M NaCl. The solution (10
mg/ml SEPHAROSE) was loaded onto the NHS-ACTIVATED HITRAP COLUMN.
The monoclonal antibody 8-86-2 was allowed to react with the
NHS-ACTIVATED SEPHAROSE at 20.degree. C. for 45 minutes, to
immobilize the antibody on the SEPHAROSE.
[0573] Because of the high reactivity of the monoclonal antibody
8-86-2 to human, mouse and rat CTGFs, the affinity column prepared
with the antibody can be used for purifying any human CTGF, mouse
CTGF, and rat CTGF.
[0574] <4-8> Purification of Mammalian CTGF by Affinity
Chromatography
[0575] Culture supernatant was collected from each culture of the
HeLa transformant cells expressing human CTGF (Example
<2-2>), the HeLa transformant cells expressing mouse CTGF
(Example 3) and the HeLa transformant cells expressing rat CTGF
(Example <11-2>). The supernatant was fractionated by
heparin-column chromatography. The column was washed with 0.3M
NaCl/PBS, and then the protein fraction of interest was eluted with
0.7M NaCl/PBS. Thus, the crude fractions of human, mouse and rat
CTGFs were obtained.
[0576] Each crude fraction was loaded onto the affinity column,
prepared in Example <4-7>, in which the anti-CTGF antibody
8-86-2 had been immobilized. The column was washed with phosphate
buffer. The fraction of interest was then eluted with 0.1M glycine
buffer (pH2.5) and then was neutralized with 0.75M Tris-HCl buffer
(pH9.0). The eluted fractions were dialyzed against phosphate
buffer. Thus, highly purified recombinant CTGFs derived from human,
mouse and rat were obtained.
[0577] The purified recombinant CTGFs were electrophoresed on a
sodium dodecylsulfate polyacrylamide gel with a concentration
gradient of 10 to 20% polyacrylamide. The separated bands on the
gel were silver-stained, and bands of about 38-kDa corresponding to
human, mouse and rat CTGFs were found on the stained gel (FIG.
3).
[0578] <4-9> Examination of the Stimulatory Activity of
Purified CTGF for Cell Proliferation
[0579] To verify whether or not each of the CTGFs purified in
Example <4-8> has a biological activity, the stimulatory
activity of purified CTGF for cell proliferation was tested.
[0580] The cells of rat kidney fibroblast cells NRK-49F (ATCC
CRL-1570; 2.times.10.sup.3 cells/well) were cultured in a DMEM
medium containing 10% fetal calf serum (FCS) in a 96-well
microplate for three days. The culture supernatant was removed and
the cells were washed once with the DMEM medium. Then the cells
were cultured in a fresh DMEM medium for one day. Subsequently, the
purified recombinant CTGF was added in various concentrations (100,
50, 25, 12.5, 6.3, and 3.1 ng/ml) into each cell culture. The
culture was continued for 2 days and then [.sup.3H]-thymidine (3.7
kBq/well) was added thereto. After a 6-hour culture, the cells were
harvested for the measurement of [.sup.3H]-thymidine uptake by the
cells. The measurement was carried out in a liquid scintillation
counter (Beckman). The cells were cultured in the same manner but
in the absence of CTGF, and [.sup.3H]-thymidine uptake by the cells
was measured as a control.
[0581] The result is shown in FIG. 4. It was evidenced that each of
the purified recombinant CTGFs derived from human, mouse, and rat
exhibited a concentration-dependent stimulatory activity for the
cell proliferation and accordingly all the recombinant CTGFs
possessed a biological function.
[0582] <4-10> Crossreactivity
[0583] The reactivities of various anti-human CTGF monoclonal
antibodies (10 .mu.g/ml) and anti-mouse CTGF monoclonal antibodies
(10 .mu.g/ml) to each of human CTGF, mouse CTGF and rat CTGF were
tested by ELISA in the same manner described in Example
<4-3>.
[0584] The microplates used in this assay were coated with each of
the recombinant human, mouse and rat CTGFs purified by using
affinity column prepared in Example <4-7>. The coating was
performed with the proteins in the concentrations of (A)100, 30,
and 10 ng/well or (B) 100, 10, and 1 ng/well.
[0585] In the assay of (B), negative control assay was carried out
by using a human monoclonal antibody against KLH (keyhole limpet
hemocyanin; Pierce Chemical Co.) in the same manner described
above. The anti-KLH antibody was prepared by immunizing the
above-described human antibody-producing transgenic mice with KLH
as an immunogen.
[0586] The assay results obtained with the concentration series (A)
are shown in FIGS. 5-7; the assay results obtained with the
concentration series (B) are shown in FIGS. 8-10.
[0587] The results shown in FIGS. 5-10 are also summarized in the
column of "crossreactivity" in FIGS. 1 and 2.
[0588] Within the column of "crossreactivity" of FIG. 1, the
results are shown in the order of 100, 30 and 10 ng/well of the
coating concentration from the left. In each coating concentration,
the reactivity represented by the fluorescence intensity of 1000 or
more is marked with ".largecircle."; 500 or more and less than
1000, ".DELTA."; less than 500, ".times.."
[0589] Within the column of "crossreactivity" of FIG. 2, the
results are shown in the order of 100, 10 and 1 ng/well of the
coating concentration from the left. In each coating concentration,
the reactivity represented by the fluorescence intensity of 1000 or
more is marked with ".largecircle."; 500 or more and less than
1000, ".DELTA."; less than 500, ".times.".
[0590] Thus, it was revealed that the monoclonal antibodies of the
present invention had a variety of characteristics in terms of the
crossreactivity.
[0591] <4-11> Activity of Inhibiting the Binding of CTGF to
Various Cells
[0592] A recent study has revealed that CTGF is involved in cell
adhesion (Exp. Cell. Res., Vol. 233, p. 63-77, 1997). The
inhibiting effect of the various monoclonal antibodies prepared
above on the CTGF-mediated cell adhesion was investigated as an
index of judging whether or not the antibodies have the activity
(neutralizing activity) of inhibiting the CTGF functions. The test
was performed by using the three distinct methods described below
in <4-11-1> to <4-11-3>.
[0593] <4-11-1> Inhibition of the Binding of CTGF with Human
Kidney-Derived Fibroblast Cell Line 293-T
[0594] The above-prepared various monoclonal antibodies (0.5
.mu.g/well) reactive to human CTGF were added into the wells of a
microplate coated with the recombinant human CTGF (the coating
concentration: 0.5 .mu.g/well). The immobilization of the human
CTGF on the plate was performed by the same procedure described in
Example <4-3>. The above-prepared various monoclonal
antibodies (0.5 .mu.g/well) reactive to mouse CTGF were added into
the wells of a microplate coated with the recombinant mouse CTGF
(the coating concentration: 0.5 .mu.g/well). The immobilization of
the mouse CTGF on the plate was performed by the same procedure
described in Example <4-3>.
[0595] The supernatants were removed from each plate, and then the
cells of human kidney-derived fibroblast cell line 293-T (ATCC
CRL1573) labeled with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
tetraacetoxymethyl ester (BCECF; Molecular Probes Inc.) were added
into each well (5.times.10.sup.4 cells/well). The plates were
allowed to stand at 4.degree. C. for 1 hour.
[0596] The floating cells were removed and then the cells adhering
to the plates were solubilized by adding phosphate buffer (100
.mu.l) containing 1% NP-40 thereto. The release of BCECF into the
culture supernatant was caused by cytolysis. The intensity of
fluorescence emitted by BCECF was measured by the FLUOROSCAN II
MICROPLATE FLUOROMETER (Flow Laboratories Inc.).
[0597] Control assay was carried out in the same manner described
above but without any antibodies added.
[0598] The results are shown in FIG. 11. The results shown in FIG.
11 are also summarized in the column of "Activity of inhibiting the
binding of 293 cells" in FIG. 1. In the column of FIG. 1, the mark
".largecircle." indicates that the cell adhesion was inhibited
significantly by the antibody, and the mark ".times." indicates
that the antibody exhibited no inhibiting activity.
[0599] <4-11-2> Inhibition of the Binding of CTGF with Rat
Kidney-Derived Fibroblast Cell Line NRK-49F
[0600] The above-prepared various monoclonal antibodies (the final
concentration: 20 .mu.g/well, 6 .mu.g/well or 2 .mu.g/well)
reactive to the human CTGF were added into the wells of a
microplate coated with the recombinant human CTGF (the coating
concentration: 1 .mu.g/well) prepared by the same procedure
described in Example <4-3>.
[0601] The cells of rat kidney-derived fibroblast cell line NRK-49F
(ATCC CRL1570) labeled with
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
tetraacetoxymethyl ester (BCECF; Molecular Probes Inc.) were added
into each well (1.times.10.sup.4 cells/well). The plates were
allowed to stand at 4.degree. C. for 1 hour.
[0602] The floating cells were removed and then the cells adhering
to the plates were solubilized by adding phosphate buffer (100
.mu.l) containing 1% NP-40 thereto. The release of BCECF into the
culture supernatant was caused by cytolysis. The intensity of
fluorescence emitted by BCECF was measured by the FLUOROSCAN II
MICROPLATE FLUOROMETER (Flow Laboratories Inc.).
[0603] Control assay was carried out in the same manner described
above but without any antibodies added. Negative control assay was
carried out by using human monoclonal antibody against KLH (keyhole
limpet hemocyanin; Pierce Chemical Co.) in the same manner
described above. The anti-KLH antibody was prepared by immunizing
the above-described human antibody-producing transgenic mice with
KLH.
[0604] Another control assay was performed in the same manner
described above but without any antibodies added and by using a
microplate on which no CTGF was immobilized.
[0605] The results are shown in FIG. 12. The result is shown as a
rate (%) of bound cells, which is calculated based on the value of
fluorescence intensity determined.
[0606] The results shown in FIG. 12 are also summarized in the
column of "Activity of Inhibiting the Binding of NRK Cells" in FIG.
2. In the column of FIG. 2, the mark ".largecircle." indicates that
the cell adhesion was significantly inhibited by the antibody, and
the mark ".times." indicates that the antibody exhibited weak
inhibiting activity or no activity.
[0607] <4-11-3> Inhibition of the Binding of CTGF with
Various Cells
[0608] The above-prepared various monoclonal antibodies (the final
concentration: 20 .mu.g/well) reactive to human CTGF were added
into the wells of microplate coated with the recombinant human CTGF
(the coating concentration: 1 .mu.g/well) prepared by the same
procedure described in Example <4-3>.
[0609] After the plates were allowed to stand for 60 minutes and
the supernatants were removed from each plate, the cells
(1.times.10.sup.4 cells/well) indicated below labeled with
2',7'-bis(2-carboxyethyl )-5(6)-carboxyfluorescein
tetraacetoxymethyl ester (BCECF; Molecular Probes Inc.) were added
into each well. The plates were allowed to stand at 4.degree. C.
for 1 hour. The following cells were used in this assay:
[0610] (1) human lung-derived fibroblast cell (NHLF2837;
Clonetics);
[0611] (2) human osteosarcoma-derived cell line MG-63 (ATCC
CRL1427); and
[0612] (3) rat kidney-derived fibroblast cell line NRK-49F (ATCC
CRL1570).
[0613] The floating cells were removed and then the cells adhering
to the plates were solubilized by adding phosphate buffer (100
.mu.l) containing 1% NP-40 thereto. The release of BCECF into the
culture supernatant was caused by cytolysis. The intensity of
fluorescence emitted by BCECF was measured in the FLUOROSCAN II
MICROPLATE FLUOROMETER (Flow Laboratories Inc.).
[0614] Control assay was carried out in the same manner described
above but without any antibodies added. Negative control assay was
carried out by using human monoclonal antibody against KLH (keyhole
limpet hemocyanin; Pierce Chemical Co.) in the same manner
described above. The anti-KLH antibody was prepared by immunizing
the above-described human antibody-producing transgenic mice with
KLH.
[0615] Another control assay was performed in the same manner
described above but without any antibodies added and by using a
microplate on which no CTGF was immobilized.
[0616] The results are shown in FIG. 13. The result is shown as a
rate (%) of bound cells, which is calculated based on the value of
fluorescence intensity determined.
