JP2000232884A - Monoclonal antibody against connective tissue growth factor and its medicinal use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new monoclonal antibody against connective tissue growth factor(CTGF) which reacts with the whole or a part of human, mouse and rat CTGF and is useful in the treatment or prevention for various diseases caused by CTGF and the determination or the like of CTGF. SOLUTION: This monoclonal antibody is a new monoclonal antibody possessing various properties, e.g. showing reactivity to all the human, mouse and rat connective tissue growth factor(CTGF), displaying reactivity to the both of human and mouse CTGF and no reactivity to rat CTGF, exerting reactivity to the both of mouse and rat CTGF and no reactivity to human CTGF, inhibiting the linkage between human ren-derived fibroblast strain 293-T and human CTGF, inhibiting the linkage between rat ren-derived fibroblast strain NRK-49F or the like and human CTGF or controlling the increase of renal hydroxypurine and is useful in the treatment or prevention for various diseases caused by human CTGF and the determination or the like of CTGF in the body fluid of mammals.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a mammalian connective tissue growth factor (CT).
GF), a monoclonal antibody or a part thereof, a cell producing the monoclonal antibody, an antibody-immobilized insoluble carrier obtained by immobilizing the monoclonal antibody or a part thereof, and labeling the monoclonal antibody with a labeling substance. Labeled antibody, kit used for detection, quantification, separation or purification of mammalian CTGF, mammalian CGF
Method for detecting, quantifying, separating or purifying TGF, pharmaceutical composition comprising said monoclonal antibody, human CTGF
Transgenic mouse and rat CTG
F polypeptide, DN encoding rat CTGF
A and antibodies reactive with rat CTGF.

[0002]

2. Description of the Related Art Tissue regeneration in tissue injury involves the removal of unnecessary tissue fragments and cell fragments or bacteria by phagocytic cells such as macrophages transferred to the injury site, restoration of the vascular system, and subsequent restoration with new tissue. This is done via substitution. In the process of tissue regeneration and repair,
Transforming growth factor β produced by macrophages and neutrophils that appear during the repair process
wth Factor β (TGF-β)) has been shown to act as the first regulator. The functions of TGF-β are diverse and regulate not only the induction of proliferation of mesenchymal cells, the suppression of proliferation of vascular endothelial cells and epithelial cells, but also the production of extracellular matrix (Extracellular Matrix (ECM)) from connective tissue cells It is known to have functions.

[0003] Platelet-derived growth factor (PDGF) and connective tissue growth factor (PDGF) are contained in the culture supernatant of mesenchymal cells which are stimulated by TGF-β to induce proliferation. CTG
F); also referred to as Hcs24), it is considered that the cell growth-inducing activity of TGF-β is indirectly exerted by other regulatory factors.

[0004] For CTGF, human and mouse C
TGF has already been identified (in addition, rat CTGF
No identification has been reported yet. ), And their physicochemical and biological properties have been analyzed ([human CTGF] J. Cell Biology, Vol. 11).
4, No. 6, p. 1285-1294, 1991; Int. J. Biochem. CellB
iol., Vol.29, No.1, p.153-161, 1997; Circulation,
Vol.95, No.4, p.831-839, 1997; Cell Growth Diffe
r., Vol. 7, No. 4, p. 469-480, 1996; J. Invest. Dermat
ol., Vol. 106, No. 4, p. 729-733, 1996; J. Invest. De
rmatol., Vol. 105, No. 2, p. 280-284, 1995; J. Inves
t. Dermatol., Vol. 105, No. 1, p. 128-132, 1995; and International Patent Application Publication No. WO 96/38172. [Mouse CTGF
(Fisp12)] JP-A-5-255597, Cell Growth Diff
er., Vol.2, No.5, p.225-233, 1991; FEBS Letters, V
ol. 327, No. 2, p. 125-130, 1993; and DNA Cell Biol.,
Vol.10, No.4, p.293-300, 1991).

[0005] CTGF is a secreted glycoprotein rich in cysteine residues having a molecular weight of about 38 kD, and its biosynthesis and secretion have been shown to be induced by TGF-β. CTGF is similar to PDGF in that it undergoes production induction by TGF-β, binds to PDGF receptor, induces proliferation of mesenchymal cell lines, and is produced from fibroblasts and epithelial cells. It is a completely different molecule that has properties but very little amino acid sequence homology (The Journal of Cell Biology, Vol. 114, No. 6,
p.1287-1294, 1991 and Molecular Biology of the Cel
l, Vol.4, p.637-645, 1993). Also, recent studies have shown that in culture supernatants of human and mouse fibroblasts and in porcine uterus-derived secretions, a biologically active molecular weight of about 10 to 1 which is considered to be a degradation product of 38 kDa CTGF.
A low molecular weight CTGF of 2 kDa has been identified (Growth
Factors, Vol. 15, No. 3, p. 199-213, 1998; J. Biol.
Chem., Vol.272, No.32, p.20275-20282, 1997).

Although the details of the physiological function of CTGF and its relation to diseases have not been completely elucidated yet, induction of production of CTGF by TGFβ, significant expression of CTGF mRNA in tissues and cells of patients with various diseases are disclosed. Expression (Int. J. Biochem. Cell. Biol., Vol. 29, No.
1, p.153-161, 1997; Circulation, Vol.95, No.4, p.83
1-839, 1997; J. Invest. Dermatol., Vol. 106, No. 4,
p.729-733, 1996; J. Invest. Dermatol., Vol.105, N
o.2, p.128-132, 1995; J. Cell Physiol., Vol.165, N
o.3, p.556-565, 1995 and Kidney Int., Vol.48, No.2,
p.5001-5009, 1995, etc.) and findings on the promotion of migration and proliferation of vascular endothelial cells by CTGF (J. Cell. Bio
l., Vol. 114, No. 6, p. 1285-1294, 1991; Exp. Cell Re
s., Vol.233, p.63-77, 1997;
8, extra number, page 463, PD0187, 1996 and 69th Abstract of the Japanese Biochemical Society, page 683, 1P0535, 1996, etc.)
It has been first suggested that CTGF may be involved in the development and / or progression of various diseases.

For the identification of specific diseases, it is necessary to wait for future research development and research results.
For example, cancer, arteriosclerosis, skin diseases (eg, psoriasis, scleroderma, atopy, keloid), kidney disease, arthritis (eg, rheumatoid arthritis), various fibrosis (arteriosclerosis, liver cirrhosis, arthritis, severe It is presumed to be involved in the development and / or progression of a wide range of diseases such as dermatosis, keloid, renal fibrosis, and tissue fibrosis seen in pulmonary fibrosis.

In elucidating the relationship between such various diseases and CTGF, CTGF contained in body fluids (such as serum) of patients suffering from various diseases and diseased mammals is considered.
And / or its fragments are detected and quantified, and its value is compared with a measured value in a normal living body (normal mammals such as a normal human, a normal mouse, a normal rat, and a normal rabbit). Means. For detection or quantification of a secretory protein such as CTGF, an immunological measurement method based on an antigen-antibody reaction using an antibody (particularly, a monoclonal antibody) against the secretory protein to be detected, Immunoassay (RIA) or enzyme immunoassay (EIA, ELISA)
Are widely used as the simplest and useful methods.

Similarly, the detection and quantification of CTGF also requires the detection and quantification by such an immunoassay, and the preparation and development of a monoclonal antibody against CTGF required for establishing the quantification method. However, regarding the antibody against CTGF, although there is a report on the production of antiserum (Exp. Cell Res., Vo
l.233, p.63-77, 1997; Cell Growth Differ., Vol.8,
No. 1, p. 61-68, 1997; and the 69th Annual Meeting of the Biochemical Society of Japan, p. 683, 1P0534, 1996), a monoclonal antibody,
In particular high affinity for CTGF and / or CT
No functional monoclonal antibody capable of neutralizing the activity of GF has been reported yet, and no quantitative system for CTGF by immunoassay has been provided.
In addition, a monoclonal antibody having the ability to neutralize the activity of CTGF as described above can be used not only as a component in such an immunoassay but also as a CT antibody as described above.
Although useful as an antibody drug in the treatment and / or prevention of various diseases caused by secretion of GF, no monoclonal antibody usable as the antibody drug has been reported at all.

[0010]

Accordingly, elucidation of the biological function of CTGF, which may be related to the onset and / or progression of various diseases as described above, and the causal relationship between the CTGF and various diseases, and There is a need for the development of monoclonal antibodies reactive with CTGF of various mammals such as humans, mice, rats, and rabbits, which can be used as active ingredients of pharmaceuticals in the treatment and prevention of diseases caused by CTGF. In particular, CT is a necessary means in elucidating the function of CTGF and the relationship between CTGF and various diseases.
Monoclonal antibodies used in immunoassays for GF include the desired affinity for the CTGF, C
The ability to neutralize the biological activity of TGF and / or the desired cross-reactivity to various mammals.
Development of a monoclonal antibody having a vity) is required. Furthermore, as a monoclonal antibody used for treatment and / or prevention of a patient suffering from the various diseases, in addition to the neutralizing activity, development of a monoclonal antibody having reduced or eliminated antigenicity for the patient is required. ing.

[0011]

In order to satisfy the above-mentioned social needs, the inventors of the present invention have proposed a CT method for various mammals.
As a result of intensive studies on monoclonal antibodies against GF, using CTGF derived from various mammals as an immunogen, antigen specificity, antigen affinity, neutralizing activity, and properties such as cross-reactivity, Various monoclonal antibodies against various mammalian CTGFs with different properties have been successfully produced. It was also the first in the world to produce various human monoclonal antibodies against human CTGF by immunizing transgenic mice produced to produce human antibodies using genetic recombination technology with human CTGF. Successful.

Furthermore, CTGF in body fluids (serum, etc.) of various mammals (humans, mice, rats, rabbits, etc.) can be used in an intact state by various immunoassay systems constructed using the former various monoclonal antibodies. The inventors have found that quantification can be performed easily and with high sensitivity, and have completed the present invention. In addition, they have found that the latter human antibody not only significantly neutralizes the activity of human CTGF, but also has a therapeutic effect on, for example, tissue fibrosis (eg, renal fibrosis). It has arrived. Since these human antibodies have no antigenicity to humans, which has been a major problem (side effect) in the treatment of antibody drugs composed of antibodies derived from non-human mammals such as antibodies derived from mice, etc. It dramatically increases its value.

That is, the present invention is useful as a drug in the treatment and prevention of patients, and as a component in an immunoassay for detecting and quantifying CTGF contained in body fluids of various mammals such as humans, mice and rats. For the first time in the field of the present invention, monoclonal antibodies against various mammals having various properties are provided. In addition, the present invention relates to CTG by using various monoclonal antibodies against such CTGF.
F provides an immunoassay method and assay kit for F for the first time.

The monoclonal antibody against human CTGF of the present invention can be used without causing antigenicity to humans.
It is extremely useful as a pharmaceutical for treating and preventing various diseases caused by CTGF. In addition, by immunoassay using the monoclonal antibody of the present invention, CTGF present in the body fluid of the normal living body of various mammals (human, mouse, rat, rabbit, etc.) and the living body suffering from the disease can be determined by the immunoassay using the monoclonal antibody of the present invention. It can be easily and highly sensitively detected and quantified in an intact state.