[0617] <4-12> Crossreactivity to Rabbit Tissues
[0618] Arteriosclerotic lesions were obtained from the
hyperlipidemia model rabbit WHHL (Oriental Yeast Co., Ltd.) by a
surgical operation. Frozen sections were prepared from the
arteriosclerotic lesions according to a usual method.
[0619] Each section was stained by using a Vectastain Elite ABC kit
(Funakoshi Ltd.) according to the procedure described below.
[0620] After being fixed with acetone for 1-2 minutes and dried,
the sections were moistened with a diluted serum (10 ml PBS/150
.mu.l serum) for 30 minutes. The sections were washed with PBS, and
then primary antibodies (10 .mu.g/ml or culture supernatant of
hybridoma) were added. The primary antibodies used are the
above-mentioned anti-human CTGF monoclonal antibodies (clone:
8-86-2 and 8-149-3) derived from normal mouse; anti-mouse CTGF
monoclonal antibody derived from normal rat (clone: 13-51-2); and
anti-human CTGF monoclonal antibodies derived from the human
antibody-producing transgenic mouse (clone: A4.3, A11.1, A29.6,
B29.6, B35.1, C26.11, and C114.4). The sections were allowed to
stand for 40 minutes.
[0621] Subsequently, each section was washed with PBS, and then a
solution of biotinylated secondary antibody (10 .mu.l) was added.
The sections were allowed to stand for 30 minutes. Biotin-labeled
horse anti-mouse immunoglobulin antibody was used as the secondary
antibody when the primary antibody added was the anti-human CTGF
monoclonal antibody derived from normal mouse; biotin-labeled
rabbit anti-rat immunoglobulin antibody was used as the secondary
antibody when the primary antibody added was the anti-mouse CTGF
monoclonal antibody derived from normal rat; biotin-labeled goat
anti-human immunoglobulin antibody was used as the secondary
antibody when the primary antibody added was the anti-human CTGF
monoclonal antibody derived from the human antibody-producing
transgenic mouse.
[0622] Each section was allowed to stand in a methanol solution
containing 3% hydrogen peroxide for 10 minutes and then washed with
PBS. Then 100 .mu.l of an avidin-peroxidase solution (PBS (5
ml)/peroxidase-labeled avidin DH (100 .mu.l)/biotinylated hydrogen
peroxide H (100 .mu.l) was added to the sections. The sections were
allowed to stand for 30 minutes.
[0623] After washing with PBS, a diaminobenzidine tetra
hydrochloride solution (DAB) (Water (5 ml)/buffer solution (100
.mu.l)/DAB solution (200 .mu.l)/hydrogen peroxide solution (100
.mu.l)) was added to the sections. The sections were allowed to
stand for 2-10 minutes.
[0624] After washing with cold water for 5 minutes, the sections
were subjected by Giemsa staining method and mounted. Control
staining was carried out in the same manner by using as a primary
antibody a monoclonal antibody, which is non-reactive to CTGF and
is identical in isotype to the corresponding monoclonal antibody to
be tested. The stained and mounted sections were observed under a
microscope with magnifications of .times.100 and .times.200. The
results are shown in FIG. 14. The results shown in FIG. 14 are also
summarized in the column of "Reactivity to Tissues from
Arteriosclerotic Lesions of WHHL rabbit" in FIG. 1. In the column
of FIG. 1, the mark ".largecircle." indicates that the tissue was
stained with the antibody, and the mark ".times." indicates that
the tissues was weakly stained or not stained with the
antibody.
[0625] Anti-human CTGF monoclonal antibody clones derived from the
human antibody-producing transgenic mouse, A4.3, A11.1, A29.6,
C26.11 and C114.4, and a clone of anti-mouse CTGF monoclonal
antibody derived from normal rat, 13-51-2, exhibited the reactivity
to the tissues from arteriosclerotic lesions of rabbit.
[0626] <4-13> Activity of Inhibiting Cell Proliferation by a
Stimulus with CTGF
[0627] As shown above in Example <4-9>, CTGF induces
proliferation of a variety of cells (for example, fibroblast cells
derived from various tissues such as the kidney and lung, a variety
of tumor cells, and vascular endothelial cells).
[0628] In the present experiment, the inhibiting effect of
the-monoclonal antibodies of the present invention on the
CTGF-stimulated cell proliferation was examined described
below.
[0629] <4-13-1> Preparation of Cell Culture Medium Containing
CTGF
[0630] Neonatal human dermal fibroblast cells (NHDF; Becton
Dickinson) were cultured in a dish. The cells were washed twice
with a fetal calf serum (FCS)-free DMEM medium and then further
cultured with a fresh DMEM medium containing human transforming
growth factor-.beta. (TGF-.beta.; 1 ng/ml; R&D Systems) for 1
day.
[0631] The culture supernatant was recovered, and loaded onto to a
heparin column (HITRAP; Pharmacia Biotech). The column was washed
with 0.2M NaCl/PBS, and then the factor trapped in the column was
eluted with 0.6M NaCl/PBS. The eluate was dialyzed with PBS and was
used for the cell proliferation assay described below.
[0632] Because CTGF is a heparin-binding protein, CTGF can be
partially purified by using a heparin column. The presence of CTGF
in the sample obtained above was confirmed according to a usual
method by Western blotting using the rabbit anti-human CTGF
polyclonal antibody prepared in Example 1.
[0633] Control assay was carried out by using pre-immune serum
prepared from the rabbit (the same rabbit described in Example 1)
prior to the immunization with human CTGF.
[0634] The results are shown in FIG. 24.
[0635] <4-13-2> Cell Proliferation by a Stimulus with
Purified CTGF
[0636] The cells of rat kidney-derived fibroblast cell line NRK-49F
(ATCC CRL-1570; 1.times.10.sup.4 cells/well) were placed in the
wells of a 96-well microtiter plate, and cultured for 1 day. The
plate was washed twice with a FCS-free DMEM medium. Then the cells
were further cultured for 1 day. Subsequently, the CTGF sample
prepared above in <4-13-1> (diluted 10, 30, 100 and 300 times
with a DMEM medium) was added into each well, the culture was
continued for 18 hour. [.sup.3H]-thymidine (3.7 kBq/well) was added
into each well, and the culture was further continued for 6 hours.
The cells were harvested for the measurement of [.sup.3H]-thymidine
uptake by the cells. The measurement was carried out in a liquid
scintillation counter (Beckman).
[0637] Positive control experiment was carried out with PDGF in the
same manner. Negative control experiment was performed in the same
manner but without CTGF added.
[0638] The results are shown in FIG. 25.
[0639] <4-13-3> Activity of Inhibiting Cell Proliferation
[0640] The CTGF sample (diluted 20 times with a DMEM medium)
prepared above in <4-13-1> was allowed to react for 30
minutes with the human anti-human CTGF monoclonal antibodies of the
present invention (the final concentration: 20 .mu.g/ml, 2
.mu.g/ml, or 0.2 .mu.g/ml) that was prepared above. The same
experiment described in <4-13-2> was carried out with the
mixture.
[0641] Positive control assay was carried out in the same manner
but without any antibodies added. Negative control assay was
performed in the same manner in the absence of any of the CTGF
samples and the antibodies.
[0642] The results are shown in FIGS. 15 and 16.
[0643] The same experiment described above was repeated several
times. The results including those shown in FIGS. 15 and 16 are
summarized in the column "Activity of Inhibiting the proliferation
of NRK Cells" of FIG. 2. In the column of FIG. 2, the mark
".largecircle." indicates that the cell proliferation was
significantly inhibited by the antibody.
[0644] Thus, it was revealed that the human anti-human CTGF
monoclonal antibodies of the present invention significantly
suppressed or inhibited the proliferation of human fibroblast
cells.
[0645] <4-14> Epitope Mapping
[0646] The experiment described below was performed to determine
the sites (epitopes) in the structure of human CTGF responsible for
the specific binding with the human anti-human CTGF monoclonal
antibodies of the present invention.
[0647] This experiment was carried out by the inventive sandwich
ELISA with two antibodies. The method of the sandwich ELISA of the
present invention is described in detail in Example 5.
Specifically, the experiment was conducted according to the
following steps:
[0648] (Step 1)
[0649] Antibody-immobilized microplates, on which each of the human
anti-human CTGF monoclonal antibodies (0.3 .mu.g/50 .mu.l/well)
listed in FIG. 2 was immobilized, were prepared in the same manner
described later in Example <5-1>.
[0650] (Step 2)
[0651] Labeled monoclonal antibodies were prepared in the same
manner described later in Example <5-2>, by using the
monoclonal antibodies, A, B, C and D, of the present invention, as
follows:
[0652] [Antibody A]
[0653] Mouse monoclonal antibody 8-64-6 reactive to human CTGF
(derived from a hybridoma identified by international deposit
accession No. FERM BP-6209);
[0654] [Antibody B]
[0655] Human anti-human CTGF monoclonal antibody A11.1 (derived
from a hybridoma identified by international deposit accession No.
FERM BP-6535);
[0656] [Antibody C]
[0657] Human anti-human CTGF monoclonal antibody C26.11 (consisting
of the heavy chain having an amino acid sequence comprising the
amino acid sequence of SEQ ID NO: 8 as well as the light chain
having an amino acid sequence comprising the amino acid sequence of
SEQ ID NO: 18);
[0658] [Antibody D]
[0659] Human anti-human CTGF monoclonal antibody C59.1 (consisting
of the heavy chain having an amino acid sequence comprising the
amino acid sequence of SEQ ID NO: 10 as well as the light chain
having an amino acid sequence comprising the amino acid sequence of
SEQ ID NO: 20).
[0660] (Step 3)
[0661] ELISA was conducted in the same manner described later in
Example <5-3>. Specifically, the purified recombinant human
CTGF prepared in the above example was added (15 ng/well) into the
wells of each antibody-immobilized microplate prepared in Step 1.
After the antigen-antibody reaction was allowed to proceed, the
respective labeled monoclonal antibodies prepared in Step 2 were
added (0.1 .mu.l/50 .mu.l/well) and reacted thereto. After the
subsequent treatment conducted in the same manner described later
in Example <5-3>, the reaction was stopped by adding a stop
solution into each well. The intensity of fluorescence emitted in
each well was measured with the fluorometer at a wavelength of 460
nm (excitation: 355 nm).
[0662] The value of fluorescence intensity obtained is expected to
depend on the positional relationship between the site (epitope) on
the human CTGF bound with the monoclonal antibody immobilized on
the microplate and the site (epitope) on the human CTGF bound with
the labeled monoclonal antibody. Accordingly, the results are
predicted to be described below in (1) to (3).
[0663] (1) When the site (epitope) on the human CTGF where the
monoclonal antibody immobilized on the microplate binds, is
identical to the site (epitope) on the human CTGF where the labeled
monoclonal antibody binds, then the labeled antibody added later
cannot react with the antigen-antibody complex consisting of the
human CTGF and the monoclonal antibody immobilized on the
microplate. Therefore, the value of fluorescence intensity to be
measured in Step 3 can be sufficiently near 0 in this case.
[0664] (2) When the site (epitope) on the human CTGF where the
monoclonal antibody immobilized on the microplate binds, is
adjacent to the site (epitope) on the human CTGF where the labeled
monoclonal antibody binds, then, because of some steric hindrance
around the site, the labeled antibody added later becomes less
reactive to the antigen-antibody complex consisting of the human
CTGF and the monoclonal antibody immobilized on the microplate.
Therefore, the value of fluorescence intensity to be measured in
Step 3 is expected to be low in this case.
[0665] (3) When the site (epitope) on the human CTGF where the
monoclonal antibody immobilized on the microplate binds, is distant
from the site (epitope) on the human CTGF where the labeled
monoclonal antibody binds, then the labeled antibody added later
can react with the antigen-antibody complex consisting of the human
CTGF and the monoclonal antibody immobilized on the microplate.
Therefore, the value of fluorescence intensity to be measured in
Step 3 can be significantly high in this case.
[0666] The result of the experiment agreed with the above
prediction. The result is shown in the column of "Epitope mapping"
of FIG. 2.
[0667] The meaning of each alphabet used in the column of FIG. 2 is
illustrated bellow.
[0668] "(A)" indicates the "Antibody A" used above as the labeled
antibody;
[0669] "(B)" indicates the "Antibody B" used above as the labeled
antibody;
[0670] "(C)" indicates the "Antibody C" used above as the labeled
antibody;
[0671] "(D)" indicates the "Antibody D" used above as the labeled
antibody;
[0672] "A" indicates an epitope identical or almost identical to
the epitope of "Antibody A";
[0673] "B" indicates an epitope identical or almost identical to
the epitope of "Antibody B";
[0674] "C" indicates an epitope identical or almost identical to
the epitope of "Antibody C";
[0675] "D" indicates an epitope identical or almost identical to
the epitope of "Antibody D";
[0676] "-" indicates an epitope which is different from any of the
epitopes of "Antibody A," "Antibody B," "Antibody C" and "Antibody
D";
[0677] "B/C" indicates an epitope identical or almost identical to
the epitope of "Antibody B" and/or the epitope of "Antibody C";
[0678] "A-/B" indicates an epitope close to the epitope of
"Antibody A" in position and identical or almost identical to the
epitope of "Antibody B."
[0679] Others are indicated in the same manner as indicated
above.
[0680] <4-15> Therapeutic Effect on Kidney Diseases and
Fibrotic Diseases in Tissues
[0681] The therapeutic effect of the human anti-human CTGF
monoclonal antibodies of the present invention on various diseases
was studied by using a mouse model of the diseases.
[0682] The mouse model used in this study is a disease model
exhibiting a part of the pathologic features or a part of clinical
findings as observed in any of the diseases and morbid conditions
indicated below. The therapeutic effect found in the present study
should be considered to represent all of the therapeutic effects on
the diseases or morbid conditions indicated below:
[0683] kidney diseases (renal failure, nephritis, kidney fibrosis,
etc.), various fibrotic diseases in tissues (kidney fibrosis,
pulmonary fibrosis, hepatic fibrosis, tissue fibrosis of skin,
etc., fibrosis of synovial tissue associated with rheumatoid
arthritis and fibrotic diseases associated with various cancers),
skin diseases (scleroderma, psoriasisa, atopic dermatitis, etc.),
liver diseases (cirrhosis, hepatic fibrosis, hepatitis, etc.), lung
diseases (pulmonary fibrosis, pneumonia, etc.) and rheumatoid
arthritis and arteriosclerosis, etc.
[0684] <4-15-1> Preparation of a Disease Model Mouse
[0685] The left side abdomen of each B6C3F1 mouse (male, 7-week
old, 6 individuals per each group, SLC) was opened by a surgical
operation under anesthesia with pentobarbital (50 mg/kg). The
ureter extending from the left kidney was ligated with sutures at
two sites and then cut between the two ligated sites (UUO,
unilateral ureteral obstruction). After this treatment, the opened
abdominal part was sutured. This operation results in the loss of
the most important function in the left kidney--the normal renal
filtering function of body fluids such as blood. A variety of
pathological manifestations as seen in various kidney diseases are
observed in the operated kidney.
[0686] <4-15-2> Therapeutic Effect of Anti-CTGF Monoclonal
Antibody
[0687] The human anti-human CTGF monoclonal antibody, M84 or M32
(prepared in the previous Example) was dissolved in a phosphate
buffer and given to the above-prepared model mice by
intraperitoneal injection (5 mg/kg). The administration of the
antibody was carried out, for the first time, immediately after the
operation, and then every third day, four times in total. After the
final administration (14 days after the operation), the left kidney
was removed from each mouse by a surgical operation. The extracted
kidneys were delipidated and dehydrated with acetone, and the
proteins were hydrolyzed by using 6N hydrochloric acid.
Subsequently, the samples were dried, while being kept warm and
blown with nitrogen gas, and dissolved in purified water to serve
as the assay samples. The contents of hydroxyproline (OH-proline)
in the samples from kidney tissues were determined according to a
previously reported method (Analytical Biochemistry, Vol. 55, p.
288-291, 1973; Kidney Lnt., Vol. 54, No. 1, p. 99-109, 1998).
[0688] The increased hydroxyproline level in the kidney is an index
of the onset of nephritis and kidney fibrosis resulting from renal
failure. Accordingly, the decrease in the hydroxyproline
concentration indicates that the monoclonal antibodies are useful
for the treatment of kidney diseases.
[0689] Control experiments were carried out as follows in the same
manner described above:
[0690] (1) Phosphate buffer alone (without any antibodies) was
given intraperitoneally to mice treated with the above-mentioned
UUO (ureter ligation treatment) in the same manner described
above.
[0691] (2) The abdomen was opened and then sutured without
performing UUO in normal mice.
[0692] (3) No surgical operation with the UUO treatment was
performed in normal mice.
[0693] (4) Mice were fed with food in which Pirfenidone (Kidney
Int. Vol. 54, No. 1, p. 99-109, 1998) was mixed, instead of the
antibody treatment described above (positive control experiment).
Pirfenidone is a drug for the treatment of fibrotic diseases such
as kidney fibrosis and is being under clinical trial.
[0694] The results are shown in FIG. 17.
[0695] These results illustrated that the monoclonal antibodies of
the present invention had significant suppressing effects and
therapeutic effects on the kidney diseases and tissue fibrotic
diseases.
[0696] Surprisingly, the efficacy of the monoclonal antibodies of
the present invention is the same as that of the drug used in a
high dose as a positive control (for example, the amount of the
drug given to mice corresponds to about 100 g, in total, if the
drug is administered four times to a patient with 50 kg of body
weight).
[0697] <4-16> Determination and Analysis of Gene Sequence and
Amino Acid Sequence of Human Anti-Human CTGF Monoclonal
Antibody
[0698] Nucleotide sequencing was carried out as described below to
determine the cDNA sequence encoding the variable region of the
heavy chain of each human monoclonal antibody against human CTGF
prepared in the above examples as well as the cDNA sequence
encoding the viable region and constant regions of the light chain
of the antibody. The structural features of the genes were also
analyzed. The procedures of sequencing analysis used in this
example is schematically illustrated in FIG. 18.
[0699] After culturing, the hybridomas (clone: A29, C26, C59, C114,
and M295; about 5.times.10.sup.7 cells), which were prepared in the
previous example, producing the human anti-human CTGF monoclonal
antibodies, were centrifuged and the resulting precipitates were
recovered. The cells were stored at -80.degree. C. for the later
polyA.sup.+ RNA extraction.
[0700] PolyA.sup.+ RNA was extracted and purified from each
hybridoma using a commercial product, FASTRACK 2.0 KIT (Invitrogen
Co.), as follows. Each of the above frozen cell samples were lysed
in a lysis buffer and solubilized by homogenizing with POLYTRON.
After incubating at 45.degree. C., the solubilized materials were
mixed with Oligo(dT) cellulose, and the mixtures were shaken gently
for about 1 hour. Subsequently, the Oligo(dT) cellulose was washed,
and then polyA.sup.+ RNAs were eluted with an elution buffer.
Eluted PolyA.sup.+ RNAs were precipitated with ethanol, and then
dissolved in 20 .mu.l of Tris-EDTA buffer. The concentrations of
obtained polyA.sup.+ RNAs were determined by measuring absorbance
at a wave length of 260 nm.
[0701] Complementary DNAs were prepared by using the obtained
polyA.sup.+ RNAs as templates by RACE-PCR with a commercially
available product, MARATHON cDNA AMPLIFICATION KIT (CLONTECH),
according to a usual procedure ("PCR Method for Gene Amplification:
Basic Techniques and Recent Advancement" Kyoritsu Shuppan Co.,
Ltd., p.13-15, 1992). Specifically, the first-strand cDNA synthesis
was performed using the polyA.sup.+ RNA (1-5 .mu.g) purified from
each of the hybridomas as a template, and then the second strand
was prepared from the first strand. The cDNAs were extracted once
with phenol/chloroform/isoamyl alcohol and then treated once with
chloroform. The cDNAs were precipitated with ethanol. An adaptor
DNA (SEQ ID NO: 25) was ligated to the cDNAs. The resultant DNA
products were diluted 250 times. The respective cDNAs encoding the
heavy chain and the light chain of the antibody were prepared by
using the dilutes as templates by PCR in a usual manner. The primer
of SEQ ID NO: 26 was used in the PCR for the antibody heavy chain.
The primer of SEQ ID NO: 27 was used in the PCR for the antibody
light chain.
[0702] Each PCR product was fractionated by agarose-gel
electrophoresis, and the DNAs of interest were recovered therefrom.
The nucleotide sequences of the respective cDNAs obtained were
determined by using a commercially available reagent, DYE
TERMINATOR CYCLE SEQUENCING FS KIT (PE-Applied Biosystems) and a
PRISM377 DNA Sequencer (PE-Applied Biosystems). Sequencing Primers
used in the sequence determination were the same as those used in
the above PCR amplification. Based on the sequences obtained,
appropriate sequencing primers were designed and prepared for
further sequencing analysis.
[0703] Sequence listing attached hereto contains the cDNA sequence
encoding the variable region of the heavy chain of each human
monoclonal antibody against human CTGF produced by the
above-mentioned hybridomas; the cDNA sequence encoding the variable
region of the light chain of each of the antibodies; and the
deduced amino acid sequences thereof.
[0704] <Clone A29>
[0705] (Variable Region of the Heavy Chain)
[0706] DNA sequence: SEQ ID NO: 5 (signal sequence: nucleotides
1-57, V region: nucleotides 58-363)
[0707] Amino acid sequence: SEQ ID NO: 6 (signal sequence: amino
acids 1-19, variable region: comprising amino acids 21-120)
[0708] (Variable Region of the Light Chain)
[0709] DNA sequence: SEQ ID NO: 15 (signal sequence: nucleotides
1-60, V region: nucleotides 61-365)
[0710] Amino acid sequence: SEQ ID NO: 16 (signal sequence: amino
acids 1-20, variable region: comprising amino acids 21-120)
[0711] <Clone C26>
[0712] (Variable Region of the Heavy Chain)
[0713] DNA sequence: SEQ ID NO: 7 (signal sequence: nucleotides
1-57, V region: nucleotides 58-357)
[0714] Amino acid sequence: SEQ ID NO: 8 (signal sequence: amino
acids 1-19, variable region: comprising amino acids 21-118)
[0715] (Variable Region of the Light Chain)
[0716] DNA sequence: SEQ ID NO: 17 (signal sequence: nucleotides
1-60, V region: nucleotides 61-364.)
[0717] Amino acid sequence: SEQ ID NO: 18 (signal sequence: amino
acids 1-20, variable region: comprising amino acids 21-121)
[0718] <Clone C59>
[0719] (Variable Region of the Heavy Chain)
[0720] DNA sequence: SEQ ID NO: 9 (signal sequence: nucleotides
1-57, V region: nucleotides 58-350)
[0721] Amino acid sequence: SEQ ID NO: 10 (signal sequence: amino
acids 1-19, variable region: comprising amino acids 21-116)
[0722] (Variable Region of the Light Chain)
[0723] DNA sequence: SEQ ID NO: 19 (signal sequence: nucleotides
1-66, V region: nucleotides 67-353)
[0724] Amino acid sequence: SEQ ID NO: 20 (signal sequence: amino
acids 1-22, variable region: comprising amino acids 23-117)
[0725] <Clone C114>
[0726] (Variable Region of the Heavy Chain)
[0727] DNA sequence: SEQ ID NO: 11 (signal sequence: nucleotides
1-57, V region: nucleotides 58-350)
[0728] Amino acid sequence: SEQ ID NO: 12 (signal sequence: amino
acids 1-19, variable region: comprising a segment of amino acids
21-116)
[0729] (Variable Region of the Light Chain)
[0730] DNA sequence: SEQ ID NO: 21 (signal sequence: comprising
nucleotides 1-47, V region: nucleotides 48-335)
[0731] Amino acid sequence: SEQ ID NO: 22 (signal sequence:
comprising amino acids 1-16, variable region: comprising amino
acids 17-111)
[0732] <Clone M295>
[0733] (Variable Region of the Heavy Chain)
[0734] DNA sequence: SEQ ID NO: 13 (signal sequence: nucleotides
1-58, V region: nucleotides 59-353)
[0735] Amino acid sequence: SEQ ID NO: 14 (signal sequence: amino
acids 1-19, variable region: comprising amino acids 21-117)
[0736] (Variable Region of the Light Chain)
[0737] DNA sequence: SEQ ID NO: 23 (signal sequence: nucleotides
1-66, V region: nucleotides 67-356)
[0738] Amino acid sequence: SEQ ID NO: 24 (signal sequence: amino
acids 1-22, variable region: comprising amino acids 23-118)
[0739] By using a gene sequence-analyzing computer software, a
library of variable region genes of human immunoglobulin, V BASE
SEQUENCE, constructed by Tomlinson et al. (Immunol. Today, Vol. 16,
No. 5, p. 237-242, 1995) was searched for the respective DNA
sequences determined.