That is, the invention of the present invention described below.
You. (1) Gender described in any of (a) to (g) below
A monoclonal antibody characterized by having a quality or
Part thereof: (a) Human, mouse and rat connective tissue growth factor (C
(B) reactive with both human and mouse CTGF
And not reactive with rat CTGF; (c) reactive with both mouse and rat CTGF
(D) human kidney-derived fibroblast cell line 293-T (ATCC CRL-157)
3) binding to human CTGF or the cell line 293-T
Inhibits binding to mouse CTGF; (e) rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-
1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427)
Or any of human lung-derived fibroblasts and human CTG
Inhibits binding to F; (f) stimulates human or mouse CTGF
Of rat kidney-derived fibroblast NRK-49F (ATCC CRL-1570)
Inhibits cell growth; or (g) shows an increase in the production of hydroxyproline
Inhibits elevation of the hydroxyproline in the kidney
You. (2) The monoclonal antibody is as described in (a) to
(C) having the property described in any one of
Or the monoclonal antibody according to the above (1).
Part: (a) Immunization of mouse with human CTGF or a part thereof
Monoclonal antibody or part thereof obtained by
Against human, mouse and rat CTGF
(B) transforming mouse CTGF or a part thereof into hamsters;
Monoclonal antibody obtained by immunization or a part thereof
It is suitable for human, mouse and rat CTGF
Is also reactive; or (c) immunizing rats with mouse CTGF or a portion thereof
Monoclonal antibody or a part thereof
Against human, mouse and rat CTGF
Responsive. (3) The monoclonal antibody is (a) to
(C) having the property described in any one of
Or the monoclonal antibody according to the above (1).
Part: (a) Immunization of mouse with human CTGF or a part thereof
A human or mouse monoclonal antibody
And reactive with both rat and rat CTGF, and
Human kidney-derived fibroblast cell line 293-T (ATCC CRL-1573)
(B) immunizing rats with mouse CTGF or a part thereof.
A monoclonal antibody obtained by
Is reactive with both mouse and rat CTGF, and
Human kidney-derived fibroblast cell line 293-T (ATCC CRL-1573)
Inhibits binding to mouse CTGF; or (c) transforming mouse CTGF or a portion thereof into hamsters
A monoclonal antibody obtained by immunization,
Reactive with both mouse and rat CTGF
And human kidney-derived fibroblast cell line 293-T (ATCC CRL-15
73) inhibits the binding of the mouse to CTGF. (4) The monoclonal antibody has an international deposit number of FERM B
Monochrome produced from the fusion cell identified by P-6208
(1) wherein the antibody is a null antibody.
Noclonal antibodies or portions thereof. (5) the monoclonal antibody is international deposit number FERM B
Monochrome produced from the fusion cell identified by P-6208
Monoclonal with substantially the same properties as null antibody
The monoclonal antibody according to the above (1), which is an antibody.
Lonal antibodies or parts thereof. (6) the monoclonal antibody is international deposit number FERM B
Monochrome produced from the fused cells identified by P-6209
(1) wherein the antibody is a null antibody.
Noclonal antibodies or portions thereof. (7) the monoclonal antibody is international deposit number FERM B
Monochrome produced from the fused cells identified by P-6209
Monoclonal with substantially the same properties as null antibody
The monoclonal antibody according to the above (1), which is an antibody.
Lonal antibodies or parts thereof. (8) Human, mouse or rat CTGF
A human monoclonal antibody or its
Part of. (9) Human monoclonal antibody transforms into human CTGF
Characterized as a monoclonal antibody with reactivity
The human monoclonal antibody according to (8) or
Part of that. (10) Any one of the following (a) to (d)
A human thing reactive with human CTGF
Clonal antibody or part thereof: (a) Human kidney-derived fibroblast cell line 293-T (ATCC CRL-157)
3) inhibits the binding of human CTGF; (b) rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-
1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427)
Or any of human lung-derived fibroblasts and human CTG
Inhibits binding to F; (c) for stimulation of human or mouse CTGF
Of rat kidney-derived fibroblast NRK-49F (ATCC CRL-1570)
Inhibits cell growth; or (d) shows a tendency toward increased production of hydroxyproline
Inhibits elevation of the hydroxyproline in the kidney
You. (11) Human monoclonal antibody produces human antibody
Transgenic non-human mammals with the ability to
Before being characterized as a monoclonal antibody of origin
The human thing according to any one of the above (8) to (10)
Clonal antibodies or parts thereof. (12) The human monoclonal antibody is a human CTGF
A transgenic gene capable of producing human antibodies.
Monoclones obtained by immunizing non-human mammals
The antibody described in (11) above, which is a nal antibody.
Human monoclonal antibody or part thereof as described above. (13) The transgenic non-human mammal is a tiger
Wherein the mouse is a transgenic mouse.
(8) A human monok according to any of (12) to (12).
Lonal antibodies or parts thereof. (14) the heavy chain variable region of the human monoclonal antibody
The coding region V region DNA is DP-5, DP-38, DP-65 and
Any of the gene segments selected from the group consisting of DP-75
(8) to the above (8), wherein
A human monoclonal antibody according to any of (13);
Or part of it. (15) the light chain variable region of the human monoclonal antibody
The coding region V region DNA comprises DPK1, DPK9, DPK12 and D
Any gene segment selected from the group consisting of PK24
(8) to (1)
The human monoclonal antibody according to any of 3) or
Part of that. (16) the heavy chain variable region of the human monoclonal antibody
The coding region V region DNA is DP-5, DP-38, DP-65 and
Any of the gene segments selected from the group consisting of DP-75
And the light chain of the human monoclonal antibody is
The V region DNA encoding the variable region comprises DPK1, DPK9, and DPK.
Any gene selected from the group consisting of K12 and DPK24
(8) to (8), which are derived from segments.
The human monoclonal antibody according to any one of the above (15)
Body or part thereof. (17) the heavy chain variable region of the human monoclonal is
An amino acid sequence containing any one of the amino acid sequences (a) to (j)
(H) having amino acid;
Monoclonal antibody or part thereof: (a) Amino acid of amino acid sequence set forth in SEQ ID NO: 6
(B) the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 6;
In the amino acid sequences of positions 21 to 120,
Or several amino acids are deleted, substituted, inserted or added
(C) the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 8
(D) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 8;
In the amino acid sequences of positions 21 to 118,
Or several amino acids are deleted, substituted, inserted or added
(E) amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 10
(F) amino acid sequence of amino acid sequence set forth in SEQ ID NO: 10
In the amino acid sequence of acid numbers 21 to 116, 1
Or deletion, substitution, insertion or addition of several amino acids
(G) amino acids of the amino acid sequence set forth in SEQ ID NO: 12
(H) an amino acid sequence represented by SEQ ID NO: 12;
In the amino acid sequence of acid numbers 21 to 116, 1
Or deletion, substitution, insertion or addition of several amino acids
(I) amino acids of the amino acid sequence set forth in SEQ ID NO: 14
(J) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 14;
In the amino acid sequence of acid numbers 21 to 117, 1
Or deletion, substitution, insertion or addition of several amino acids
Amino acid sequence added. (18) The light chain variable region of the human monoclonal is
An amino acid sequence containing any one of the amino acid sequences (a) to (j)
(H) having amino acid;
Or a part thereof: (a) amino acid having the amino acid sequence of SEQ ID NO: 16
(B) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 16;
In the amino acid sequence of acid numbers 21 to 120, 1
Or deletion, substitution, insertion or addition of several amino acids
(C) amino acid sequence of amino acid sequence set forth in SEQ ID NO: 18
(D) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 18;
In the amino acid sequence of acid numbers 21 to 121, 1
Or deletion, substitution, insertion or addition of several amino acids
(E) the amino acid sequence of SEQ ID NO: 20;
(F) the amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 20;
In the amino acid sequence of acid numbers 23 to 117, 1
Or deletion, substitution, insertion or addition of several amino acids
(G) amino acid sequence of amino acid sequence set forth in SEQ ID NO: 22
(H) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 22;
In the amino acid sequence of acid numbers 17 to 111, 1
Or deletion, substitution, insertion or addition of several amino acids
(I) amino acids of the amino acid sequence set forth in SEQ ID NO: 24
(J) the amino acid sequence of the amino acid sequence represented by SEQ ID NO: 24;
In the amino acid sequence of positions 23 to 118, 1
Or deletion, substitution, insertion or addition of several amino acids
Amino acid sequence added. (19) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6535.
Monoclonal antibodies produced from the fused cells or
Part of that. (20) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6535.
And monoclonal antibodies produced from fused cells
Monoclonal antibodies with identical properties or their
part. (21) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6598.
Monoclonal antibodies produced from the fused cells or
Part of that. (22) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6598.
And monoclonal antibodies produced from fused cells
Monoclonal antibodies with identical properties or their
part. (23) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6599.
Monoclonal antibodies produced from the fused cells or
Part of that. (24) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6599.
And monoclonal antibodies produced from fused cells
Monoclonal antibodies with identical properties or their
part. (25) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6600.
Monoclonal antibodies produced from the fused cells or
Part of that. (26) Monochrome reactive to human CTGF
Antibody, identified by International Deposit Number FERM BP-6600.
And monoclonal antibodies produced from fused cells
Monoclonal antibodies with identical properties or their
part. (27) Monochrome reactive to human CTGF
A null antibody or a portion thereof, wherein the monoclonal antibody
Body reacts the monoclonal antibody to human CTGF
(17) or (18) above having
Consists of some monoclonal antibodies and human CTGF
When reacted with the antigen-antibody complex, it reacts with the antigen-antibody complex.
Monoclonal antibodies characterized by lack of reactivity
Or part of it. (28) The monoclonal antibody is a human monoclonal
The antibody described in (27) above,
Noclonal antibodies or portions thereof. (29) Monochrome reactive to rat CTGF
Or a partial antibody thereof. (30) The variable region is as described in (2) to (7),
(27) The monoc according to any of (29) or (29).
The variable region derived from the nal antibody and the constant region
Characterized by being a constant region derived from immunoglobulin
Chimera mono-reactive with human CTGF
Clonal antibodies. (31) Part or all of the complementarity determining region of the hypervariable region
The part is (2) to (7), (27) or
The monoclonal antibody according to any one of (29) to (29).
And the framework region of the hypervariable region is human
A framework region derived from an immunoglobulin and a constant region
Is a constant region derived from human immunoglobulin.
Recombinant human form reactive to human CTGF
Monoclonal antibody. (32) Any of (1) to (29) above
Cells that produce the indicated monoclonal antibodies. (33) The set according to (30) or (31).
Cells that produce recombinant monoclonal antibodies. (34) The cell produces the monoclonal antibody
Capability of B cells derived from mammals and myelo derived from mammals
Characterized as a fusion cell obtained by fusing with a malignant cell
(32). (35) The cell binds the heavy chain of the monoclonal antibody.
Of the DNA to be loaded or the DNA encoding the light chain thereof
Either DNA, or both DNAs can be
Transgenic cells transformed by introduction
(32) or (3),
The cell according to 3). (36) The fusion cell as described in International Deposit No. FERM BP-6535.
(3) the fusion cell to be identified;
The fused cell according to 4). (37) The fusion cell is obtained under International Deposit No. FERM BP-6598.
(3) the fusion cell to be identified;
The fused cell according to 4). (38) The fusion cell has the International Deposit No. FERM BP-6599.
(3) the fusion cell to be identified;
The fused cell according to 4). (39) The fusion cell has the International Deposit No. FERM BP-6600.
(3) the fusion cell to be identified;
The fused cell according to 4). (40) The fusion cell is obtained under International Accession Number FERM BP-6208.
(3) the fusion cell to be identified;
The fused cell according to 4). (41) The fusion cell is obtained under International Deposit No. FERM BP-6209.
(3) the fusion cell to be identified;
The fused cell according to 4). (42) Any of (1) to (31) above.
The monoclonal antibody described above is immobilized
And an insoluble carrier immobilized thereon. (43) When the insoluble carrier is a plate, test tube,
Beads, beads, balls, filters and membranes.
Before being characterized by being an insoluble carrier selected from the group
The insoluble carrier immobilized on the antibody according to the above (42). (44) If the insoluble carrier is a filter or membrane
Or affinity column chromatography
(4) The insoluble carrier used
2. The antibody-immobilized insoluble carrier according to 2). (45) Any of the above (1) to (31)
Monoclonal antibody, alone or in combination with other substances.
Responding can result in a detectable signal
A labeled antibody that is labeled with a possible labeling substance. (46) The labeling substance is an enzyme, a fluorescent substance, a chemiluminescent substance
Quality, biotin, avidin, or radioisotope
The labeled antibody according to the above (45), wherein: (47) Any of the above (1) to (31)
The monoclonal antibody described in (42) or the above
(43) the antibody-immobilized insoluble carrier according to (43);
5) or a group consisting of the labeled antibody according to (46).
At least one monoclonal selected from the group consisting of:
Do not include antibodies, insoluble carriers immobilized on antibodies or labeled antibodies.
Detection or determination of CTGF in mammals
Kit used for quantity. (48) The method according to (42) or (43).
The antibody-immobilized insoluble carrier, and the above (45) or the above
(46) It is characterized by comprising the labeled antibody of (46).
The detection of mammalian CTGF according to (47) above,
Is a kit used for quantification. (49) Any of (1) to (31) above.
The monoclonal antibody described in (42) or the above
(43) the antibody-immobilized insoluble carrier according to (43);
5) or a group consisting of the labeled antibody according to (46).
At least one monoclonal selected from the group consisting of:
Use antibody, antibody-immobilized insoluble carrier, or labeled antibody
Mammalian CT by immunoassay characterized by the following:
A method for detecting or quantifying GF. (50) At least the following steps (a) and (b)
Detection of mammalian CTGF by immunoassay
Is the method according to the above (49) for quantification: (a) the antibody solid according to the above (42) or (43)
Reacting a sample with the immobilized insoluble carrier; and (b) reacting the antibody immobilized insoluble carrier with the antibody contained in the sample.
Antigen-antibody complex formed by binding to CTGF in mammals
The label according to (45) or (46) above,
A step of reacting the antibody. (51) At least the following steps (a) and (b)
Detection of mammalian CTGF by immunoassay
The method according to the above (49) for quantifying: (a) the labeled antibody according to the above (45) or (46)
Reacting the sample with a body; and (b) the labeled antibody and mammalian CTG contained in the sample.
The antigen-antibody complex formed by bonding with F
(42) or the insolubility of the antibody immobilized according to the above (43)
A step of reacting the carrier. (52) An immunooa including at least the following step (a):
Detect or quantify mammalian CTGF by assay
The method according to the above (49): (a) the antibody according to the above (42) or (43)
Immobilized insoluble carrier, said (45) or (46)
React the mixture containing the labeled antibody and the sample described in
Process. (53) An immunooa including at least the following step (a):
Detect or quantify mammalian CTGF by assay
The method according to the above (49): (a) the antibody solid according to the above (42) or (43);
Samples, as well as alone or other substances, on the immobilized insoluble carrier
Can produce a detectable signal by reacting with
Of mammalian CTGF labeled with a labeling substance capable of
The step of reacting a standard substance. (54) At least the following steps (a) and (b)
Detection of mammalian CTGF by immunoassay
The method according to the above (49) for quantifying: (a) reacting with a sample, alone or with another substance;
And a marker that can result in a detectable signal
Of a mammalian CTGF standard labeled with
The mixture described in any of (1) to (31) above
Reacting the above monoclonal antibody; and (b) mammalian CTGF or mammalian CTGF contained in the sample.
The labeled mammalian CTGF standard,
Antigen-antibody complex formed by binding to clonal antibody
Mammals having a reactivity with the monoclonal antibody in the body
A step of reacting the antiserum derived therefrom. (55) At least the following steps (a) to (c)
Detection of mammalian CTGF by immunoassay
Is a method according to the above (49), wherein: (a) any one of the above (1) to (31) is added to the sample
(B) reacting the reaction system in which the step (a) has been carried out, alone or with another
Has a detectable signal by reacting with
CT of mammals labeled with a labelable substance
Reacting a standard substance of GF; and (c) mammalian CTGF or mammalian CTGF contained in the sample.
The labeled mammalian CTGF standard,
Antigen-antibody complex formed by binding to clonal antibody
Mammals having a reactivity with the monoclonal antibody in the body
A step of reacting the antiserum derived therefrom. (56) The anti-drug according to (42) or (44).
Mammalian CTGF comprising a body-immobilized insoluble carrier
Kit used for separation or purification. (57) The anti-drug according to (42) or (44).
Affinity Chromatography Using Immobilized Insoluble Carrier
Mammalian CTGF characterized by using luffy
A method for separating or purifying (58) Affinity chromatography
In the above (57) which is a tea column chromatography
A method for purifying mammalian CTGF according to the above. (59) DNA encoding human CTGF is endogenous
Transcript characterized by being integrated at the sex locus
Sgenic mouse. (60) the amino acid sequence represented by SEQ ID NO: 2 or
Has an amino acid sequence substantially identical to the amino acid sequence
Rat CTGF. (61) having the amino acid sequence of SEQ ID NO: 2
DNA encoding rat CTGF. (62) a base sequence represented by SEQ ID NO: 1
The base sequence from 213 to 1256 is included.
The DNA according to (61), which is a feature. (63) Any of (2) to (31) above.
Monoclonal antibody or part thereof, and pharmaceutical
A pharmaceutical composition comprising a pharmaceutically acceptable carrier. (64) The above (9) to (18) or the above (2)
8) The human monoclonal antibody according to any one of
Or a part thereof, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising: (65) The above (14) to (18) or the above
(28) The human monoclonal antibody according to any of (28)
A pharmaceutical composition comprising the body or a part thereof. (66) The pharmaceutical composition grows upon stimulation of CTGF
Used to inhibit the growth of cells with the ability to
(63) to (65).
A pharmaceutical composition according to any of the preceding claims. (67) The pharmaceutical composition is proliferated by stimulation of CTGF.
To treat or predict diseases involving the proliferation of cells capable of
Any of the above (63) to (65) for preventing
A pharmaceutical composition according to claim 1. (68) The proliferation of the cell is brain, neck, lung, heart, liver
Gut, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, offspring
Shrine, ovary, testis, prostate, skin, oral cavity, tongue, and blood vessels
Cell proliferation in a tissue selected from the group consisting of
(66) or (67).
Pharmaceutical composition. (69) The tissue is lung, liver, kidney, or skin.
The pharmaceutical composition according to the above (68), wherein (70) The tissue is a kidney,
The pharmaceutical composition according to (69). (71) The disease is further associated with tissue fibrosis.
The pharmaceutical composition according to the above (67), wherein
object. (72) The fibrosis of the tissue is lung, liver, kidney or skin
(71) the fibrosis in the skin.
A pharmaceutical composition according to claim 1. (73) The fibrosis of the tissue is fibrosis in the kidney.
The pharmaceutical composition according to the above (72), wherein (74) a CTGF inhibitor or a CTGF production inhibitor,
And a pharmaceutically acceptable carrier.
A pharmaceutical composition for treating or preventing a disease in the subject. (75) the inhibitor has a reactivity with CTGF;
(74) wherein the antibody is a clonal antibody.
The pharmaceutical composition according to any one of the preceding claims. (76) The inhibitor according to any of (9) to (31),
It is a monoclonal antibody described in any of the above
The pharmaceutical composition according to the above (74), wherein (77) The inhibitor as described in (14) to (18) above.
Or the human monochrome according to any of (28) above.
(76) wherein the antibody is a null antibody.
Pharmaceutical composition. (78) The disease is a disease associated with tissue fibrosis.
Any one of the above (74) to (77),
A pharmaceutical composition according to any one of the above. (79) capable of proliferating by stimulation of CTGF
Has the ability to suppress the proliferation of cells induced by stimulation of CTGF
And a pharmaceutically acceptable carrier.
And a pharmaceutical composition for inhibiting the proliferation of the cells in the kidney
Adult. (80) The substance, wherein the substance is reactive with CTGF
(79) characterized in that it is a clonal antibody.
The pharmaceutical composition described above. (81) The inhibitor according to any one of (9) to (31),
It is a monoclonal antibody described in any of the above
The pharmaceutical composition according to the above (79), wherein (82) The inhibitor according to any one of (14) to (18),
Or the human monochrome according to any of (28) above.
(81) wherein the antibody is a null antibody.
Pharmaceutical composition.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail by clarifying the meaning of words used in the present invention.
"Mammal" in the present invention, human, cow, goat,
Rabbit, mouse, rat, hamster, guinea pig, etc., are preferably human, rabbit, rat, hamster or mouse, and particularly preferably human, rat, hamster or mouse. The terms "non-human mammal" and "non-human mammal" used in the present invention are synonymous with each other and mean any non-human mammal among the mammals defined above.