[0740] The result showed that the respective V region genes of the
heavy and light chains of the above-mentioned human monoclonal
antibodies are composed of the segments indicated below.
[0741] <Gene for Heavy-Chain V Region>
[0742] Clone A29: DP-38
[0743] Clone C26: DP-75
[0744] Clone C59: DP-5
[0745] Clone C114: DP-5
[0746] Clone M295: DP-65
[0747] <Gene for Light-Chain V Region>
[0748] Clone A29: DPK24
[0749] Clone C26: DPK12
[0750] Clone C59: DPK1
[0751] Clone C114: DPK1
[0752] Clone M295: DPK9
[0753] It is assumed that N-additions are located between the V
region and the downstream D region as well as between the D region
and the further downstream J region in the cDNA sequences encoding
the heavy chains of the above human monoclonal antibodies.
EXAMPLE 5
Establishment of a Sandwich ELISA System for Assaying Human CTGF
and Mouse CTGF
[0754] <5-1> Preparation of a Antibody-Immobilized
Microplate
[0755] In this example, the monoclonal antibody 8-64-6 (derived
from a hybridoma identified by international deposit accession No.
FERM BP-6209), which was derived from normal mouse and prepared in
the above-described manner, was used as a monoclonal antibody which
is immobilized on a microplate. This monoclonal antibody is highly
reactive to human CTGF and crossreactive to mouse CTGF.
[0756] The monoclonal antibody 8-64-6 was diluted with phosphate
buffer and added at a concentration of 1 .mu.g/50 .mu.l/well into
each well of a 96-well ELISA microplate (Corning Costar Co.). The
plate was incubated at room temperature for 1 hour for adsorbing
the antibody onto the plate.
[0757] Subsequently, the plate was washed with a phosphate buffer
and then a phosphate buffer (200 .mu.l/well) containing 3% bovine
serum albumin (BSA) was added into each well. The plate was
incubated at room temperature for 2 hours for the blocking of
antibody-free sites on the microplate. The plate was washed three
times with phosphate buffer.
[0758] <5-2> Preparation of a Labeled Monoclonal Antibody
[0759] In this example, the monoclonal antibody 8-86-2 (derived
from a hybridoma identified by international deposit accession No.
FERM BP-6208), which was derived from normal mouse and prepared in
the above-described manner, was used as a monoclonal antibody for
the labeling. This monoclonal antibody is highly reactive to human
CTGF, mouse CTGF, and rat CTGFs.
[0760] One milliliter of the monoclonal antibody 8-86-2 (20 mg/ml)
was dialyzed with 0.1M NaHCO.sub.3 (pH8.2-8.3) solution (at
4.degree. C. for 24 hours). Then 100 .mu.l of NHS-biotin (2 mg/ml;
Pierce Chemical Co.) was added thereto and stirred vigorously.
After the reaction was continued at room temperature for 30
minutes, the mixture was dialyzed with phosphate buffer (at
4.degree. C. for 24 hours).
[0761] <5-3> Establishment of a Assay Method Using Sandwich
ELISA
[0762] The sandwich ELISA system for assaying human CTGF and mouse
CTGF, which was established in the present invention, is as
follows.
[0763] Samples (50 .mu.l/well) to be assayed were added into each
well of the antibody-immobilized microplate prepared in Example
<5-1> and incubated at room temperature for 1 hour. The
microplate was washed three times with phosphate buffer containing
0.1% Tween 20. The biotin-labeled monoclonal antibody prepared in
Example <5-2> was diluted with a phosphate buffer containing
1% BSA and 0.1% Tween 20 and added (0.3 .mu.l/50 .mu.l/well) into
the respective wells. The plate was incubated at room temperature
for 1 hour.
[0764] The microplate was washed three times with phosphate buffer
containing 0.1% Tween 20. A solution of
streptavidin-.beta.-galactosidase (50 .mu.l; Gibco-BRL) diluted
1000 times with a solution (pH7.0) containing 20 mM HEPES, 0.5M
NaCl and BSA (1 mg/ml), was added into each well. The plate was
incubated at room temperature for 30 minutes.
[0765] The microplate was washed three times with phosphate buffer
containing 0.1% Tween 20. A solution of 1%
4-Methyl-umbelliferyl-.beta.-D- -galactoside (50 .mu.l; Sigma) in a
phosphate buffer (10 mM, pH7.0, containing Na and K ions)
containing 100 mM NaCl, 1 mM MgCl.sub.2 and 1 mg/ml BSA, was added
into each well. The plate was incubated at room temperature for 10
minutes.
[0766] 1M Na.sub.2CO.sub.3 (100 .mu.l) was added to each well to
stop the reaction. The fluorescence intensity was measured by the
FLUOROSCAN II MICROPLATE FLUOROMETER (Flow Laboratories Inc.) at a
wavelength of 460 nm (excitation wavelength: 355 nm). The amount of
human CTGF or mouse CTGF in the sample were determined by using the
calibration curves as prepared in the following example.
[0767] <5-4> Preparation of the Calibration Curve
[0768] The calibration curve was prepared by using the sandwich
ELSA established in Example <5-3>, in which the labeled CTGF
standard used was the affinity-purified recombinant human CTGF or
recombinant mouse CTGF, which were prepared in Example <4-8>.
The result is shown in FIG. 19.
[0769] The calibration curve for human CTGF was obtained with a
significant difference even within an extremely low concentration
range of 3 ng/ml to 1000 ng/ml. The calibration curve for mouse
CTGF was also obtained with a significant difference for a
concentration range of 30 ng/ml to 1000 ng/ml. However, rat CTGF
was not measurable in the sandwich ELISA system established in
Example <5-3>.
EXAMPLE 6
Establishment of a Sandwich ELISA System for Assaying Mouse CTGF
and Rat CTGF
[0770] <6-1> Preparation of Antibody-Immobilized
Microplate
[0771] In this example, the monoclonal antibody 13-51-2, which was
derived from normal rat and prepared in the above-described manner,
was used as a monoclonal antibody which is immobilized on a
microplate. This monoclonal antibody is highly reactive to mouse
CTGF and crossreactive to rat CTGF.
[0772] The monoclonal antibody 13-51-2 was diluted with phosphate
buffer and added at a concentration of 1 .mu.g/50 .mu.l/well into
each well of a 96-well ELISA microplate (Corning Costar Co.). The
plate was incubated at room temperature for 1 hour for adsorbing
the antibody onto the plate.
[0773] Subsequently, the plate was washed with a phosphate buffer
and then a phosphate buffer (200 .mu.l/well) containing 3% Bovine
serum albumin (BSA) was added into each well. The plate was
incubated at room temperature for 2 hours for the blocking of
antibody-free sites on the microplate. The plate was washed three
times with phosphate buffer.
[0774] <6-2> Preparation of a Labeled Monoclonal Antibody
[0775] In this example, labeled monoclonal antibody was the
biotin-labeled monoclonal antibody prepared in Example <5-2>.
Specifically, the labeled monoclonal antibody was prepared by
labeling, with biotin, the antibody 8-86-2 (derived from a
hybridoma identified by international deposit accession No. FERM
BP-6208) highly reactive to all of human CTGF, mouse CTGF, and rat
CTGF.
[0776] <6-3> Establishment of a Assay Method Using Sandwich
ELISA
[0777] The sandwich ELISA system for assaying mouse CTGF and rat
CTGF, which was established in the present invention, is as
follows.
[0778] Samples (50 .mu.l/well) to be assayed were added into each
well of the antibody-immobilized microplate prepared in Example
<6-1> and incubated at room temperature for 1 hour. The
microplate was washed three times with phosphate buffer containing
0.1% Tween 20. The biotin-labeled monoclonal antibody prepared in
Example <6-2> was diluted with a phosphate buffer containing
1% BSA and 0.1% Tween 20 and added (0.3 .mu.l/50 .mu.l/well) into
the respective wells. The plate was incubated at room temperature
for 1 hour.
[0779] The microplate was washed three times with phosphate buffer
containing 0.1% Tween 20. A solution of
streptavidin-.beta.-galactosidase (50 .mu.l; Gibco-BRL), diluted
1000 times with a solution (pH7.0) containing 20 mM HEPES, 0.5M
NaCl and BSA (1 mg/ml), was added into each well. The plate was
incubated at room temperature for 30 minutes.
[0780] The microplate was washed three times with phosphate buffer
containing 0.1% Tween 20. A solution of 1%
4-Methyl-umbelliferyl-.beta.-D- -galactoside (50 .mu.l; Sigma) in a
phosphate buffer (10 mM, pH7.0, containing Na and K ions)
containing 100 mM NaCl, 1 mM MgCl.sub.2 and 1 mg/ml BSA, was added
into each well. The plate was incubated at room temperature for 10
minutes.
[0781] 1M Na.sub.2CO.sub.3 (100 .mu.l) was added to each well to
stop the reaction. The fluorescence intensity was measured by the
FLUOROSCAN II MICROPLATE FLUOROMETER (Flow Laboratories Inc.) at a
wavelength of 460 nm (excitation wavelength: 355 nm). The amount of
mouse CTGF or rat CTGF in the sample were determined by using the
calibration curve as prepared in the following example.
[0782] <6-4> Preparation of the Calibration Curve
[0783] The calibration curve was prepared by using the sandwich
ELSA established in Example <6-3> in which the labeled CTGF
standard used was the affinity-purified recombinant mouse CTGF or
recombinant rat CTGF, which were prepared in Example <4-8>.
The result is shown in FIG. 20.
[0784] The calibration curve for mouse CTGF was obtained with a
significant difference even for an extremely low concentration
range of 1 ng/ml to 1000 ng/ml. The calibration curve for rat CTGF
was also obtained with a significant difference for a concentration
range of 10 ng/ml to 1000 ng/ml. However, human CTGF was not
measurable in the sandwich ELISA system established in Example
<6-3>.
EXAMPLE 7
Assay of CTGF in the Sera from Patients Affected with Various
Diseases
[0785] CTGF in the sera from patients affected with various
diseases was assayed by the sandwich ELISA established in Example
<5-3>.
[0786] <7-1> Biliary Atresia, Rheumatic Vasculitis, Malignant
Rheumatoid Arthritis, Psoriasis, and Atopic Dermatitis
[0787] Human sera used in this experiment were collected from
normal healthy persons (33 samples), patients affected with biliary
atresia and submitted to a surgical operation (post-operative
sample; <Group 1> patients with normal clinical findings (17
samples); <Group 2> patients with progressing symptoms (14
samples); <Group 3> patients with severe symptoms in need of
liver transplantation (8 samples)), patients with rheumatic
vasculitis (10 samples), patient with malignant rheumatoid
arthritis (MRA) (17 samples), patients with psoriasis (24 samples)
and patients with atopic dermatitis (34 samples).
[0788] The results are shown in FIG. 21 (biliary atresia) and FIG.
22 (rheumatic vasculitis, malignant rheumatoid arthritis,
psoriasis, and atopic dermatitis).
[0789] It was evidenced that, among patients affected with biliary
atresia, CTGF was significantly expressed in Group 2 patients (with
symptoms at progressive stage). In addition, as compared with
normal healthy persons, patients affected with rheumatic vasculitis
or malignant rheumatoid arthritis exhibited significantly higher
expression of CTGF.
[0790] <7-2> Rheumatoid Arthritis and Osteoarthritis
[0791] Samples used in this experiment were synovial fluids
collected from patients affected with rheumatoid arthritis (RA; 36
patients) and patients with osteoarthritis (OA; 19 patients). The
result is shown in FIG. 23.
[0792] It was found that the synovial fluid CTGF levels of patients
with rheumatoid arthritis were significantly higher than those of
patients with osteoarthritis.
[0793] Based on this result, it can be stated that the expression
of CTGF in patients with various diseases as well as normal healthy
persons can be highly sensitively quantified by the assay system of
the present invention and that the system can be utilized as a
clinical diagnosis for accurate clinical judgment of the degree of
illness.
EXAMPLE 8
Preparation of Antibody Fragments F(ab').sub.2 and Fab
[0794] The antibody fragments F(ab').sub.2 and Fab derived from
various monoclonal antibodies prepared above, are prepared as
follows.
[0795] A sodium acetate buffer (20 mM; pH3.5) containing monoclonal
antibody (5 mg/ml) is incubated at 37.degree. C. for 30 minutes.
Insolubilized pepsin (1 ml; Pierce Chemical Co.) is added thereto.
The mixture is then incubated at 37.degree. C. for 12 hours while
being shaken on a rotator. The reaction solution is centrifuged
(3000 rpm, for 10 minutes) and the resulting supernatant is
recovered.
[0796] Protein A-affinity chromatography is performed by using a
Protein A column kit (Amersham) according to the supplier's
protocol, as follows. A binding buffer is added to the precipitate
obtained by centrifugation. The solution is centrifuged (3000 rpm,
for 10 minutes) again, and then the resulting supernatant is
recovered. The first and second supernatants obtained are combined
together and an equal volume of the binding buffer is added
thereto. The mixture is adjusted to pH 8.9 by adding 1N sodium
hydroxide thereto. The mixed solution is loaded onto the Protein A
column equilibrated with the binding buffer. Then the column is
washed twice with the binding buffer (5 ml) to elute and collect
the fraction of interest. The fraction is dialyzed with 5 mM
phosphate buffer (2 L, pH6.8) (at 4.degree. C., for 24 hours).
[0797] Further purification is performed by high performance liquid
chromatography (HPLC) using hydroxyapatite column (BioRad). The
sample solution after dialysis is loaded onto the hydroxyapatite
column. 5 mM phosphate buffer is allowed to flow through the column
for 15 minutes, the antibody fragments are eluted with a linear
gradient of 5 mM-0.4 M phosphate buffer. The eluate is collected on
a fraction collector and the absorbance is monitored at a
wavelength of 280 nm for the recovery of F(ab').sub.2-containing
fractions. The fractions collected are dialyzed with 2L of
phosphate buffer (at 4.degree. C., for 24 hours). The purified
F(ab').sub.2 of monoclonal antibody is thus obtained.
EXAMPLE 9
Preparation of Human-CTGF-Expressing Transgenic Mouse
[0798] The human CTGF-encoding cDNA obtained in Example 2 was
blunted by using a DNA blunting kit (Takara Shuzo Co.) and inserted
into an expression vector, pCAGGS (Gene, Vol. 108, p. 193-200,
1991), containing chicken .beta.-actin promoter to obtain the
plasmid phCTGF. Human kidney-derived fibroblast cell line 293-T
(ATCC CRL1573) was transformed by electroporation with phCTGF. It
was verified that the transformant expressed and secreted human
CTGF into the culture supernatant by the sandwich ELISA established
in Example 5.
[0799] The plasmid phCTGF was linearized by the treatment with
restriction enzyme for the subsequent preparation of transgenic
mouse.
[0800] White ICR females with a copulatory plug were selected as
foster mothers that were obtained by mating white ICR female
mouse(Japan SLC) with a vasectomized white ICR male mouse(Japan
SLC). Donor female mice giving fertilized eggs to be used for human
CTGF gene transfer were prepared by mating a female BDF-1 mouse
(Japan SLC), which were superovulated by the administration of
PEAMEX (5 units; Sankyo Zohki Co.) and PREGNYL (5 units; Organon
Co.), with a BDF-1 male mouse (Japan SLC). After the mating, the
oviduct was removed from the BDF-1 mouse (female), and was
hyaluronidase-treated to obtain only fertilized eggs. The eggs were
stored in a medium.
[0801] The human CTGF gene was introduced into the fertilized eggs
by using a manipulator under a microscope according to the usual
method. Fertilized eggs were held-in-place with a holding needle.
The above-mentioned linear human CTGF gene, which was dissolved in
Tris-EDTA buffer, was microinjected into the male pronucleus of the
egg with a DNA injection needle at 37.degree. C.
[0802] After the gene transfer, only fertilized eggs with a normal
appearance were selected. The eggs, to which the human CTGF was
introduced, were transferred into the fimbria of the oviduct in the
ovary of the mouse (white ICR mouse) used as a foster mother.
[0803] Genomic DNA was extracted form the tails of the resulting
offspring (chimeric mice) born from the foster mother. The presence
of the transferred human CTGF gene in mouse genome was confirmed by
PCR. In addition, it was confirmed that human CTGF was expressed
and secreted into blood serum of the mouse, by the sandwich ELISA
established by Example 5. Then the chimeric mice were mated with
normal mice to prepare heterozygous transgenic mice expressing
human CTGF at high levels. The heterozygous mice were mated to each
other to prepare homozygous transgenic mice.
EXAMPLE 11
Preparation of Rat CTGF
[0804] <11-1> cDNA Cloning
[0805] (1) Preparation of Rat cDNA Library and Probe
[0806] Cells of rat kidney-derived fibroblast strain NRK-49F (ATCC
CRL-1570; about 1.times.10.sup.6 cells/ml) were centrifuged (at
4.degree. C., 2,000.times.g, for 5 minutes). The cells precipitated
were suspended in ISOGEN (Nippon Gene) and then chloroform was
added thereto. After the mixture was shaken, the upper layer was
recovered. Isopropanol was added to the obtained upper solution.
The mixture was allowed to stand at room temperature for 10 minutes
and centrifuged (at 4.degree. C., 12,000.times.g, for 10 minutes)
for RNA precipitation. After washing with ethanol, the precipitated
RNA was dissolved in TE buffer. Poly(A).sup.+ RNA was purified from
the total RNA using an mRNA Purification Kit (Pharmacia).
[0807] Complementary DNA synthesis was performed by using the
poly(A)+ RNA (5 .mu.g) as a template and a SUPERSCRIPT 1 SYSTEM FOR
cDNA SYNTHESIS KIT (GIBCO-BRL). An oligo dT primer with NotI site
(GIBCO-BRL) was used for improved screening efficiency. After
linking to a SalI adaptor, cDNA was digested with NotI to give
unidirectional cDNA. The heterogeneous cDNA was fractionated by
using a cDNA size fractionation column (GIBCO-BRL).
[0808] The nucleotide sequences of the human and mouse cDNAs
obtained in Example 2 were compared to each other. A pair of 5'
(SEQ ID NO: 3) and 3' (SEQ ID NO: 4) primers were designed and
synthesized based on a highly homologous region shared by the human
and mouse CTGF cDNAs.
[0809] PCR (polymerase chain reaction) amplification was performed
by using the cDNA library prepared above as a template and by using
the above-mentioned primers and Ex Taq DNA polymerase (Takara Shuzo
Co.). The reaction was carried out on a DNA THERMAL CYCLER (Perkin
Elmer Cetus). The final concentration of each primer was 0.4 .mu.M
and that of Mg.sup.2+ was 1.5 mM. The cycling profile was 35 cycles
of the following cycle: denaturing at 94.degree. C. for 1 minute,
annealing at 55.degree. C. for 1 minute and extension at 72.degree.
C. for 1 minute. After electrophoresed on an agarose gel, the
amplified DNA was purified by using a QUIAEX DNA extraction kit
(QUIAGEN).
[0810] The recovered DNA fragment was ligated with a vector pCRII
(Invitrogen Co.) by using the TA CLONING KIT (Invitrogen Co.).
Nucleotide sequence of the resulting cDNA was sequenced on an
A.L.F. DNA SEQUENCER (Pharmacia) by using an AUTO READ SEQUENCING
KIT (Pharmacia) according to the dideoxy method. The nucleotide
sequence of the cDNA fragment was compared with those of human and
mouse CTGF cDNAs obtained in Examples 2 and 3. It was confirmed
that the cDNA fragment contained the coding region for rat
homologue (rat CTGF) corresponding to the human and mouse CTGF
gene.
[0811] A probe for plaque hybridization was prepared by labeling
the cDNA (about 0.8 kb) with FITC by using an ECL RANDOM PRIME
LABELING KIT (Amersham).
[0812] (2) Ligation of cDNA to Vector and Packaging
[0813] The cDNA obtained above in (1) was ligated to a vector
1ZipLox NotI-SalI arm (GIBCO-BRL). The ligation reaction was
performed by using a DNA ligation Kit (Takara Shuzo Co.). The
ligated DNA was packaged in vitro by using a GIGA PACK II GOLD
(Stratagene). A cDNA library comprising recombinant
phage-containing plaques was prepared with the obtained phage
particles and E. coli host Y1090 strain (GIBCO-BRL).
[0814] (3) Screening of a cDNA Library
[0815] According to the plaque hybridization method described in
"Molecular Cloning: A Laboratory Manual (Maniatis et al., Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)", screening of
the cDNA library prepared above in (2) was carried out by using
RAPID HYBRIDIZATION BUFFER (Amersham), as follows.
[0816] After the cDNA (1.times.10.sup.4 plaques) prepared above in
(2) was seeded on agar plates, filter replicas were prepared with
HYBOND-N-NYLON MEMBRANES (Amersham). Plaque hybridization was
performed in the RAPID HYBRIDIZATION BUFFER (Amersham) by using the
FITC-labeled probe prepared above in (1) and the replicas. The
primary screening and the secondary screening yielded 13 positive
clones. Each clone obtained by single-plaque isolation was treated
by in vivo excision method according to the manual provided by the
supplier GIBCO-BRL. Thirteen clones were obtained as plasmid
DNAs.
[0817] (4) Determination of Nucleotide Sequence
[0818] Nucleotide sequence of the 13 cDNA cones were determined on
an A.L.F. DNA SEQUENCER (Pharmacia) by using an AUTO READ
SEQUENCING KIT (Pharmacia) according to the dideoxy method. All 13
clones shared the same nucleotide sequence. Comparison of the cDNA
sequences with those of human and mouse CTGFs revealed that an
obtained clone, r311, contained a full-length cDNA encoding rat
CTGF. The full-length cDNA sequence (comprising the nucleotide
sequence of the 5' end and the 3' end thereof) of rat CTGF is shown
in SEQ ID NO: 1, and the deduced amino acid sequence thereof is
shown in SEQ ID NO: 2.
[0819] <11-2> Preparation of Recombinant Rat CTGF
[0820] The clone r311 prepared in Example <11-1>, which
contained a cDNA encoding rat CTGF, was digested with SalI and
DraI, to obtain the fragment of rat CTGF-encoding cDNA. The DNA
fragment was inserted into a plasmid pcDNA3.1(-) (Invitrogen Co.)
to prepare an expression vector. Human epithelioid cell line Hela
cell (ATCC CCL-2) was transformed with the vector by
electroporation. GENETICIN-resistant transformants were selected by
culturing the transformed cells in an RPMI1640 medium containing
GENETICIN (0.8 mg/ml; GIBCO-BRL) and 10% fetal calf serum for about
two weeks. The selected transformants were cultured in a serum-free
medium ASF104 (Ajinomoto Co. Inc.) for the expression of the
recombinant rat CTGF. The expression of rat CTGF was confirmed by
Western blotting using the monoclonal antibody, which was prepared
above in Example 4, having the crossreactivity to rat CTGF.
[0821] The culture supernatant was recovered and treated by
ammonium sulfate precipitation method. The precipitated proteins
were fractionated by heparin-column chromatography. The column was
washed with 0.3M NaCl/PBS, and then the protein fraction of
interest was eluted with 0.5M NaCl/PBS. Thus, a fraction with
partially purified rat CTGF was obtained.
INDUSTRIAL APPLICABILITY
[0822] The present invention provides previously unavailable
various monoclonal antibodies derived from a variety of mammals.
The antibodies are reactive to CTGFs from a variety of mammals such
humans, mice, rats and rabbits, and are different from one another
in respect to the properties such as antigenic specificity,
affinity for the antigen, neutralizing activity, and
crossreactivity. Particularly, the present invention leads the way
in the world in providing various human monoclonal antibodies
against human CTGF by using, as an immune animal, the transgenic
mouse prepared to produce human antibodies by recombinant
technology.