The term "amino acid" as used in the present invention means any naturally occurring amino acid, and is preferably as follows, according to the three-letter or single-letter notation of the alphabet used to represent amino acids. Means the amino acid represented by Glycine (Gly / G), alanine (Ala / A), valine (Va
l / V), leucine (Leu / L), isoleucine (Ile /
I), serine (Ser / S), threonine (Thr / T), aspartic acid (Asp / D), glutamic acid (Glu / E), asparagine (Asn / N), glutamine (Glu / Q), lysine (Lys / L K), arginine (Arg / R), cysteine (Cys
/ C), methionine (Met / M), phenylalanine (Phe
/ F), tyrosine (Tyr / Y), tryptophan (Trp /
W), histidine (His / H), proline (Pro / P).

The term "connective tissue growth factor" (Connec
"tive Tissue Growth Factor (CTGF)""is the mammalian CTGF, and includes, for example, human and mouse CTGF having the structure and function reported in the previous report as described above (for example, Journal of Cel
l Biology, Vol.114, No.6, p.1287-1294, 1991, Molec
ular Biology of the Cell, Vol. 4, p. 637-645, 1993,
And Biochem. Biophys. Res. Comm., Vol. 234, p. 206-2
10, 1997). Needless to say, the present invention also includes rat CTGF, which is one of the present invention.

The connective tissue growth factor referred to in the present invention includes CTGF having a molecular weight of about 38 kDa described in the literature.
(For example, human CTGF), as well as a low-molecular-weight CTG having a molecular weight of about 10 to 12 kDa, which is considered to be a degradation product of full-length CTGF (for example, human CTGF) having a molecular weight of about 38 kDa.
Includes F protein (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 has not been elucidated yet, in human CTGF, leucine (Leu246) at position 246 and glutamic acid (Glu24) at position 247 of full-length human CTGF consisting of 349 amino acids.
It is thought to be caused by cutting during 7) 103
C-terminal protein (molecular weight: about 11,800D)
a) Alternatively, glutamic acid (Glu247) at position 247 and glutamic acid (Glu248) at position 248 of the same full-length human CTGF
Is thought to be caused by a disconnection between
C-terminal protein (molecular weight: about 11,671D)
a) has the possibility of being: Furthermore, as long as the “monoclonal antibody” according to the present invention described below is reactive with CTGF (particularly human CTGF) having a primary protein primary structure (amino acid sequence) or a part thereof, the CTGF referred to in the present invention is used. Also includes CTGF having an amino acid sequence substantially identical to the amino acid sequence of the native protein or a part thereof.

As used in the present invention, the term "having substantially the same amino acid sequence" refers to naturally occurring CT
As long as it has substantially the same biological properties as GF, a plurality of amino acids in the amino acid sequence, preferably 1 to 1
A protein having an amino acid sequence in which 0 amino acids, particularly preferably 1 to 5 amino acids have been substituted, deleted and / or modified, and a plurality of amino acids, preferably 1 to 10 amino acids, It is meant to include proteins having an amino acid sequence to which amino acids, particularly preferably 1 to 5 amino acids are added. Furthermore, the case of a plurality of combinations of such substitution, deletion, modification and addition may be used. CTGF in the present invention
Can be produced by appropriately using known methods known in the technical field such as a chemical synthesis method, a cell culture method, or a modification method thereof, in addition to a gene recombination technique.

The CTGF of the present invention includes the C
"Parts" of TGF are also included. Here, “part of CTGF” is defined as CTGF as defined above (the molecular weight of about 10
(Including a low molecular weight CTGF of about 12 kDa to about 12 kDa), specifically a CTGF peptide fragment having 5 to 100 amino acid residues (for example, C-terminal side), Specifically, it includes a CTGF peptide fragment having 5 to 50 amino acid residues, and more specifically, a peptide fragment having 5 to 30 amino acid residues. Preferably, a site where CTGF binds or interacts with its receptor (such as a receptor binding site) or a site required for CTGF to exert its biological function (such as an active site)
Is a partial structure of CTGF containing These polypeptides (partial structures and fragments) can be produced by a genetic recombination technique or a chemical synthesis method according to a known method or a modification method thereof known in the technical field described later, or a cell culture. It can be produced by appropriately cleaving CTGF isolated by the method using a proteolytic enzyme or the like.

The "monoclonal antibody" in the present invention is a monoclonal antibody reactive to mammalian connective tissue growth factor (CTGF) or a part thereof. Specifically, it is a monoclonal antibody having the features described in any one of the inventions (1) to (31). More specifically, they are various monoclonal antibodies having various properties and industrial utility as described in the examples and drawings below.

Preferred embodiments of the monoclonal antibody of the present invention include, for example, the following monoclonal antibodies (a) to (d). (A) A monoclonal antibody having any of the properties (d) to (g) of the monoclonal antibody according to (1). (B) The monoclonal antibody according to (2). (C) The monoclonal antibody according to any one of (4) to (7). (D) The monoclonal antibody according to any one of (9) to (31). In this embodiment, for the treatment or prevention of various diseases, that is, for use for the purpose of application as a pharmaceutical, human monoclonal antibodies included in the monoclonal antibodies described in (a) to (d) above are preferable. In this embodiment, for the purpose of quantifying, detecting, separating or purifying mammalian CTGF, which is another subject of the present invention, any of the monoclonal antibodies described in (a) to (d) above can be used. obtain.

Further preferred embodiments of the monoclonal antibody of the present invention include, for example, the following (e) or (f)
Monoclonal antibody. (E) a monoclonal antibody having any of the properties (d) to (g) of the monoclonal antibody according to (1). (F) The monoclonal antibody according to any of (4) to (7). G) The monoclonal antibody according to any of (10), (14) to (28). In this embodiment, for the treatment or prevention of various diseases, that is, for use for the purpose of application as a pharmaceutical, human monoclonal antibodies included in the monoclonal antibodies described in (e) to (f) above are preferable. In this embodiment, for the purpose of quantifying, detecting, separating or purifying mammalian CTGF, which is another subject of the present invention, any of the monoclonal antibodies described in (e) to (f) above can be used. obtain.

Particularly preferred embodiments of the monoclonal antibody of the present invention include, for example, the following monoclonal antibodies (g) and (h). (G) The monoclonal antibody according to (D) to (G). (H) The above (14) to (26) or the above (28)
The monoclonal antibody according to any one of the above. In this embodiment, for the treatment or prevention of various diseases, that is, for use for the purpose of application as a pharmaceutical, the monoclonal antibody described in (h) above is preferable. In this aspect, another subject of the present invention is a mammalian CTG.
For use for the purpose of quantifying, detecting, separating or purifying F, any of the monoclonal antibodies described in (G) to (H) above can be used.