[0823] Among the monoclonal antibodies of the present invention,
the anti-human CTGF monoclonal antibody and the pharmaceutical
composition thereof, suppress and inhibit the onset and advancement
of various diseases which are believed to be caused by CTGF; such
diseases include, for example, kidney diseases (kidney fibrosis,
nephritis, and renal failure, etc.), lung diseases (for example,
pulmonary fibrosis and pneumonia), liver diseases (for example,
hepatic fibrosis, cirrhosis, and hepatitis), skin diseases (for
example, injuries, scleroderma, psoriasis, and keloid), arthritis
(for example, rheumatoid arthritis and osteoarthritis), and
vascular diseases (for example, rheumatic vasculitis), tissue
fibrosis developed as a complication in various cancers, and
arteriosclerosis (specifically, tissue fibrosis which occurs as a
complication). The anti-human CTGF monoclonal antibody and the
pharmaceutical composition thereof are thus useful as
pharmaceuticals for treating or preventing these diseases.
[0824] The utility value of the antibody as a pharmaceutical is
dramatically elevated, because the human monoclonal antibodies and
the pharmaceutical composition thereof are nonantigetic in humans,
the antigenicity being a major therapeutic problem (side effect) in
the treatment with antibody pharmaceuticals comprising antibodies
derived from non-human mammals such as mice.
[0825] By using immunoassay with the monoclonal antibodies of the
present invention, it is possible to provide various immunoassay
systems (methods and kits) for conveniently and highly sensitively
assaying intact CTGF in the body fluids (such as serum) from
mammals (human, mouse, rat and rabbit). It is also possible to
easily purify CTGFs of high purity from various mammals by using
affinity column chromatography with an insoluble carrier on which
the monoclonal antibody is immobilized.
[0826] Moreover, the inventive non-human transgenic mammal
(transgenic mouse, etc.) expressing human CTGF is extremely useful
as a tool for screening candidate pharmaceutical agents (low
molecular weight compounds, antibodies, antisense nucleotides, and
polypeptides except human CTGF) having the activity of regulating
human CTGF functions (inhibition, suppression, activation,
stimulation, etc.) as well as being useful as an animal model for
studying physiological functions of human CTGF. Specifically, it is
possible to assess the effect of such a pharmaceutical agent on
human CTGF by administering the agent to the non-human transgenic
mammal and assaying the levels of human CTGF expressed in the
animal, by using the assay system (sandwich ELISA, etc.) of the
present invention.
Sequence CWU 1
1
27 1 2338 DNA Rat 5'UTR (1)..(212) CDS (213)..(1256) 3'UTR
(1257)..(2338) polyA_signal (2297)..(2302) 1 ctccaagaag actcagccag
acccactcca gctccgaccc taggagaccg acctcctcca 60 gacggcagca
gccccagccc agtggacaac cccaggagcc accacctgga gcgtccggac 120
accaacctcc gccccgagac cgagtccagg ctccggccgc gcccctcgtc gcctctgcac
180 cccgctgtgc gtcctcctgc cgcgccccga cc atg ctc gcc tcc gtc gcg ggt
233 Met Leu Ala Ser Val Ala Gly 1 5 ccc gtt agc ctc gcc ttg gtg ctc
ctc ctc tgc acc cgg cct gcc acc 281 Pro Val Ser Leu Ala Leu Val Leu
Leu Leu Cys Thr Arg Pro Ala Thr 10 15 20 ggc cag gac tgc agc gcg
cag tgt cag tgc gca cgt gaa gcg gcg ccg 329 Gly Gln Asp Cys Ser Ala
Gln Cys Gln Cys Ala Arg Glu Ala Ala Pro 25 30 35 cgc tgc ccc gcc
ggc gtg agc ctg gtg ctg gac ggc tgc ggc tgc tgc 377 Arg Cys Pro Ala
Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys 40 45 50 55 cgc gtc
tgc gcc aag cag ctg gga gaa ctg tgc acg gag cgt gat ccc 425 Arg Val
Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro 60 65 70
tgc gac cca cac aag ggt ctc ttc tgc gac ttc ggc tcc ccc gcc aac 473
Cys Asp Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn 75
80 85 cgc aag att ggc gtg tgc cct gcc aaa gat ggt gca ccc tgt gtc
ttc 521 Arg Lys Ile Gly Val Cys Pro Ala Lys Asp Gly Ala Pro Cys Val
Phe 90 95 100 ggt ggg tcc gtg tac cgc agc ggc gag tcc ttc caa agc
agt tgc aaa 569 Gly Gly Ser Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser
Ser Cys Lys 105 110 115 tac cag tgc act tgc ctg gat ggg gcc gtg ggc
tgt gtg ccc ctg tgc 617 Tyr Gln Cys Thr Cys Leu Asp Gly Ala Val Gly
Cys Val Pro Leu Cys 120 125 130 135 agc atg gac gtg cgc ctg ccc agc
cct gac tgc ccc ttc ccg aga agg 665 Ser Met Asp Val Arg Leu Pro Ser
Pro Asp Cys Pro Phe Pro Arg Arg 140 145 150 gtc aag ctg ccc ggg aaa
tgc tgt gag gag tgg gtg tgt gat gag ccc 713 Val Lys Leu Pro Gly Lys
Cys Cys Glu Glu Trp Val Cys Asp Glu Pro 155 160 165 aag gac cgc aca
gtg gtt ggc cct gcc cta gct gcc tac cga ctg gaa 761 Lys Asp Arg Thr
Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu 170 175 180 gac aca
ttt ggc cct gac cca act atg atg cga gcc aac tgc ctg gtc 809 Asp Thr
Phe Gly Pro Asp Pro Thr Met Met Arg Ala Asn Cys Leu Val 185 190 195
cag acc aca gag tgg agc gcc tgt tct aag acc tgt ggg atg ggc atc 857
Gln Thr Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile 200
205 210 215 tcc acc cgg gtt acc aat gac aat acc ttc tgc agg ctg gag
aag cag 905 Ser Thr Arg Val Thr Asn Asp Asn Thr Phe Cys Arg Leu Glu
Lys Gln 220 225 230 agt cgt ctc tgc atg gtc agg ccc tgt gaa gct gac
cta gag gaa aac 953 Ser Arg Leu Cys Met Val Arg Pro Cys Glu Ala Asp
Leu Glu Glu Asn 235 240 245 att aag aag ggc aaa aag tgc atc cgg acg
cct aaa att gcc aag cct 1001 Ile Lys Lys Gly Lys Lys Cys Ile Arg
Thr Pro Lys Ile Ala Lys Pro 250 255 260 gtc aag ttt gag ctt tct ggc
tgc acc agt gtg aag acc tac cgg gct 1049 Val Lys Phe Glu Leu Ser
Gly Cys Thr Ser Val Lys Thr Tyr Arg Ala 265 270 275 aag ttc tgt ggg
gtg tgc acg gac ggc cgc tgc tgc aca ccg cac aga 1097 Lys Phe Cys
Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg 280 285 290 295
acc acc aca ctg ccg gtg gag ttc aag tgc ccc gat ggc gag atc atg
1145 Thr Thr Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Ile
Met 300 305 310 aaa aag aac atg atg ttc atc aag acc tgt gcc tgc cat
tac aac tgt 1193 Lys Lys Asn Met Met Phe Ile Lys Thr Cys Ala Cys
His Tyr Asn Cys 315 320 325 ccc ggg gac aat gac atc ttt gag tcc ttg
tac tac agg aag atg tat 1241 Pro Gly Asp Asn Asp Ile Phe Glu Ser
Leu Tyr Tyr Arg Lys Met Tyr 330 335 340 gga gac atg gcg taa
agccagggag taagggacac gaactcattt agactataac 1296 Gly Asp Met Ala
345 ttgaactgag ttacatctca ttttcttctg taaaaaaaac aaaaagggtt
acagtagcac 1356 attaatttaa atctgggttc ctaactgctg tgggagaaaa
caccccaccg aagtgagaac 1416 cgtgtgtcat tgtcatgcaa atagcctgtc
aatctcagac actggtttcg agacagttta 1476 gacttgacag ttgttcacta
gcgcacagtg acagaacgca cactaaggtg agcctcctgg 1536 aagagtggag
atgccaggag aaagacaggt actagctgag gtcattttaa aagcagcgat 1596
atgcctactt tttggagtgt gacaggggag ggacattata gcttgcttgc agacagacct
1656 gctctagcaa gagctgggtg tgtgtcctcc actcggtgag gctgaagcca
gctattcttt 1716 cagtaagaac agcagtttca gcgctgacat tctgattcca
gygacactgg tcgggagtca 1776 gaaccttgtc tattagactg gacagcttgt
ggcaagtgaa tttgccggta acaagccaga 1836 tttttatgga tcttgtaaat
attgtggata aatatatata tttgtacagt tatctargtt 1896 aatttaaaga
cgtttgtgcc tattgttctt gttttaagtg cttttggaat ttttaaactg 1956
atagcctcaa actccaaaca ccatcgatag gacataaagc ttgtctgtga ttcaaaacaa
2016 aggagatact gcagtggaaa ctgtaacctg agtgactgtc tgtcagaaca
tatggtacgt 2076 agacggtaaa gcaatggatc agaagtcaga tttctagtag
gaaatgtaaa atcactgttg 2136 gcgaacaaat ggcctttatt aagaaatggc
ttgctcaggg taactggtca gatttccacg 2196 aggaagtgtt tgctgcttct
ttgactatga ctggtttggg aggcagttta tttgttgaga 2256 gtgtgaccaa
aagttacatg tttgcacctt tctagttgaa aataaagtat atatattttt 2316
tatatgaaaa aaaaaaaaaa aa 2338 2 347 PRT Rat 2 Met Leu Ala Ser Val
Ala Gly Pro Val Ser Leu Ala Leu Val Leu Leu 1 5 10 15 Leu Cys Thr
Arg Pro Ala Thr Gly Gln Asp Cys Ser Ala Gln Cys Gln 20 25 30 Cys
Ala Arg Glu Ala Ala Pro Arg Cys Pro Ala Gly Val Ser Leu Val 35 40
45 Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu
50 55 60 Leu Cys Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu
Phe Cys 65 70 75 80 Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val
Cys Pro Ala Lys 85 90 95 Asp Gly Ala Pro Cys Val Phe Gly Gly Ser
Val Tyr Arg Ser Gly Glu 100 105 110 Ser Phe Gln Ser Ser Cys Lys Tyr
Gln Cys Thr Cys Leu Asp Gly Ala 115 120 125 Val Gly Cys Val Pro Leu
Cys Ser Met Asp Val Arg Leu Pro Ser Pro 130 135 140 Asp Cys Pro Phe
Pro Arg Arg Val Lys Leu Pro Gly Lys Cys Cys Glu 145 150 155 160 Glu
Trp Val Cys Asp Glu Pro Lys Asp Arg Thr Val Val Gly Pro Ala 165 170
175 Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro Thr Met
180 185 190 Met Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala
Cys Ser 195 200 205 Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr
Asn Asp Asn Thr 210 215 220 Phe Cys Arg Leu Glu Lys Gln Ser Arg Leu
Cys Met Val Arg Pro Cys 225 230 235 240 Glu Ala Asp Leu Glu Glu Asn
Ile Lys Lys Gly Lys Lys Cys Ile Arg 245 250 255 Thr Pro Lys Ile Ala
Lys Pro Val Lys Phe Glu Leu Ser Gly Cys Thr 260 265 270 Ser Val Lys
Thr Tyr Arg Ala Lys Phe Cys Gly Val Cys Thr Asp Gly 275 280 285 Arg
Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro Val Glu Phe Lys 290 295
300 Cys Pro Asp Gly Glu Ile Met Lys Lys Asn Met Met Phe Ile Lys Thr
305 310 315 320 Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile
Phe Glu Ser 325 330 335 Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala
340 345 3 20 DNA Artificial Sequence Description of Artificial
Sequence Artificially synthesized primer sequence 3 tgcggctgct
gccgcgtctg 20 4 21 DNA Artificial Sequence Description of
Artificial Sequence Artificially synthesized primer sequence 4
gcacaggtct tgatgaacat c 21 5 444 DNA Homo sapiens CDS (1)..(444)
sig_peptide (1)..(57) V_region (58)..(363) 5 atg gag ttt ggg ctg
agc tgg att ttc ctt gct gct att tta aaa ggt 48 Met Glu Phe Gly Leu
Ser Trp Ile Phe Leu Ala Ala Ile Leu Lys Gly 1 5 10 15 gtc cag tgt
gag gtg cag ctg gtg gag tct ggg gga ggc ttg gta aag 96 Val Gln Cys
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 cct
ggg ggg tcc ctt aag acc tct cct gtg cag cct ctg gat tca act 144 Pro