Particularly preferred embodiments of the monoclonal antibody of the present invention include, for example, the following (i) to (f):
Can be mentioned. (I) The monoclonal antibody according to (4) or (6). (Nu) The monoclonal antibody according to any of (14) to (16). (R) A monoclonal antibody having the characteristics described in (a), (c), (e), (g) or (i) of the monoclonal antibody according to (17). (ヲ) A monoclonal antibody having the characteristics described in (a), (c), (e), (g) or (i) of the monoclonal antibody described in (18). (W) The monoclonal antibody according to any one of (19), (21), (23) and (25). (F) The monoclonal antibody according to (28). In this embodiment, the monoclonal antibody described in any of (nu) to (f) above is preferable for use in the treatment or prevention of various diseases, that is, for use as a medicament. In this embodiment, in the use for the purpose of quantifying, detecting, separating or purifying mammalian CTGF, which is another subject of the present invention, the above (i) to (f) are used.
Although any of the monoclonal antibodies described in (1) above can be used, the monoclonal antibody described in (1) above is particularly preferable.

The "monoclonal antibody" of the present invention uses the connective tissue growth factor (including natural, recombinant, synthetic, and cell culture supernatant) defined above or a part thereof as an antigen (immunogen). Natural antibodies obtained by immunizing mammals such as mice, rats, hamsters, guinea pigs or rabbits, chimeric antibodies and human antibodies (CDR-grafted antibodies) that can be produced using genetic recombination techniques, and, for example, Human monoclonal antibodies that can be produced using human antibody-producing transgenic animals and the like are also included. Moreover. Recombinant monoclonal antibodies, such as those produced from "cells producing recombinant monoclonal antibodies" of the present invention described below, are also included in the monoclonal antibodies of the present invention. In the case of a monoclonal antibody, Ig
G, IgM, IgA (IgA1, IgA2), IgD or I
Includes monoclonal antibodies having any isotype such as gE. Preferably, IgG (IgG1, IgG2,
IgG3, IgG4, preferably IgG2 or IgG4) or IgM
And more preferably IgG. .

The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method. That is, for example,
Antigen, if necessary, Freund's adjuvant (Freun
d's Adjuvant), together with mammals (including transgenic animals created to produce antibodies from other animals, such as human antibody-producing transgenic mice described below), preferably mice, rats, hamsters, and guinea pigs Immunize rabbits, cats, dogs, pigs, goats, horses or cows, more preferably mice, rats, hamsters, guinea pigs or rabbits. Polyclonal antibodies can be obtained from serum obtained from the immunized animal. In addition, a monoclonal antibody is prepared by preparing a fusion cell (hybridoma) from the antibody-producing cell obtained from the immunized animal and a myeloma cell line (myeloma cell) having no autoantibody-producing ability, cloning the hybridoma, It is produced by selecting a clone that produces a monoclonal antibody showing specific affinity for the antigen used for immunization. It can also be produced by the "cells producing recombinant monoclonal antibodies" of the present invention described below.

A monoclonal antibody can be specifically produced as follows. That is, the aforementioned connective tissue growth factor (CTGF; including natural, recombinant, synthetic, and cell culture supernatant) or a part thereof is used as an immunogen,
The immunogen is optionally added to Freund's adjuvant (Fr
together with a non-human mammal, specifically, a mouse, rat, hamster, guinea pig or rabbit, preferably mouse, rat or hamster (an antibody derived from another animal such as a human antibody-producing transgenic mouse described below). Immunization is performed by one or several injections or subcutaneous, intramuscular, intravenous, intravenous, footpad or intraperitoneal injections or transplantation of transgenic animals (including transgenic animals produced to produce). Usually, immunization is performed 1 to 4 times about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization. The number of immunizations and the time interval can be appropriately changed depending on the nature of the immunogen used.

The preparation of fusion cells (hybridomas) secreting monoclonal antibodies was carried out according to the method of Kohler and Milstein et al. (Nature, Vol. 256, No. 4).
95-497, 1975) and modifications according to it. That is, an antibody-producing cell contained in a spleen, lymph node, bone marrow, tonsil, or the like, and preferably in the spleen, obtained from a non-human mammal immunized as described above, and preferably a mouse, rat, guinea pig, hamster, rabbit Alternatively, it is prepared by cell fusion with a myeloma cell having no autoantibody-producing ability derived from a mammal such as a human, more preferably a mouse, a rat or a human.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (AT
CC No. CRL-1580), P3 / NSI / 1-Ag4-1 (NS-1), P3 /
X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI,
F0 or BW5147, rat myeloma 210RCY3-Ag.
2.3., Human-derived myeloma U-266AR1, GM1500-6TG-A1-
2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used. Screening for cells producing monoclonal antibodies (eg, hybridomas) is performed by culturing the cells in, for example, a microtiter plate, and using the culture supernatant of the wells in which proliferation has been observed in the immunization antigen used in the mouse immunization described above. For RIA, E
The measurement can be performed by an enzyme immunoassay such as LISA.

The production of the monoclonal antibody from the hybridoma is carried out by in vitro or by using a mouse, rat, guinea pig, hamster or rabbit, etc.
Preferably, it is performed in vivo in a mouse or rat, more preferably in ascites of a mouse, or the like, and can be performed by isolation from the obtained culture supernatant or ascites of a mammal. In addition, a gene encoding a monoclonal antibody is cloned from the hybridoma or a “cell producing a recombinant monoclonal antibody” of the present invention described below, and the gene is incorporated into an endogenous gene using a transgenic animal production technique. Genetic cows, goats, sheep or pigs can be produced and a large amount of a monoclonal antibody derived from the antibody gene can be obtained from the milk of the transgenic animal (Nikkei Science, April 1997, No. 78 pages to 84
page). When the cells are cultured in vitro, the hybridoma is grown, maintained, and stored in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culture method, and the monoclonal antibody is added to the culture supernatant. It is possible to use any known nutrient medium derived from a known nutrient medium or a known basal medium as used for production.

As the basic medium, for example, a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium and MCDB104 medium, MEM medium, D-MEM medium,
Examples include a high calcium medium such as a PMI1640 medium, an ASF104 medium, and an RD medium. The basic medium can contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances depending on the purpose.
For isolation and purification of the monoclonal antibody, the above-mentioned culture supernatant or ascites is obtained by using a saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), an anti-immunoglobulin column or It can be performed by subjecting it to affinity column chromatography such as a protein A column.

The monoclonal antibody of the present invention has a heavy chain having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of each of the heavy and / or light chains constituting the antibody. A monoclonal antibody consisting of a chain and / or a light chain is also encompassed, but the "several amino acids" herein means a plurality of amino acids, specifically 1 to 10 amino acids, and is preferably Is one to five amino acids. The partial modification (deletion, substitution, insertion, addition) of the amino acid as described above is introduced into the amino acid sequence of the monoclonal antibody of the present invention by partially modifying the base sequence encoding the amino acid sequence. be able to. This partial modification of the nucleotide sequence can be performed by a known site-specific mutagenesis method (Site specific mutagenesis).
Genes) can be introduced by a conventional method (Proc. Natl. Acsd. Sci. USA, Vol. 81, p. 5662-5666,
1984).

In the present invention, "human monoclonal antibody" refers to a mammalian CTGF as defined above.
(Preferably human CTGF). For example, various human monoclonal antibodies having various properties as described in the examples and drawings below can be mentioned. In particular,
Variable region of heavy chain (H chain) constituting immunoglobulin and constant region of H chain (Constant Reg)
ion) and all regions including the light chain (L chain) variable region and the L chain constant region are human immunoglobulins derived from genes encoding human immunoglobulins. The light chain includes a human κ chain or a human λ chain.

[0036] The human monoclonal antibody of the present invention can be produced, for example, according to a previously reported method, "Transgenic mouse capable of producing human antibody".
"A transgenic non-human mammal capable of producing a human antibody", as represented by, for example, can be produced by immunizing with a mammalian CTGF as defined above. The production and screening of the immunized and antibody-producing fusion cells (hybridomas) to the non-human mammal, and the large-scale preparation of the human monoclonal antibody,
It can be performed using the general method described above (Na
Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetic
s, Vol. 15, p. 146-156, 1997; Japanese Patent Publication No. 4-504365;
Japanese Patent Publication No. Hei 7-509137; Nikkei Science, June, No. 4
0 to 50, 1995; International Application Publication WO 94/2
No. 5585; Nature, Vol. 368, p. 856-859, 1994;
And Japanese Patent Application Laid-Open No. 6-500233. The human antibody-producing transgenic mouse can be specifically produced, for example, by using a method comprising the following steps. Other human antibody-producing transgenic non-human mammals can be produced in a similar manner.

(1) The mouse endogenous immunoglobulin heavy chain gene functions by substituting at least a part of the mouse endogenous immunoglobulin heavy chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination. Producing knockout mice that have been specifically inactivated. (2) By replacing at least a part of the mouse endogenous immunoglobulin light chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination, the mouse endogenous immunoglobulin light chain gene (particularly κ
A knockout mouse in which the chain gene is functionally inactivated. (3) Yeast artificial chromosome,
YAC) a step of producing a transgenic mouse in which a desired region of a human immunoglobulin heavy chain locus is integrated into a mouse chromosome using a vector capable of carrying a large gene such as a vector. (4) Human immunoglobulin light chains (particularly κ
Chain) producing a transgenic mouse in which the desired region of the locus has been integrated into the mouse chromosome. (5) By breeding the knockout mouse and transgenic mouse of (1) to (4) in any order, both the mouse endogenous immunoglobulin heavy chain locus and the mouse endogenous immunoglobulin light chain locus function. Producing a transgenic mouse that has been specifically inactivated and has both the desired region of the human immunoglobulin heavy chain locus and the desired region of the human immunoglobulin light chain locus integrated on the mouse chromosome.

In the knockout mouse, the locus cannot be rearranged (rearranged) by substituting an appropriate region of the mouse endogenous immunoglobulin locus with a foreign marker gene (such as a neomycin resistance gene) by homologous recombination. Can be produced by inactivating as described above. Inactivation using the homologous recombination includes, for example, positive negative selection (Positive
A method called Negative Selection (PNS) can be used (Nikkei Science, May, p.52-62, 199).
Four). For functional inactivation of the immunoglobulin heavy chain locus, for example, a J region or a C region (eg, a Cμ region)
Can be achieved by introducing obstacles to some of them. Functional inactivation of an immunoglobulin light chain (for example, a κ chain) can be achieved by, for example, introducing a disorder into a part of the J region or C region, or a region including a region spanning the J region and C region. It is.

The transgenic mouse can be prepared by a conventional method such as that usually used in the production of transgenic animals (for example, the latest manual for animal cell experiments, L.I.
(See C Publishing, Chapter 7, pp. 361-408, 1990). Specifically, for example, an HPRT-negative (lacking the hypoxanthine guanine phosphoribosyltransferase gene) ES cell (embryonic stem cell) derived from a normal mouse blastcyst is ligated to the human immunoglobulin heavy chain. The gene encoding the locus or the light chain locus or a part thereof and the yeast containing the YAC vector into which the HPRT gene has been inserted are fused by spheroplast fusion. ES cells in which the exogenous gene has been integrated on the mouse endogenous gene are selected by the HAT selection method. Next, the selected ES cells are microinjected into fertilized eggs (blastocysts) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12,
pp. 7380-7384, 1980; U.S. Patent No. 4,873,191).
The blastocysts are implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse is obtained. The heterogeneic
By crossing transgenic mice,
According to Mendel's law, homogenic transgenic mice are obtained.

The "chimeric monoclonal antibody" in the present invention is a monoclonal antibody produced by genetic engineering, and specifically, the variable region thereof comprises a non-human mammal (mouse, rat, hamster, etc.) A chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody, which is a variable region derived from a globulin and whose constant region is derived from a human immunoglobulin. The constant regions derived from human immunoglobulin include IgG (IgG1, IgG2, IgG3, IgG4),
Although it has a unique amino acid sequence depending on isotypes such as gM, IgA, IgD, and IgE, the constant region of the recombinant chimeric monoclonal antibody in the present invention may be a human immunoglobulin constant region belonging to any isotype. Preferably, it is a human IgG constant region.

The chimeric monoclonal antibody of the present invention can be produced, for example, as follows. However, it is needless to say that the present invention is not limited to such a manufacturing method. For example, a mouse / human chimeric monoclonal antibody is described in Experimental Medicine (Special Issue), Vol. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-732.
It can be manufactured with reference to Japanese Patent Publication No. 80 and the like. That is, a human immunoglobulin is downstream of an active _VH gene (rearranged VDJ gene encoding a heavy chain variable region) obtained from DNA encoding a mouse monoclonal antibody isolated from a hybridoma producing the mouse monoclonal antibody. C obtained from DNA encoding glomlin
_{An H} gene (C gene encoding the H chain constant region) and an active _VL gene obtained from DNA encoding a mouse monoclonal antibody isolated from the hybridoma (rearranged VJ encoding the L chain variable region) gene)
Of the (C gene encoding the L chain constant region) C _L gene obtained from the DNA encoding human immunoglobulin Guromu phosphorus downstream and inserted in one or separate expression vectors are arranged so as to respectively allow expression, the It can be produced by transforming a host cell with an expression vector and culturing the transformed cell.

Specifically, first, DNA is extracted from a mouse monoclonal antibody-producing hybridoma by an ordinary method, and the DNA is subjected to an appropriate restriction enzyme (for example, EcoRI, HindIII).
) And subjected to electrophoresis (for example, using 0.7% agarose gel) to perform Southern blotting. The electrophoresed gel is stained with, for example, ethidium bromide or the like, and after photographing, the position of the marker is attached, and the gel is washed twice with water.
Soak in M HCl solution for 15 minutes. It is then immersed in a 0.4N NaOH solution for 10 minutes while gently shaking. By the usual method,
Transfer to the filter and after 4 hours collect the filter and
Wash twice with SSC. After sufficiently drying the filter, baking (75 ° C., 3 hours) is performed. After baking, put the filter into 0.1 × SSC / 0.1% SDS solution,
Treat at 5 ° C for 30 minutes. Then, immerse in 3 × SSC / 0.1% SDS solution. The obtained filter is put in a plastic bag together with the prehybridization solution, and treated at 65 ° C. for 3 to 4 hours.

Next, the probe DN labeled with ³² P was added thereto.
A and hybridization solution at 65 ° C for 12 hours.
Let react for about an hour. After hybridization,
Appropriate salt concentration, reaction temperature and time (eg, 2 × SSC
-0.1% SDS solution, room temperature, 10 minutes). Put the filter in a plastic bag, 2 × SSC
Is added, sealed and autoradiographed.
Rearranged VDJs encoding the H and L chains of the mouse monoclonal antibody, respectively, by the Southern blot method described above.
Gene and VJ gene are identified. The region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation, and phage vectors (eg, Charon 4A, Charon 28, λEMBL)
3, λEMBL4, etc.) and transform Escherichia coli (eg, LE392, NM539, etc.) with the phage vector to prepare a genomic library. Using the genomic library with an appropriate probe (H chain J gene, L chain (κ) J gene, etc.), for example, the Benton Davis method (Science
(Science), vol. 196, pp. 180-182, 19
177), and plaque hybridization is performed to obtain rearranged VDJ genes or positive clones each containing the VJ gene. A restriction enzyme map of the obtained clone is prepared, the nucleotide sequence is determined, and it is confirmed that the desired rearranged gene containing the rearranged _VH (VDJ) gene or _VL (VJ) gene has been obtained. .

Meanwhile, isolated Separately, human C _H gene and human C _L gene used for chimerization. For example, human IgG1
When a chimeric antibody with the isolating Cκ gene is C gamma ₁ gene and the C _L gene is C _H gene.
These genes using mouse C gamma ₁ gene and mouse Cκ gene, corresponding to human C gamma ₁ gene and human Cκ gene by utilizing high homology in nucleotide sequence of the mouse immunoglobulin gene and the human immunoglobulin genes as a probe, the human genome live It can be obtained by isolation from a rally.

Specifically, for example, clone Ig146 (Proceedings National Academy of Sciences)
(Proc. Natl. Acad. Sci. USA), Vol. 75, No. 4709
-4713, 1978).
-BamHI fragment and clone MEP10 (Proceedings National Academy of Sciences (Proc. Natl. Acad. Sc)
i. USA), 78, 474-478, 1981.
Using a 6.8 kb EcoRI fragment from E. coli as a probe, a HaeIII-AluI genomic library of human lambda Charon 4A (Cell, Vol. 15, Nos. 1157-1174).
1978), a DNA fragment containing the human Cκ gene and retaining the enhancer region is isolated. Moreover, the human C gamma ₁ gene, for example, human fetal liver cells DNA
Is cut with HindIII, fractionated by agarose gel electrophoresis, a 5.9 Kb band is inserted into λ778, and isolated using the aforementioned probe.

[0046] In this way the mouse V _H gene was isolated and mouse V _L gene, and using the human C _H gene and human C _L gene, mouse V _H gene while considering the promoter region and enhancer region the human C _H gene downstream, and human C _L gene downstream of a mouse V _L gene, with appropriate restriction enzymes and DNA ligase, incorporated in a conventional manner, for example, in the expression vector such as pSV2gpt or pSV2neo. In this case, chimeric genes of mouse V _H gene / human C _H gene and mouse V _L gene / human C _L gene can be arranged simultaneously on one expression vector,
Each can also be arranged on a separate expression vector.

The chimeric gene insertion / expression vector thus prepared can be used, for example, in P3X63 / Ag8 / 653 cells or SP2
The cells are introduced into myeloma cells, such as 10 cells, which do not themselves produce antibodies by a protoplast fusion method, a DEAE-dextran method, a calcium phosphate method, an electroporation method, or the like. The transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain a desired chimeric monoclonal antibody-producing cell. The desired chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.

The "human monoclonal antibody (CDR-grafted antibody)" in the present invention is a monoclonal antibody produced by genetic engineering, and specifically, a part of the complementarity determining region of the hypervariable region. Alternatively, all are the complementarity determining regions of a hypervariable region derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), and the framework region of the variable region is the framework region of the variable region derived from human immunoglobulin. And its constant region is a human immunoglobulin-derived constant region.

Here, the complementarity-determining regions of the hypervariable region are three regions (complementary regions) which are present in the hypervariable region in the variable region of the antibody and which directly bind complementarily to the antigen.
CDR1, CDR2, CDR3), and the framework region of the variable region refers to four relatively conserved regions (Fram) intervening before and after the three complementarity-determining regions.
ework; FR1, FR2, FR3, FR4). In other words, all regions other than part or all of the complementarity determining region of the hypervariable region of a monoclonal antibody derived from a non-human mammal,
A monoclonal antibody that has replaced the corresponding region of human immunoglobulin. The constant regions derived from human immunoglobulin include IgG (IgG1, IgG2, IgG3, IgG4), and IgG.
Although each has a unique amino acid sequence depending on the isotype, such as M, IgA, IgD and IgE, the constant region of the humanized monoclonal antibody in the present invention may be the constant region of human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. Further, the framework region of the variable region derived from human immunoglobulin is not limited.

The humanized monoclonal antibody of the present invention can be produced, for example, as follows. However, it is needless to say that the present invention is not limited to such a manufacturing method. For example, a recombinant humanized monoclonal antibody derived from a mouse monoclonal antibody,
JP-T-4-506458 and JP-A-62-296.
It can be produced by genetic engineering with reference to Japanese Patent Publication No. 890 or the like. That is, from a hybridoma producing a mouse monoclonal antibody, at least one mouse H chain C
A DR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene, and a human immunoglobulin gene encoding all regions other than the human H chain CDR corresponding to the mouse H chain CDR The human L chain gene encoding the H chain gene and all regions other than the human L chain CDR corresponding to the pre-mouse L chain CDR is isolated.

The isolated mouse H chain CDR gene and human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse L chain CDR gene and human L chain gene can be expressed. Into another suitable expression vector as described above. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can be introduced so that they can be expressed in the same expression vector. By transforming host cells with the expression vector thus prepared, human-type monoclonal antibody-producing transformed cells are obtained, and the target human-type monoclonal antibody is obtained from the culture supernatant by culturing the transformed cells. obtain.

The "monoclonal antibody" in the present invention includes "a part" of the monoclonal antibody. The “part of the monoclonal antibody” refers to a partial region of the monoclonal antibody in the present invention described above, and specifically, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment
of antibody), sFv, dsFv (disulphide stabil
ised Fv) or dAb (single domain antibody)
(Expert Opin. Ther. Patents), No. 6
Vol. 5, No. 5, pp. 441-456, 1996).

Here, "F (ab ')_Two"And" Fab '"
Proteolysis of immunoglobulins (monoclonal antibodies)
Treatment with enzymes such as pepsin or papain
And exists between the two heavy chains in the hinge region.
Produced by digestion before and after disulfide bonds
Means fragment. For example, IgG with papain
Upon treatment, the dimer present between the two heavy chains in the hinge region
Is cleaved upstream of the sulfide bond and V_L(L chain variable area
Area) and C_L(L chain constant region) and V _H(H
Chain variable region) and C_Hγ1 (γ1 region in H chain constant region)
Chain fragment consisting of a disulfide at the C-terminal region
Produce two homologous antibody fragments joined by ligation
Can be built. These two homologous antibody fragments
Are called Fab '. In addition, IgG is treated with pepsin.
As a result, the disulfate existing between the two H chains in the hinge region
The two Fab's are cleaved downstream of the
Antibody fragment that is slightly larger than
Can be manufactured. F (ab ')
_TwoThat.

The term "cell producing a monoclonal antibody" or "cell producing a recombinant monoclonal antibody" of the present invention means any cell producing the aforementioned monoclonal antibody of the present invention. Specifically, for example, cells described in any of the following (1) to (3) can be mentioned. (1) Mammalian CTGF (preferably human CTGF) as defined above, or a part thereof, or a cell secreting the CTGF, etc., has the ability to produce the aforementioned non-human mammal or the aforementioned human antibody. Monoclonal antibody-producing B cells obtained by immunizing a transgenic mouse (or other transgenic non-human mammal) and capable of being collected from the animal that produces a monoclonal antibody reactive with the CTGF. (2) The aforementioned fused cells (hybridomas) obtained by cell fusion of the antibody-producing B cells thus obtained with myeloma cells derived from mammals. (3) a gene encoding the monoclonal antibody (either a heavy chain encoding gene or a light chain encoding gene, or both, isolated from the monoclonal antibody producing B cell or the monoclonal antibody producing fused cell (hybridoma)) Of the B cells and cells other than the hybridoma (for example, CHO (Chinese hamst
(E. ovarian) cells), BHK (Baby hamster kydney) cells, etc.). Here, the monoclonal antibody-producing transformed cell (genetically modified cell) described in the above (3) is the same as the above (1).
B) or a transgenic cell producing a recombinant of a monoclonal antibody produced by the hybridoma of (2). These antibody-producing transformed cells are:
It can be produced using the general gene recombination technique used in the production of the above-mentioned chimeric monoclonal antibody and humanized monoclonal antibody.

The “monoclonal antibody having substantially the same properties” in the present invention means at least the following points when the biological properties of the monoclonal antibody are compared with those of other monoclonal antibodies. Means no significant difference. (1) Reactivity to CTGF derived from a specific animal used for immunization of a non-human mammal to prepare the monoclonal antibody. (2) Reactivity to CTGF of another animal species corresponding to CTGF of the specific animal species (so-called cross-reactivity). (3) Characteristics required by various tests described in Examples described later.

"Mammal-derived antiserum" in the present invention
Means a serum containing an antibody reactive with the monoclonal antibody of the present invention or a part thereof. The antiserum is, for example, a mammal such as mouse, rat, guinea pig, rabbit, goat, pig or cow, preferably rat, guinea pig, rabbit or goat, the monoclonal antibody of the present invention or a part thereof, It can be produced by immunization using the method described in the method for producing an antibody.

The “insoluble carrier” in the present invention includes the monoclonal antibody of the present invention or a part thereof (antibody fragment), or a sample (eg, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant). Means a support for carrying CTGF by physical adsorption or chemical bonding. For example, the following (A) and (B) can be mentioned. (A) Plastics made of polystyrene resin, polycarbonate resin, silicon resin, nylon resin, etc., plates, test tubes, tubes, etc., made of water-insoluble substances typified by glass, etc., beads (Especially microbeads), balls, filters, membranes, etc. (B) Insoluble used in affinity chromatography such as a cellulose-based carrier, an agarose-based carrier, a polyacrylamide-based carrier, a dextran-based carrier, a polystyrene-based carrier, a polyvinyl alcohol-based carrier, a polyamino acid-based carrier, or a porous silica-based carrier. Carriers can be mentioned.

The “antibody-immobilized insoluble carrier” of the present invention includes:
On the insoluble carrier as described above, the monoclonal antibody of the present invention (or a part of the antibody, that is, an antibody fragment)
Means an insoluble carrier that is supported by physical adsorption or chemical bonding. These antibody-immobilized insoluble carriers can be used for CTG contained in a sample (for example, a body fluid sample such as serum or plasma, a culture supernatant or a centrifugal supernatant).
It can be used to detect, quantify, separate or purify F. For the purpose of the detection or quantification, the insoluble carriers exemplified in the above (A) can be used. 96
It is preferable to use a multiwell microtiter plate made of plastic or the like having a large number of wells (wells), such as a well microtiter plate or a 48-well microtiter plate. For the purpose of the separation or purification, the filter or membrane exemplified in the above (A) or the insoluble carrier exemplified in the above (B) can be used.

In the present invention, “a labeling substance capable of producing a detectable signal alone or by reacting with another substance” refers to a monoclonal antibody or a part thereof (antibody fragment) as described above. Or a substance used to make the presence thereof detectable by binding to a standard substance of CTGF by physicochemical bonding or the like. Specific examples include enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, and radioisotopes. More specifically, peroxidase (eg, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase Enzymes such as glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, phycobiliprotein, rare earths metal chelate, dansyl chloride or fluorescent substances such as tetramethylrhodamine isothiocyanate, ³ H, ¹⁴
C, a radioisotope such as ¹²⁵ I or ¹³¹ I, biotin,
Avidin, or a chemiluminescent substance.