Gly Gly Ser Leu Lys Thr Ser Pro Val Gln Pro Leu Asp Ser Thr 35 40
45 ttc agt aac gcc tgg atg agc tgg gtc cgc cag gct cca gga agg ggc
192 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg Gly
50 55 60 tgg agt ggg ttg gcc gta tta aaa gca aaa ctg atg gtg gga
cac aca 240 Trp Ser Gly Leu Ala Val Leu Lys Ala Lys Leu Met Val Gly
His Thr 65 70 75 80 gac tac gct gca ccc gtg aaa ggc aga ttc acc atc
tca aga gat gat 288 Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp 85 90 95 tca aaa aac acg ctg tat ctg caa atg aac
agc ctg aaa acc gag gac 336 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
Ser Leu Lys Thr Glu Asp 100 105 110 aca gcc gtg tat tac tgt acc aca
aaa tgg gtg gct acg gac tac ttt 384 Thr Ala Val Tyr Tyr Cys Thr Thr
Lys Trp Val Ala Thr Asp Tyr Phe 115 120 125 gac tac tgg ggc cag gga
acc ctg gtc acc gtc tcc tca gcc tcc acc 432 Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 aag ggc cca tcg
444 Lys Gly Pro Ser 145 6 148 PRT Homo sapiens 6 Met Glu Phe Gly
Leu Ser Trp Ile Phe Leu Ala Ala Ile Leu Lys Gly 1 5 10 15 Val Gln
Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30
Pro Gly Gly Ser Leu Lys Thr Ser Pro Val Gln Pro Leu Asp Ser Thr 35
40 45 Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln Ala Pro Gly Arg
Gly 50 55 60 Trp Ser Gly Leu Ala Val Leu Lys Ala Lys Leu Met Val
Gly His Thr 65 70 75 80 Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met
Asn Ser Leu Lys Thr Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Thr
Thr Lys Trp Val Ala Thr Asp Tyr Phe 115 120 125 Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro
Ser 145 7 447 DNA Homo sapiens CDS (1)..(447) sig_peptide (1)..(57)
V_region (58)..(357) 7 atg gac tgg acc tgg agg atc tct ttc ttg gtg
gca gca gcc aca gga 48 Met Asp Trp Thr Trp Arg Ile Ser Phe Leu Val
Ala Ala Ala Thr Gly 1 5 10 15 gcc cac tcc cag gtg cag ctg gtg cag
tct ggg gct gag gtg aag aag 96 Ala His Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aag gtc
tcc tgc aag gct ttc tgg cta cac ctt 144 Pro Gly Ala Ser Val Lys Val
Ser Cys Lys Ala Phe Trp Leu His Leu 35 40 45 tca ccc ggc tac tat
atg cac tgg gtg cga cag gcc cct gga caa ggg 192 Ser Pro Gly Tyr Tyr
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60 ctt gag tgg
atg gga tgg atc aac cct aac agt agt ggc aca cac tat 240 Leu Glu Trp
Met Gly Trp Ile Asn Pro Asn Ser Ser Gly Thr His Tyr 65 70 75 80 gca
cag atg ttt cag ggc agg gtc acc gtg acc agg gac acg tcc atc 288 Ala
Gln Met Phe Gln Gly Arg Val Thr Val Thr Arg Asp Thr Ser Ile 85 90
95 agc aca gcc tac atg gag ctg agc agg ctg aga tct gac gac acg gcc
336 Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala
100 105 110 gtg tat tac tgt gcg aga gag ggg ata gca gca gct gcc atc
tac ggt 384 Val Tyr Tyr Cys Ala Arg Glu Gly Ile Ala Ala Ala Ala Ile
Tyr Gly 115 120 125 atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc
tcc tca gcc tcc 432 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Ala Ser 130 135 140 acc aag ggc cca tcg 447 Thr Lys Gly Pro
Ser 145 8 149 PRT Homo sapiens 8 Met Asp Trp Thr Trp Arg Ile Ser
Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Ala His Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser
Val Lys Val Ser Cys Lys Ala Phe Trp Leu His Leu 35 40 45 Ser Pro
Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly 50 55 60
Leu Glu Trp Met Gly Trp Ile Asn Pro Asn Ser Ser Gly Thr His Tyr 65
70 75 80 Ala Gln Met Phe Gln Gly Arg Val Thr Val Thr Arg Asp Thr
Ser Ile 85 90 95 Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser
Asp Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Glu Gly Ile Ala
Ala Ala Ala Ile Tyr Gly 115 120 125 Met Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro Ser 145
9 438 DNA Homo sapiens CDS (1)..(438) sig_peptide (1)..(57)
V_region (58)..(350) 9 atg gac tgc acc tgg agg atc ctc ttc ttg gtg
gca gca gct aca ggc 48 Met Asp Cys Thr Trp Arg Ile Leu Phe Leu Val
Ala Ala Ala Thr Gly 1 5 10 15 acc cac gcc cag gtc cag ctg gta cag
ttt ggg gct gag gtg aag aag 96 Thr His Ala Gln Val Gln Leu Val Gln
Phe Gly Ala Glu Val Lys Lys 20 25 30 cct ggg gcc tca gtg aag gtc
tcc tgc aag gtt tcc gga tac acc ctc 144 Pro Gly Ala Ser Val Lys Val
Ser Cys Lys Val Ser Gly Tyr Thr Leu 35 40 45 act gaa tta tcc atg
cac tgg gtg cga cag gct cct gga aaa ggg ctt 192 Thr Glu Leu Ser Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg atg
gga agt ttt gat cct gaa gat ggt gaa aca atc tac gca 240 Glu Trp Met
Gly Ser Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala 65 70 75 80 cag
aag ttc cag ggc aga gtc acc atg acc gag gac aca tct aca gac 288 Gln
Lys Phe Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp 85 90
95 aca gcc tac atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg
336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
100 105 110 tat tac tgt gca acc tct acg gtg gta act ccg tgg tac ttt
gac tac 384 Tyr Tyr Cys Ala Thr Ser Thr Val Val Thr Pro Trp Tyr Phe
Asp Tyr 115 120 125 tgg ggc cag gga acc ctg gtc acc gtc tcc tca gcc
tcc acc aag ggc 432 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly 130 135 140 cca tcg 438 Pro Ser 145 10 146 PRT Homo
sapiens 10 Met Asp Cys Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala
Thr Gly 1 5 10 15 Thr His Ala Gln Val Gln Leu Val Gln Phe Gly Ala
Glu Val Lys Lys 20 25 30 Pro Gly Ala Ser Val Lys Val Ser Cys Lys
Val Ser Gly Tyr Thr Leu 35 40 45 Thr Glu Leu Ser Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Met Gly Ser Phe
Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala 65 70 75 80 Gln Lys Phe Gln
Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp 85 90 95 Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110
Tyr Tyr Cys Ala Thr Ser Thr Val Val Thr Pro Trp Tyr Phe Asp Tyr 115
120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly 130 135 140 Pro Ser 145 11 438 DNA Homo sapiens CDS (1)..(438)
sig_peptide (1)..(57) V_region (58)..(350) 11 atg gac tgc acc tgg
agg atc ttc ttc ttg gtg gca gca gct aca ggc 48 Met Asp Cys Thr Trp
Arg Ile Phe Phe Leu Val Ala Ala Ala Thr Gly 1 5 10
15 acc cac gcc cag gtc cag ctg gta cag tct ggg gct gag gtg aag aag
96 Thr His Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30 cct ggg gcc tca gtg aag gtc tcc tgc aag gtt tcc gga tac
acc ctc 144 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr
Thr Leu 35 40 45 act gaa tta tcc atg cac tgg gtg cga cag gct cct
gga aaa ggg ctt 192 Thr Glu Leu Ser Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 50 55 60 gag tgg atg gga agt ttt gat cct gaa gat
ggt gaa aca atc tac gca 240 Glu Trp Met Gly Ser Phe Asp Pro Glu Asp
Gly Glu Thr Ile Tyr Ala 65 70 75 80 cag aag ttc cag ggc aga gtc acc
atg acc gag gac aca tct aca gac 288 Gln Lys Phe Gln Gly Arg Val Thr
Met Thr Glu Asp Thr Ser Thr Asp 85 90 95 aca gcc tac atg gag ctg
agc agc ctg aga tct gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 tat tac tgt gca
acc tct acg gtg gta act ccg tgg tac ttt gac tac 384 Tyr Tyr Cys Ala
Thr Ser Thr Val Val Thr Pro Trp Tyr Phe Asp Tyr 115 120 125 tgg ggc
cag gga acc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 432 Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140
cca tcg 438 Pro Ser 145 12 146 PRT Homo sapiens 12 Met Asp Cys Thr
Trp Arg Ile Phe Phe Leu Val Ala Ala Ala Thr Gly 1 5 10 15 Thr His
Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu 35
40 45 Thr Glu Leu Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60 Glu Trp Met Gly Ser Phe Asp Pro Glu Asp Gly Glu Thr
Ile Tyr Ala 65 70 75 80 Gln Lys Phe Gln Gly Arg Val Thr Met Thr Glu
Asp Thr Ser Thr Asp 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Thr Ser Thr
Val Val Thr Pro Trp Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser 145
13 450 DNA Homo sapiens CDS (1)..(450) sig_peptide (1)..(58)
V_region (59)..(353) 13 atg aaa cac ctg tgg ttc ttc ctt cct gct ggt
ggc agc tcc cag atg 48 Met Lys His Leu Trp Phe Phe Leu Pro Ala Gly
Gly Ser Ser Gln Met 1 5 10 15 ggt cct gtc cca ggt gca gct gca gga
gtc ggg ccc agg act ggt gaa 96 Gly Pro Val Pro Gly Ala Ala Ala Gly
Val Gly Pro Arg Thr Gly Glu 20 25 30 gcc ttc aca gac cct gtc ctc
acc tgc act gtc tct ggt ggc tcc atc 144 Ala Phe Thr Asp Pro Val Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 agc agt ggt ggt tac
tac tgg agc tgg atc cgc cag cac cca ggg aag 192 Ser Ser Gly Gly Tyr
Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys 50 55 60 ggc ctg gag
tgg att ggg tac atc tat tac agt ggg agc acc tac tac 240 Gly Leu Glu
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr 65 70 75 80 aac
ccg tcc ctc aag agt cga gtt acc ata tca gta gac acg tct aag 288 Asn
Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys 85 90
95 aac cag ttc tcc ctg aag ctg agc tct gtg act gcc gcg gac acg gcc
336 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
100 105 110 gtg tat tac tgt gcg agc tat tac tat gat agt ggt ggt tat
tac gac 384 Val Tyr Tyr Cys Ala Ser Tyr Tyr Tyr Asp Ser Gly Gly Tyr
Tyr Asp 115 120 125 tac ttt gac tac tgg ggc cag gga acc ctg gtc acc
gtc tcc tca gcc 432 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Ala 130 135 140 tcc acc aag ggc cca tcg 450 Ser Thr Lys
Gly Pro Ser 145 150 14 150 PRT Homo sapiens 14 Met Lys His Leu Trp
Phe Phe Leu Pro Ala Gly Gly Ser Ser Gln Met 1 5 10 15 Gly Pro Val
Pro Gly Ala Ala Ala Gly Val Gly Pro Arg Thr Gly Glu 20 25 30 Ala
Phe Thr Asp Pro Val Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40
45 Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys
50 55 60 Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Tyr Tyr 65 70 75 80 Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Ser Tyr Tyr
Tyr Asp Ser Gly Gly Tyr Tyr Asp 115 120 125 Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala 130 135 140 Ser Thr Lys Gly
Pro Ser 145 150 15 423 DNA Homo sapiens CDS (1)..(423) sig_peptide
(1)..(60) V_region (61)..(365) 15 atg gtg ttg cag acc cag gtc ttc
att tct ctg ttg ctc tgg atc tct 48 Met Val Leu Gln Thr Gln Val Phe
Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 ggt gcc tac ggg gac atc