Here, the radioisotope and the fluorescent substance alone can provide a detectable signal. On the other hand, enzymes, chemiluminescent substances, biotin and avidin alone cannot produce a detectable signal, and thus react with one or more other substances to produce a detectable signal. For example, in the case of an enzyme, at least a substrate is required, and a method for measuring enzyme activity (colorimetric method, fluorescent method, bioluminescent method, chemiluminescent method, etc.)
A variety of substrates are used depending on the. For example, in the case of peroxidase, hydrogen peroxide is used as a substrate.
In the case of biotin, at least avidin or enzyme-modified avidin is generally reacted, but not limited thereto. If necessary, various coloring substances depending on the substrate may be used.

The “labeled antibody” and “labeled standard substance of mammalian CTGF” in the present invention mean a monoclonal antibody (or antibody fragment) and CTGF labeled with various standard substances as described above, respectively. .
These labeled antibodies and labeled standards are used for a sample (for example,
It can be used for detecting, quantifying, separating or purifying CTGF contained in a body fluid sample such as serum or plasma, a culture supernatant or a centrifugal supernatant. In the present invention,
Although any of the above-mentioned labeling substances can be used, it is preferable to label with an enzyme such as peroxidase or biotin in consideration of high detection sensitivity or quantitative sensitivity and convenience of operation.

Here, the “standard substance of CTGF” means, in contrast to CTGF of unknown concentration (content) contained in a sample,
Refers to CTGF that has been isolated in advance, and refers to a standard whose concentration can be freely controlled according to the purpose of the assay. The standard substance can be used, for example, for preparing a calibration curve.

The “immunoassay” in the present invention is
In the present invention, this means a method for detecting or quantifying an antigen contained in a sample (for example, a body fluid sample such as plasma, a culture supernatant or a centrifugal supernatant, etc.) based on the principle of an antigen-antibody reaction. The antibody in the antibody reaction, wherein the monoclonal antibody (or a fragment thereof) having reactivity with the mammalian CTGF of the present invention, the insoluble carrier immobilized with the antibody (or the insoluble carrier immobilized with the antibody fragment) as described above Or one or more of said monoclonal antibodies (or antibody fragments) selected from labeled antibodies (or labeled antibody fragments) as described above, and that the antigen is mammalian CTGF. Known immunoassays can also be applied.

For example, as described in the enzyme immunoassay (3rd edition, edited by Eiji Ishikawa et al., Published by The Medical Shoin, 1987), for example, the solid phase method using one antibody and the liquid phase method using two antibodies ,
Two antibody solid phase method, sandwich method, EMIT method (Enzymem
ultiplied immunoassay technique), enzyme channeling assay (Enzyme channeling immunoassa
y), enzyme activity modifier labeled immunoassay (Enzyme mo
dulator mediated enzyme immunoassay, EMMI
A), enzyme inhibitor labeling immunoassay (Enzyme inhib
Itor immunoassay, immunoenzymometric assay, Enzyme enhanced immunoassay, or Proximal linkage immunoassay
inkage immunoassay), etc.
For example, a one-pot method as described in JP-B-47 can be mentioned.

In the present invention, such an immunoassay can be appropriately selected and used according to the purpose, but it is convenient and / or economically convenient, and particularly, is clinically versatile. Considering the points, it is preferable to use the sandwich method, the one-pot method, the one-antibody solid phase method, or the two-antibody liquid phase method, and more preferably, the sandwich method or the one-pot method. Particularly preferably, the monoclonal antibody of the present invention is immobilized on a multiwell microplate having a large number of wells, such as a 96-well microplate, and an insoluble carrier immobilized thereon, and a labeled antibody labeled with an enzyme or biotin. Or a one-pot method using an antibody-immobilized insoluble carrier in which the monoclonal antibody of the present invention is immobilized on beads or microballs such as microbeads and a labeled antibody labeled with an enzyme or biotin. .

In a particularly preferred embodiment, to give a specific example, the monoclonal antibody “8-64-6” or “13-51-2” shown in FIG. 1 was immobilized on a microplate or microbeads. The antibody-immobilized insoluble carrier and the monoclonal antibody "8-86" described in FIG.
-2 "is a sandwich method or a one-pot method in combination with a labeled antibody labeled with an enzyme or biotin.
An antibody-immobilized insoluble carrier on which “8-64-6” has been immobilized, and “8-64-6”
86-2 "in combination with a labeled antibody labeled with an enzyme or biotin, the human and mouse C
TGF can be detected and quantified with high sensitivity. Also,
An antibody-immobilized insoluble carrier immobilized with `` 13-51-2 '' and `` 8
-86-2 "can be detected and quantified with high sensitivity in rat (disclosed for the first time in the present application) and mouse CTGF by a combination with a labeled antibody labeled with an enzyme or biotin.

The sandwich method, the one-pot method,
The one-antibody solid phase method and the two-antibody liquid phase method will be described in detail. The sandwich method is an immunoassay method including at least the following steps (a) and (b) described in the above (50) of the present invention. (A) reacting a sample with the antibody-immobilized insoluble carrier of the present invention; and (b) antigen-antibody complex formed by binding of the antibody-immobilized insoluble carrier to mammalian CTGF contained in the sample. A step of allowing the body to react with the labeled antibody of the present invention.

In accordance with the present invention, the monoclonal antibody “8-64-6” shown in FIG.
Is immobilized on a microplate, and the “labeled antibody” is obtained by labeling the monoclonal antibody “8-86-2” shown in FIG. 1 with an enzyme such as peroxidase or biotin as the “labeled antibody”. ], The method for quantifying human or mouse CTGF is specifically described, for example, by the following steps, but is not limited to the specific examples. Also, as the “antibody-immobilized insoluble carrier”, the “antibody-immobilized microplate” in which the monoclonal antibody “13-51-2” shown in FIG.
Further, the monoclonal antibody “8-86-2” shown in FIG. 1 was labeled with an enzyme such as peroxidase or biotin as a “labeled antibody” and operated in the same manner as described below to obtain only mouse CTGF. But not rat CTGF (disclosed for the first time in this application).

(Step 1) A step of immobilizing the monoclonal antibody “8-64-6” of the present invention on a microplate to prepare an antibody-immobilized microplate; (Step 2) A human or mouse is immobilized on the antibody-immobilized microplate. Adding a sample such as serum, and reacting the sample with the monoclonal antibody immobilized on the microplate; (Step 3) washing the microplate and removing unreacted sample from the microplate; (Step 4) labeling the monoclonal antibody “8-86-2” of the present invention with an enzyme such as biotin or peroxidase to prepare a labeled antibody; (Step 5) labeling the microplate washed in Step 3 with the label The antibody was added, and the monoclonal antibody immobilized on the microplate and human or mouse CTGF contained in the sample were mixed. Reacting the labeled antibody with an antigen-antibody complex formed by the reaction;

(Step 6) washing the microplate and removing unreacted labeled antibody from the microplate; (Step 7) using the microplate washed in step 6 together with a coloring material, if necessary. Depending on the type of enzyme used, various substrates (provided that a labeled antibody labeled with an enzyme such as peroxidase is used in step 5) or avidin or enzyme-modified avidin (provided that labeled antibody labeled with biotin in step 5) (If used), and reacting the substrate, avidin or enzyme-modified avidin with a labeling substance on the labeled antibody; (Step 8) when the enzyme-modified avidin was added in step 7, it was used for the modification. Adding various substrates depending on the type of enzyme, and reacting the enzyme bound to avidin with the substrate; (Step 9) adding a reaction stop solution to the microplate Step stop the enzymatic reaction and coloring reaction; and (Step 10) colorimetric intensity, the step of measuring fluorescence intensity or luminescence intensity.

The one-pot method is the method of (5) of the present invention described above.
0), (51) or (52). That is, the first is an immunoassay method including at least the following steps (a) and (b). (A) reacting a sample with the antibody-immobilized insoluble carrier of the present invention; and (b) antigen-antibody complex formed by binding of the antibody-immobilized insoluble carrier to mammalian CTGF contained in the sample. A step of allowing the body to react with the labeled antibody of the present invention.

Second, at least the following (a) and (b)
An immunoassay method comprising the steps of: (A) reacting the labeled antibody of the present invention with a sample;
And (b) the labeled antibody and mammalian CTG contained in the sample
Reacting the antibody-immobilized insoluble carrier of the present invention with the antigen-antibody complex formed by binding to F.

The third is an immunoassay method including at least the following step (a). (A) a step of reacting a mixture containing the antibody-immobilized insoluble carrier of the present invention, the labeled antibody of the present invention, and a sample; In accordance with the present invention, FIG.
Using `` antibody-immobilized microbeads '' in which the monoclonal antibody `` 8-64-6 '' described in
In addition, a method for quantifying human or mouse CTGF using a labeled antibody in which the monoclonal antibody “8-86-2” shown in FIG. 1 is labeled with an enzyme such as peroxidase or biotin as the “labeled antibody” is specifically described. To be more specific, for example, it is constituted by the following steps, but is not limited only to the specific example.

As the “antibody-immobilized insoluble carrier”, “antibody-immobilized microbeads” in which the monoclonal antibody “13-51-2” shown in FIG. 1 was immobilized on microbeads were used, and as the “labeled antibody” The monoclonal antibody “8-86-2” shown in FIG. 1 was treated in the same manner as described below using a “labeled antibody” labeled with an enzyme such as peroxidase or biotin, thereby obtaining not only mouse CTGF but also rat (in the present application). CTGF (disclosed for the first time) can also be quantified.

The first method comprises the following steps. (Step 1) Immobilizing the monoclonal antibody “8-64-6” of the present invention on microbeads to prepare antibody-immobilized microbeads; (Step 2) Internal volume such as a test tube, microplate or tube Adding the antibody-immobilized microbeads and a sample such as human or mouse serum together with a buffer together with a buffer to a container having the following, and reacting the sample with the monoclonal antibody immobilized on the microbeads; (Step 4) A step of labeling the monoclonal antibody “8-86-2” of the present invention with an enzyme such as biotin or peroxidase to prepare a labeled antibody; (Step 5) )) Adding the labeled antibody to the container containing the beads washed in step 3 and mixing the monoclonal antibody immobilized on the beads with the human or human contained in the sample; Is the CT of the mouse
Reacting the labeled antibody with an antigen-antibody complex formed by reaction with GF;

(Step 6) The step of removing the unreacted labeled antibody by removing the internal solution in the container and washing the beads; (Step 7) The container containing the beads washed in Step 6 Depending on the type of enzyme used, various substrates (provided that a labeled antibody labeled with an enzyme such as peroxidase is used in step 5) or avidin or enzyme-modified avidin (provided in step 5), depending on the type of enzyme used. (In the case of using a labeled antibody labeled with biotin), and reacting the substrate, avidin or enzyme-modified avidin with a labeling substance on the labeled antibody; Is a step of adding various substrates depending on the type of the enzyme used for the modification, and reacting the enzyme bound to avidin with the substrate; (Step 9) reacting with the reaction system of Step 7 or Step 8 Stop solution was added, steps to stop the enzyme reaction and coloring reaction; and (Step 10) colorimetric intensity, the step of measuring fluorescence intensity or luminescence intensity.

The second method comprises the following steps. (Step 1) a step of labeling the monoclonal antibody "8-86-2" of the present invention with an enzyme such as biotin or peroxidase to prepare a labeled antibody; (Step 2) contents such as a test tube, a microplate or a tube; A step of adding the labeled antibody and a sample of human or mouse serum or the like together with a buffer solution to a container having a volume, and allowing the labeled antibody to react with the sample; (Step 3) the monoclonal antibody “8-64-6” of the present invention; Is immobilized on microbeads to produce antibody-immobilized microbeads. (Step 4) The beads are added to the reaction system in Step 2, and the labeled antibody reacts with human or mouse CTGF contained in the sample. Reacting the antigen-antibody complex thus formed with the monoclonal antibody immobilized on the beads; (Step 5) removing the internal solution in the container and washing the beads Thereby removing unreacted labeled antibody;

(Step 6) Into the container containing the beads washed in step 5, together with a coloring material, if necessary, various substrates depending on the type of enzyme used (however, in step 2, an enzyme such as peroxidase is used). A labeled antibody, or avidin or enzyme-modified avidin (when using a labeled antibody labeled with biotin in step 2), and the substrate, avidin or enzyme-modified avidin and a labeling substance on the labeled antibody are added. (Step 7) When enzyme-modified avidin is added in Step 6, various substrates are added depending on the type of the enzyme used for the modification, and the enzyme bound to avidin reacts with the substrate. (Step 8) a step of adding a reaction stop solution to the reaction system of Step 6 or Step 7 to stop the enzyme reaction and the color-forming reaction; and (Step 9) colorimetric intensity, fluorescent intensity or emission The step of measuring the intensity.

The third method comprises the following steps. (Step 1) immobilizing the monoclonal antibody "8-64-6" of the present invention on microbeads to prepare antibody immobilized microbeads; (Step 2) preparing the monoclonal antibody "8-86-2" of the present invention Step of labeling with an enzyme such as biotin or peroxidase to prepare a labeled antibody; (Step 3) immobilizing the antibody prepared in Step 1 together with a buffer solution in a container having an internal volume such as a test tube, plate, or tube. Adding microbeads, the labeled antibody prepared in step 2, and a sample such as human or mouse serum, and reacting the monoclonal antibody, labeled antibody, and sample immobilized on the beads simultaneously; (step 4) Removing the unreacted labeled antibody by removing the internal solution in the container and washing the beads;

(Step 5) Into the container containing the beads washed in step 4, together with a coloring material, if necessary, various substrates depending on the type of enzyme used (however, in step 3, an enzyme such as peroxidase is used). A labeled antibody, or avidin or an enzyme-modified avidin (if a labeled antibody labeled with biotin in step 3) is added, and the substrate, avidin or enzyme-modified avidin and a labeling substance on the labeled antibody are added. (Step 6) When enzyme-modified avidin is added in Step 5, various substrates are added depending on the type of the enzyme used for the modification, and the enzyme bound to avidin reacts with the substrate. (Step 7) a step of adding a reaction stop solution to the reaction system of Step 5 or Step 6 to stop the enzymatic reaction and the color-forming reaction; and (Step 8) a colorimetric intensity, a fluorescence intensity, or a light-emitting intensity. The step of measuring the intensity.

The one-antibody solid phase method is the same as the method (53) of the present invention described above.
In other words, the immunoassay method includes at least the following step (a). (A) A sample and a standard substance of mammalian CTGF labeled with a labeling substance capable of producing a detectable signal by reacting with a sample alone or with another substance are added to the antibody-immobilized insoluble carrier of the present invention. The step of reacting.

In accordance with the present invention, the monoclonal antibody “8-64-6” shown in FIG.
A method for quantifying human or mouse CTGF using an “antibody-immobilized microplate” in which is immobilized on a microplate and using a particularly common enzyme such as peroxidase or biotin as a “labeling substance”. To be more specific, for example, it is constituted by the following steps, but is not limited only to the specific example. As the “antibody-immobilized insoluble carrier”, an “antibody-immobilized microplate” in which the monoclonal antibody “13-51-2” shown in FIG. 1 is immobilized on a microplate is used. In the same manner as described below using an enzyme such as peroxidase or biotin, which is a target, not only mouse CTGF but also rat CTGF (disclosed for the first time in the present application) can be used.
Can also be quantified.

(Step 1) Immobilizing the monoclonal antibody "8-64-6" of the present invention on a microplate to prepare an antibody-immobilized microplate; (Step 2) using biotin as a standard substance for human or mouse CTGF Alternatively, a step of preparing a labeled CTGF standard substance by labeling with an enzyme such as peroxidase; (Step 3) adding a sample such as human or mouse serum and the labeled CTGF standard substance to the microplate; With a monoclonal antibody immobilized on the microplate; (Step 4) washing the microplate and removing unreacted labeled standard from the microplate;

(Step 5) The microplate washed in step 4 may be labeled with various substrates depending on the type of the enzyme used, if necessary, together with a coloring material (however, in step 3, an enzyme such as peroxidase was used to label the microplate). When using a labeled standard)
Or a step of adding avidin or enzyme-modified avidin (when using a labeled standard substance labeled with biotin in step 3), and reacting the substrate, avidin or enzyme-modified avidin with a labeled substance on the labeled standard substance; Step 6) When enzyme-modified avidin is added in Step 5, various substrates are added depending on the type of enzyme used for the modification, and the enzyme bound to avidin reacts with the substrate; (Step 7) Adding a reaction stop solution to the microplate to stop the enzymatic reaction and the coloring reaction; and (Step 8) measuring the colorimetric intensity, the fluorescence intensity or the luminescence intensity.

The two-antibody liquid phase method is applicable to the above-mentioned (54) of the present invention.
And (55). That is, the first is an immunoassay method including at least the following steps (a) and (b). (A) The monoclonal antibody of the present invention is added to a sample and a mixture thereof with a standard substance of mammalian CTGF labeled with a labeling substance capable of producing a detectable signal either alone or by reacting with another substance. (B) reacting the antigen-antibody complex formed by binding the mammalian CTGF or the labeled mammalian CTGF standard substance contained in the sample with the monoclonal antibody to the monoclonal antibody; A step of reacting a mammal-derived antiserum reactive with the antibody.

Second, at least the following (a) to (c)
An immunoassay method comprising the steps of: (A) reacting the sample with the monoclonal antibody of the present invention; (b) reacting the reaction system, which has been subjected to the step (a), alone or with another substance to provide a detectable signal. CT of mammals labeled with a possible labeling substance
Reacting a GF standard substance; and (c) an antigen-antibody complex formed by binding of the mammalian CTGF or the labeled mammalian CTGF standard substance contained in the sample to the monoclonal antibody. A step of allowing the body to react with a mammal-derived antiserum reactive with the monoclonal antibody.

According to the present invention, “monoclonal antibody”
Using the monoclonal antibody “8-64-6” or “8-86-2” described in FIG. 1 and using a particularly common enzyme such as peroxidase or biotin as a “labeling substance”, The method for quantifying mouse CTGF will be specifically described. For example, the method comprises the following steps, but is not limited to the specific examples. Further, the monoclonal antibody "13-51-2" shown in FIG. 1 was used as the "monoclonal antibody", and an enzyme such as peroxidase or biotin was used as the "labeling substance" in the same manner as described below. Not only CTGF but also rat (disclosed for the first time in this application) CTGF can be quantified.

The first method includes the following steps. (Step 1) A step of labeling a human or mouse CTGF standard substance with an enzyme such as biotin or peroxidase to prepare a labeled CTGF standard substance; (Step 2) Having an internal volume such as a test tube, plate or tube A mixture of a sample such as mouse or human serum and the labeled CTGF standard prepared in step 1 is added to the container, and then the monoclonal antibody “8-64” of the present invention is added.
Adding "-6" or "8-86-2" to competitively react the sample with a labeled CTGF standard with the monoclonal antibody; (Step 3) mouse-derived such as goat anti-mouse gamma globulin serum Adding an antiserum derived from an animal other than a mouse that is reactive to the monoclonal antibody, and adding mammalian CTGF or labeled C contained in the sample formed in step 2.
Reacting the antiserum with an antigen-antibody complex of a TGF standard substance and the monoclonal antibody to aggregate and precipitate a complex aggregate comprising the complex and the antiserum; (step 4) centrifuging the reaction system of step 3 Separating the separated and aggregated and precipitated complex;

(Step 5) The composite aggregate separated in step 4 may be labeled with various substances depending on the type of the enzyme used (however, in step 2, labeled with an enzyme such as peroxidase, etc.) , Or avidin or enzyme-modified avidin (when using a labeled standard labeled with biotin in step 2),
Reacting the substrate, avidin or enzyme-modified avidin with a labeling substance on a labeling standard; (step 6) when adding enzyme-modified avidin in step 5, depends on the type of enzyme used for the modification. (Step 7) a step of adding a reaction stop solution to the reaction system of Steps 5 and 6 to stop the enzyme reaction and the color-forming reaction; and Step 8) a step of measuring a colorimetric intensity, a fluorescence intensity or a light emission intensity.

The second method comprises the following steps. (Step 1) A step of labeling a human or mouse CTGF standard substance with an enzyme such as biotin or peroxidase to prepare a labeled CTGF standard substance; (Step 2) Having an internal volume such as a test tube, plate or tube A sample such as human or mouse serum is added to the container, and then the monoclonal antibody of the present invention "8-64-
(6) or (8-86-2) and reacting the sample with the monoclonal antibody; (Step 3) adding the labeled C prepared in step 1
Adding a TGF standard, and reacting the labeled CTGF standard with the remaining monoclonal antibody that has not reacted with the sample in step 2; (step 4) mouse-derived such as goat anti-mouse gamma globulin serum; An antiserum derived from an animal other than a mouse that is reactive to the monoclonal antibody is added, and an antigen-antibody complex between the mammalian CTGF contained in the sample formed in step 2 and the monoclonal antibody, and / or an antigen-antibody complex formed in step 3 Reacting the antiserum with the antigen-antibody complex of the labeled CTGF standard substance and the monoclonal antibody to be aggregated to precipitate and precipitate a complex aggregate comprising the complex and the antiserum; (Step 5) Step 4 Centrifuging the reaction system to separate the aggregated precipitate;

(Step 6) The composite aggregate separated in step 5 may be labeled with various substances depending on the type of the enzyme used (provided with an enzyme such as peroxidase in step 3), together with a coloring substance as necessary. , Or avidin or enzyme-modified avidin (when using a labeled standard labeled with biotin in step 3),
Reacting the substrate, avidin or enzyme-modified avidin with a labeling substance on a labeling standard; (step 7) when adding enzyme-modified avidin in step 6, depends on the type of enzyme used for the modification. (Step 8) a step of adding a reaction stop solution to the reaction system of Steps 6 and 7 to stop the enzyme reaction and the color-forming reaction; and Step 9) a step of measuring a colorimetric intensity, a fluorescence intensity or a light emission intensity.

The term “affinity chromatography” in the present invention refers to the interaction (affinity) between substances such as an antigen and an antibody, an enzyme and a substrate, or a receptor and a ligand.
Means a method for separating or purifying a target substance contained in a sample (for example, a body fluid sample such as serum and plasma, a culture supernatant or a centrifugal supernatant, etc.). The method of the present invention uses a sample (for example, Body fluid samples such as serum and plasma,
Culture supernatant or centrifugal supernatant).

Specifically, (1) after immobilizing the monoclonal antibody (or antibody fragment) of the present invention having reactivity with mammalian CTGF on a filter or a membrane which is an insoluble carrier as described above, A method of separating CTGF contained in a sample by contacting the sample with a filter or a membrane, and (2) a cellulose-based carrier, an agarose-based carrier, a polyacrylamide-based carrier, a dextran-based carrier,
A monoclonal antibody (or antibody fragment) having a reactivity with mammalian CTGF of the present invention is inoculated on an insoluble carrier such as a polystyrene carrier, a polyvinyl alcohol carrier, a polyamino acid carrier or a porous silica carrier by a conventional method. Immobilization (polymerization by physical adsorption, crosslinking, immobilization by sealing in a matrix or non-covalent bonding, etc.), filling the insoluble carrier into a glass, plastic or stainless steel column, A sample (eg, a body fluid sample such as serum or plasma, a culture supernatant, a centrifugal supernatant, or the like) is eluted onto a column (eg, a column) to separate or purify CTGF contained in the sample. is there. The latter method (2) is particularly called affinity column chromatography.

As the insoluble carrier used for the affinity column chromatography, any insoluble carrier can be used as long as the monoclonal antibody (or antibody fragment) of the present invention can be immobilized thereon. Pharmacia
ia) Sepharose 2B, Sepharose 4B, Sepharose 6
B, CNBr-activated Sepharose 4B, AH-Sepharose 4B, C
H-Sepharose 4B, Activated CH-Sepharose 4B, Epoxy-a
ctivated Sepharose 6B, Activated thiol-Sepharose 4
B, Sephadex, CM-Sephadex, ECH-Sepharose 4B, EAH-Se
pharose 4B, NHS-activated Sepharose or Thiopro
Bio-Rad (Bio-Rad) such as pyl Sepharose 6B
-Gel A, Cellex, Cellex AE, Cellex-CM, Cellex PAB,
Bio-Gel P, Hydrozide Bio-Gel P, Aminoethyl Bio-Gel
P, Bio-Gel CM, Affi-Gel 10, Affi-Gel 15, Affi-Pre
p 10, Affi-Gel Hz, Affi-Prep Hz, Affi-Gel 102, CM
Bio-Gel A, Affi-Gel heparin, Affi-Gel 501 or A
ffi-Gel 601, Chromagel A, Chromagel P, Enzafix P-HZ, Enzafix P-SH or Enzafix P-AB manufactured by Wako Pure Chemical Industries, Ltd.
a) AE-Cellurose, CM-Cellurose or PAB Cell
urose and the like.

The “pharmaceutical composition” according to the present invention comprises the monoclonal antibody of the present invention or a part thereof as an active ingredient, a “CTGF inhibitor”, a “CTGF production inhibitor” or “stimulation of CTGF stimulation” described later. And a "pharmaceutically acceptable carrier", which is a composition useful as a pharmaceutical comprising any one of "substances capable of suppressing the proliferation of cells having the ability to inhibit CTGF stimulation". Here, "pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, coloring agents, sweeteners, thickeners. , A flavoring agent, a solubilizer or other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, liquids, capsules, troches, elixirs, suspensions, emulsions, syrups, etc. A composition can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions, suppositories and pessaries for enteral administration which contain one or more active substances and are formulated in a conventional manner.

The dose may be determined by the patient's age, sex, weight and condition, therapeutic effect, administration method, treatment time, or the active ingredient (such as the protein or antibody) contained in the pharmaceutical composition.
The dose can vary depending on the type and the like, but it can be generally administered in the range of 10 μg to 1000 mg (or 10 μg to 500 mg) per adult per dose. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be necessary. In particular, in the case of an injection, 0.1 μg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection is used.
It can be produced by dissolving or suspending to a concentration of mg antibody / ml carrier.