gtg atg acc cag tct cca gac tcc ctg gct 96 Gly Ala Tyr Gly Asp Ile
Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 gtg tct ctg ggc
gag agg gcc acc atc aac tgc aag tcc agc cag act 144 Val Ser Leu Gly
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr 35 40 45 gtt tta
tac agc tcc aac aat aag aac tac tta gct tgg tac cag cag 192 Val Leu
Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln 50 55 60
aaa cca gga cag cct cct aag ctg ctc att tac tgg gca tct acc cgg 240
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg 65
70 75 80 gaa tcc ggg gtc cct gac cga ttc agt ggc agc ggg tct ggg
aca gat 288 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp 85 90 95 ttc act ctc acc atc agc agc ctg cag gct gac gat
gtg gca gtt tat 336 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Asp Asp
Val Ala Val Tyr 100 105 110 tac tgt cag caa tat tat agt act cct ccg
tgg acg ttc ggc caa ggg 384 Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro Pro
Trp Thr Phe Gly Gln Gly 115 120 125 acc aag gtg gaa atc aaa cga act
gtg gct gca cca tct 423 Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
Pro Ser 130 135 140 16 141 PRT Homo sapiens 16 Met Val Leu Gln Thr
Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 15 Gly Ala Tyr
Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 20 25 30 Val
Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Thr 35 40
45 Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser
Thr Arg 65 70 75 80 Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp 85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
Asp Asp Val Ala Val Tyr 100 105 110 Tyr Cys Gln Gln Tyr Tyr Ser Thr
Pro Pro Trp Thr Phe Gly Gln Gly 115 120 125 Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala Pro Ser 130 135 140 17 420 DNA Homo sapiens CDS
(1)..(420) sig_peptide (1)..(60) V_region (61)..(364) 17 atg aag
gat ctg ctc agc ttc ctg ggg ctg cta atg ctc tgg ata cct 48 Met Lys
Asp Leu Leu Ser Phe Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15
gga tcc agt gca gat att gtc atg acc cag acg cca ctc ttc tgt ccg 96
Gly Ser Ser Ala Asp Ile Val Met Thr Gln Thr Pro Leu Phe Cys Pro 20
25 30 tca ccc ctg gac agc cga gcc tcc atc tcc tgc aag tct ggt ctg
agc 144 Ser Pro Leu Asp Ser Arg Ala Ser Ile Ser Cys Lys Ser Gly Leu
Ser 35 40 45 ctc ctg cac agt gat gga aag acc tat ttg cat tgg tac
ctg cag aag 192 Leu Leu His Ser Asp Gly Lys Thr Tyr Leu His Trp Tyr
Leu Gln Lys 50 55 60 cca ggc cag cct cca cag ctc ctg atc tat gag
agt ttc caa ccg gtt 240 Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Glu
Ser Phe Gln Pro Val 65 70 75 80 ctc ctg gag tgc cag ata ggc tca gtg
gca gcg ggt cag gac aga ttt 288 Leu Leu Glu Cys Gln Ile Gly Ser Val
Ala Ala Gly Gln Asp Arg Phe 85 90 95 cac act gaa aat cag ccg ggt
gga agg ctg agg aat gtt ggg gtt tat 336 His Thr Glu Asn Gln Pro Gly
Gly Arg Leu Arg Asn Val Gly Val Tyr 100 105 110 tac tgc atg caa agt
tta cag ctt ccg ctc act ttc ggc gga ggg acc 384 Tyr Cys Met Gln Ser
Leu Gln Leu Pro Leu Thr Phe Gly Gly Gly Thr 115 120 125 aag gtg gag
atc aaa cga act gtg gct gca cca tct 420 Lys Val Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser 130 135 140 18 140 PRT Homo sapiens 18 Met Lys
Asp Leu Leu Ser Phe Leu Gly Leu Leu Met Leu Trp Ile Pro 1 5 10 15
Gly Ser Ser Ala Asp Ile Val Met Thr Gln Thr Pro Leu Phe Cys Pro 20
25 30 Ser Pro Leu Asp Ser Arg Ala Ser Ile Ser Cys Lys Ser Gly Leu
Ser 35 40 45 Leu Leu His Ser Asp Gly Lys Thr Tyr Leu His Trp Tyr
Leu Gln Lys 50 55 60 Pro Gly Gln Pro Pro Gln Leu Leu Ile Tyr Glu
Ser Phe Gln Pro Val 65 70 75 80 Leu Leu Glu Cys Gln Ile Gly Ser Val
Ala Ala Gly Gln Asp Arg Phe 85 90 95 His Thr Glu Asn Gln Pro Gly
Gly Arg Leu Arg Asn Val Gly Val Tyr 100 105 110 Tyr Cys Met Gln Ser
Leu Gln Leu Pro Leu Thr Phe Gly Gly Gly Thr 115 120 125 Lys Val Glu
Ile Lys Arg Thr Val Ala Ala Pro Ser 130 135 140 19 405 DNA Homo
sapiens CDS (1)..(405) sig_peptide (1)..(66) V_region (67)..(353)
19 atg gac atg agg gtc cct gct cag ctc ctg ggg ctc ctg ctg ctc tgg
48 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 ctc tca ggt gcc aga tgt gac atc cag atg acc cag tct cca
tcc ttc 96 Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Phe 20 25 30 cct gtc tgc atc tgt agg aga cag agt cac cat cac
ttg cca ggc gag 144 Pro Val Cys Ile Cys Arg Arg Gln Ser His His His
Leu Pro Gly Glu 35 40 45 tca gga cat tca cca cta ttt aaa ttg gta
tca gca gaa acc agg gaa 192 Ser Gly His Ser Pro Leu Phe Lys Leu Val
Ser Ala Glu Thr Arg Glu 50 55 60 agc cct aag ctc ctg atc tac gat
gca tcc aat ttg gaa aca ggg tcc 240 Ser Pro Lys Leu Leu Ile Tyr Asp
Ala Ser Asn Leu Glu Thr Gly Ser 65 70 75 80 cat cac ggt tca gtg gaa
gtg gat ctg gga cag att tta ctt tca cca 288 His His Gly Ser Val Glu
Val Asp Leu Gly Gln Ile Leu Leu Ser Pro 85 90 95 tca gca gcc tgc
agc tct gaa gat att gca aca tat tac tgt caa cag 336 Ser Ala Ala Cys
Ser Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln 100 105 110 tat aat
aat ctc atc acc ttc ggc caa ggg aca cga ctg gag att aaa 384 Tyr Asn
Asn Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120 125
cga act gtg gct gca cca tct 405 Arg Thr Val Ala Ala Pro Ser 130 135
20 135 PRT Homo sapiens 20 Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Ser Gly Ala Arg Cys Asp Ile
Gln Met Thr Gln Ser Pro Ser Phe 20 25 30 Pro Val Cys Ile Cys Arg
Arg Gln Ser His His His Leu Pro Gly Glu 35 40 45 Ser Gly His Ser
Pro Leu Phe Lys Leu Val Ser Ala Glu Thr Arg Glu 50 55 60 Ser Pro
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Thr Gly Ser 65 70 75 80
His His Gly Ser Val Glu Val Asp Leu Gly Gln Ile Leu Leu Ser Pro 85
90 95 Ser Ala Ala Cys Ser Ser Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln 100 105 110 Tyr Asn Asn Leu Ile Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser 130 135 21 387
DNA Homo sapiens CDS (1)..(387) sig_peptide (1)..(47) Initiation
codon and a portion of a signal sequence are lacked. 21 gat agg gtc
cta ggg gtc ctg atg gtt ggg ttt tcg gtg ccg gat gag 48 Asp Arg Val
Leu Gly Val Leu Met Val Gly Phe Ser Val Pro Asp Glu 1 5 10 15 aac
atc cag atg acc cag tat cca tct ccc tgt ctg cat acc tgt agg 96 Asn
Ile Gln Met Thr Gln Tyr Pro Ser Pro Cys Leu His Thr Cys Arg 20 25
30 aga cag agt cac cat cac ttg cca gag cga gct cag gac att cac cac
144 Arg Gln Ser His His His Leu Pro Glu Arg Ala Gln Asp Ile His His
35 40 45 tat cta aat tgg tat cag cag aaa cca ggg aaa gcc cta agc
tct gat 192 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Leu Ser
Ser Asp 50 55 60 cta cga tgc atc caa ttt gga aac agg gtc cca tca
cgg ttc agt gga 240 Leu Arg Cys Ile Gln Phe Gly Asn Arg Val Pro Ser
Arg Phe Ser Gly 65 70 75 80 agt gga tct ggg aca gat tct act tca cca
tca gca gcc tgc agc tct 288 Ser Gly Ser Gly Thr Asp Ser Thr Ser Pro
Ser Ala Ala Cys Ser Ser 85 90 95 gaa gat att gca aca tat tac tgt
caa cag tat aat aat ctc atc acc 336 Glu Asp Ile Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Asn Leu Ile Thr 100 105 110 ttc ggc caa ggg aca cga
ctg gag att aaa cga act gtg gct gca cca 384 Phe Gly Gln Gly Thr Arg
Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 115 120 125 tct 387 Ser 22
129 PRT Homo sapiens 22 Asp Arg Val Leu Gly Val Leu Met Val Gly Phe
Ser Val Pro Asp Glu 1 5 10 15 Asn Ile Gln Met Thr Gln Tyr Pro Ser
Pro Cys Leu His Thr Cys Arg 20 25 30 Arg Gln Ser His His His Leu
Pro Glu Arg Ala Gln Asp Ile His His 35 40 45 Tyr Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Leu Ser Ser Asp 50 55 60 Leu Arg Cys
Ile Gln Phe Gly Asn Arg Val Pro Ser Arg Phe Ser Gly 65 70 75 80 Ser
Gly Ser Gly Thr Asp Ser Thr Ser Pro Ser Ala Ala Cys Ser Ser 85 90
95 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Leu Ile Thr
100 105 110 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala
Ala Pro 115 120 125 Ser 23 411 DNA Homo sapiens CDS (1)..(411)
sig_peptide (1)..(66) V_region (67)..(356) 23 atg gac atg agg gtc
cct gct cag ctc ctg ggg ctc ctg ctg ctc tgg 48 Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 ctc tca ggt
gcc aga tgt gac atc cag atg acc cag tct cca tcc tcc 96 Leu Ser Gly
Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 ctg
tct gca tct gta gga gac aga gtc acc atc act tgc cgg gca agt 144 Leu
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40
45 cag agc att agc agc tat tta aat tgg tat cag cag aaa cca ggg aaa
192 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60 gcc cct aag ctc ctg att tat gct gca tcc agt ttg caa agt
ggg tcc 240 Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser
Gly Ser 65 70 75 80 cat caa ggt tca gtg gca gtg gat tat gcg aca gat
ttc cat ttc tca 288 His Gln Gly Ser Val Ala Val Asp Tyr Ala Thr Asp
Phe His Phe Ser
85 90 95 cca tca gca gtt tgc cac ctg acg att ttg caa ctt act act
gtc cac 336 Pro Ser Ala Val Cys His Leu Thr Ile Leu Gln Leu Thr Thr
Val His 100 105 110 aga gtt aca gta tcc cat tca ctt tcg gcc ctg ggg
acc aaa gtg gat 384 Arg Val Thr Val Ser His Ser Leu Ser Ala Leu Gly
Thr Lys Val Asp 115 120 125 agc aaa cga act gtg gct gca cca tct 411
Ser Lys Arg Thr Val Ala Ala Pro Ser 130 135 24 137 PRT Homo sapiens
24 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15 Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser 35 40 45 Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Ser 65 70 75 80 His Gln Gly Ser Val Ala
Val Asp Tyr Ala Thr Asp Phe His Phe Ser 85 90 95 Pro Ser Ala Val
Cys His Leu Thr Ile Leu Gln Leu Thr Thr Val His 100 105 110 Arg Val
Thr Val Ser His Ser Leu Ser Ala Leu Gly Thr Lys Val Asp 115 120 125
Ser Lys Arg Thr Val Ala Ala Pro Ser 130 135 25 27 DNA Artificial
Sequence Description of Artificial Sequence Artificially
synthesized adaptor sequence 25 ccatcctaat acgactcact atagggc 27 26
25 DNA Artificial Sequence Description of Artificial Sequence
Artificially synthesized primer sequence 26 ccagggccgc tgtgctctcg
gaggt 25 27 23 DNA Artificial Sequence Description of Artificial
Sequence Artificially synthesized primer sequence 27 gggggtcagg
ctggaactga gga 23
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