[0097] The thus-produced injection can be administered to a human patient in need of treatment at a dose of 1k per administration.
1 μg to 100 mg per g body weight, preferably 50 μg
It can be administered once to several times a day at a rate of g to 50 mg. Examples of the administration form include medically appropriate administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and intraperitoneal injection. Preferably, it is an intravenous injection. Injectables can also be prepared as non-aqueous diluents (eg, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions and emulsions. . Such injections can be sterilized by filtration sterilization through a bacteria retaining filter, blending of a bactericide or irradiation. Injectables can be manufactured in the form of preparation for use. That is, a sterile solid composition can be prepared by freeze-drying or the like, and dissolved in sterile distilled water for injection or another solvent before use.

The pharmaceutical composition of the present invention comprises cells capable of proliferating by stimulation of CTGF derived from various tissues of a living body (eg, various fibroblasts, various vascular endothelial cells, various cells, etc.). Useful for inhibiting growth. Such tissues include, for example, brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovaries, testes, prostate, skin, oral cavity, tongue, and blood vessels. . Preferably, lung, liver, kidney or skin can be mentioned. As described above, since the pharmaceutical composition of the present invention can suppress the proliferation of cells having the ability to proliferate by the stimulation of CTGF as described above, the pharmaceutical composition of the present invention can also be used for various tissues as described above. It is useful as a medicament for treating or preventing various diseases associated with the proliferation of the cells in the above. Examples of tissues associated with cell proliferation in the disease include brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate, skin, oral cavity, and tongue , And blood vessels. Preferably, lung, liver, kidney or skin can be mentioned.

The disease includes, for example, fibrosis in various tissues (renal fibrosis, pulmonary fibrosis, fibrosis in liver,
Fibrosis in the skin, etc.), kidney diseases (eg, renal fibrosis, nephritis, renal failure, etc.), diseases in the lungs (eg, pulmonary fibrosis, pneumonia, etc.), skin diseases (eg, psoriasis, scleroderma, atopy, Keloids), liver disease (eg, fibrosis in the liver, hepatitis, cirrhosis, etc.), arthritis (eg,
It can be applied to treatment or prevention of rheumatoid arthritis), various cancers, arteriosclerosis and the like. Preferably,
Kidney disease (eg, renal fibrosis, nephritis, renal failure, etc.), lung disease (eg, pulmonary fibrosis, pneumonia, etc.), skin disease (eg, psoriasis, scleroderma, atopy, keloid, etc.), or liver disease (Eg, fibrosis in the liver, hepatitis, cirrhosis, etc.). More preferably, kidney disease (eg, renal fibrosis, nephritis, renal failure, etc.) can be mentioned.

The pharmaceutical composition of the present invention includes “CTG”
F inhibitor "," CTGF production inhibitor "or" CT
C of cells capable of proliferating upon stimulation of GF stimulation
A substance that has the ability to suppress the growth caused by TGF stimulation "
Also included are pharmaceutical compositions comprising Here, "C
"TGF inhibitor", "CTGF production inhibitor" or "substance" refers to a substance having an activity of suppressing or inhibiting the biological function of CTGF or CTGF from various cells.
Means a substance having an activity of suppressing or inhibiting the production of For example, substances having any of the following activities can be mentioned. (1) Human kidney-derived fibroblasts (eg, cell line 293-T
(ATCC CRL-1573)) and the binding of human CTGF or the binding of the cells to mouse CTGF. (2) Rat kidney-derived fibroblast cell line (for example, cell line NR
K-49F (ATCC CRL-1570)), human osteosarcoma-derived cell line MG-6
3 (ATCC CRL-1427) or human lung derived fibroblasts to suppress or inhibit the binding of human CTGF. (3) Rat kidney-derived fibroblast cell lines (for example, cell line NRK-) stimulated by human CTGF or mouse CTGF
49F (ATCC CRL-1570)) is suppressed or inhibited. (4) It suppresses or inhibits the increase in the production of hydroxyproline in the kidney in which the production of hydroxyproline is increasing.

The above-mentioned "substance" specifically includes, for example, the following substances. (A) The monoclonal antibody of the present invention (not limited to a naturally occurring antibody or a recombinant antibody) or a part of the antibody. (B) antisense DNA. (C) antisense RNA. (D) Low molecular chemical substances (chemically synthesized or natural products) other than the above (a) to (c).

The antisense DNA in the present invention is
DNA containing a partial nucleotide sequence in the nucleotide sequence of DNA encoding mammalian (preferably human) CTGF protein
Or a DNA in which a part of the DNA is chemically modified,
Or a DNA comprising a base sequence complementary to the partial base sequence
Alternatively, a DNA in which a part of the DNA is chemically modified can be given.

Here, the “partial nucleotide sequence” refers to the DTG encoding the mammalian (preferably human) CTGF protein.
It means a partial base sequence consisting of an arbitrary number of bases at an arbitrary site included in the base sequence of NA. The DNA
Can inhibit the production of CTGF protein by inhibiting the transcription of the DNA into mRNA or the translation of the mRNA into protein by hybridizing to DNA or RNA encoding the protein.

The partial base sequence includes a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, and more preferably a continuous partial base sequence of 5 to 50 bases. A sequence, more preferably a continuous partial base sequence of 5 to 30 bases is exemplified. Further, when this DNA is used as an antisense drug, the DNA has an increased half-life in blood (stability) when administered to the body of a patient, an increased permeability of an intracellular membrane,
Alternatively, a part of the base sequence of the DNA can be chemically modified for the purpose of increasing the resistance to degradation in the digestive tract or increasing absorption in the case of oral administration. The chemical modification includes, for example, a phosphate bond, ribose, nucleobase, sugar moiety, 3 ′ and / or
Or chemical modification of the 5 'end or the like.

The modification of the phosphate bond may be one or more of the following: a phosphodiester bond (D-oligo), a phosphorothioate bond, a phosphorodithioate bond (S-oligo), a methylphosphonate bond (MP-oligo), Changes to any of the phosphoramidate, non-phosphate and methylphosphonothioate linkages or combinations thereof may be included. Ribose modifications include:
Changes to 2′-fluororibose or 2′-O-methylribose can be mentioned. Modifications of nucleobases include changes to 5-propynyluracil or 2-aminoadenine and the like.

The antisense RNA in the present invention is
RNA containing a partial nucleotide sequence in the nucleotide sequence of RNA encoding mammalian (preferably human) CTGF protein
Or RNA in which part of the RNA is chemically modified,
Or RNA containing a base sequence complementary to the partial base sequence
Alternatively, an RNA in which a part of the RNA is chemically modified can be mentioned.

As used herein, the term “partial nucleotide sequence” refers to the R (SEQ ID NO: 1) encoding a mammalian (preferably human) CTGF protein.
It means a partial base sequence consisting of an arbitrary number of bases at an arbitrary site included in the base sequence of NA. The RNA
Can inhibit the production of CTGF protein by inhibiting the transcription of the DNA into mRNA or the translation of the mRNA into protein by hybridizing to DNA or RNA encoding the protein.

The partial base sequence includes a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, and more preferably a continuous partial base sequence of 5 to 50 bases. A sequence, more preferably a continuous partial base sequence of 5 to 30 bases is exemplified. Further, when this RNA is used as an antisense drug, when the RNA is administered into a patient's body, an increase in blood half-life (stability), an increase in intracellular membrane permeability,
Alternatively, a part of the base sequence of the RNA can be chemically modified for the purpose of increasing the resistance to degradation in the digestive tract or increasing the absorption in the case of oral administration. The chemical modification includes, for example, a phosphate bond, ribose, nucleobase, sugar moiety, 3 ′ and / or
Or chemical modification of the 5 'end or the like.

The modification of the phosphate bond may be such that one or more of the bonds is a phosphodiester bond (D-oligo), a phosphorothioate bond, a phosphorodithioate bond (S-oligo), a methylphosphonate bond (MP-oligo), Changes to any of the phosphoramidate, non-phosphate and methylphosphonothioate linkages or combinations thereof may be included. Ribose modifications include:
Changes to 2′-fluororibose or 2′-O-methylribose can be mentioned. Modifications of nucleobases include changes to 5-propynyluracil or 2-aminoadenine and the like.

The therapeutic effect of the pharmaceutical composition of the present invention on various disease symptoms can be tested and examined by administering to a known disease model animal according to a conventional method. For example, when examining the therapeutic effect of renal fibrosis, which is one of tissue fibrosis and one of renal diseases, one of the ureters of a mouse is ligated to stop the renal filtration function of blood and the like. Mouse model (UUO model, Unilateral ureteral obstruction) induced renal dysfunction
In addition, a method of administering the pharmaceutical composition of the present invention, and measuring the degree of suppressing the increase in the production of hydroxyproline, which is an index of the onset of nephritis and renal fibrosis caused by renal dysfunction, can be used. . A decrease in the concentration of the hydroxyproline indicates that the pharmaceutical composition is effective for treating the kidney disease.

Kidney diseases, for example, microglomerular abnormalities (eg, nephrotic microchange group (MCNS)), focal glomerulosclerosis (FGS), membranous glomerulonephritis (membranous nephropathy, MN), IgA Models detailed in the previous report include nephropathy, mesangial proliferative nephritis, acute glomerulonephritis after streptococcal infection (APSGN), crescent-forming (extravascular) nephritis, interstitial nephritis, and acute renal failure Animals can be used (“Testing and experimental methods for production of disease-specific animal models and development of new drugs”, p.34
-46, 1993, Technical Information Association (published). For skin diseases, for example, wounds, keloids, atopy, dermatitis, scleroderma, psoriasis, etc., model animals detailed in the previous report can be used ("Production of disease-specific model animals and development of new drugs"). Test and Experimental Methods ”, p.229-235, 1993, Technical Information Association (published)).

For liver diseases, for example, hepatitis (for example, viral hepatitis (type A, B, C, E, etc.)), cirrhosis or drug-induced liver injury, model animals detailed in the previous report were used. Can be used (“Testing and experimental methods for the production of disease-specific animal models and the development of new drugs”, p.119-12
9 and p.349-358, 1993, Technical Information Association (published). For example, when examining the effects on arteriosclerosis and restenosis,
A pseudo-restenosis model created by inserting a balloon catheter into the rat aorta and performing PTCA can be used.

For example, when confirming the effects on tumor growth and metastasis, commercially available mice such as normal mice such as Balb / c mice, nude mice or model mice such as SCID mice, for example, subcutaneous, tail vein, A cancer metastasis model prepared by implanting cancer cells in the spleen, under the capsule of the kidney, in the abdominal cavity or in the wall of the cecum can be used.

The "rat CTGF" of the present invention (specifically, having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence) and "DNA encoding rat CTGF" (Specifically, base number 213 in the base sequence described in SEQ ID NO: 1)
DNA having a base sequence from 1 to 1256) are defined with the following meanings and can be produced according to a conventional method as described below.

Note that the term “substantially the same” has the same meaning as described above. "CTGF of rat" of the present invention
Can be produced by appropriately using a known method known in the technical field such as a chemical synthesis method or a cell culture method, or a modification method thereof, in addition to a genetic recombination technique described below. "DNA" of the present invention
Is a DNA encoding the rat CTGF of the present invention, and includes any base sequence capable of encoding the rat CTGF of the present invention. Specifically, a DNA encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 can be mentioned. A preferred embodiment is a DNA containing the nucleotide sequence of base numbers 213 to 1256 in the nucleotide sequence shown in SEQ ID NO: 1 (for example, a DNA having the nucleotide sequence shown in SEQ ID NO: 1).

The DNA encoding rat CTGF in the present invention includes both cDNA and genomic DNA. In the present invention, any codon that encodes the same amino acid is included in the present invention. The DNA of the present invention may be obtained by any method. For example, mRN
A, complementary DNA (cDNA) prepared from A, genomic DN
DNA prepared from A, D obtained by chemical synthesis
It also includes DNAs obtained by amplifying by PCR using NA, RNA or DNA as templates and DNAs constructed by appropriately combining these methods.

The DNA encoding rat CTGF according to the present invention can be obtained by a method of cloning cDNA from mRNA encoding rat CTGF according to a conventional method,
A method of isolating and splicing genomic DNA,
PC using the cDNA sequence or mRNA sequence as a template
It can be obtained by a method of preparing with R, a method of chemical synthesis, or the like. The rat CTGF-encoding DNA of the present invention can be obtained by digesting each of the thus prepared rat CTGF-encoding DNAs with an appropriate restriction enzyme, and digesting the obtained DNA fragment as necessary. Together with the linker DNA or tag (Tag)
It can be prepared by ligation using an appropriate DNA polymerase or the like.

As a method for cloning cDNA encoding the rat CTGF (hereinafter referred to as target protein) from mRNA, the following method is exemplified. First, mRNA encoding the protein of interest is obtained from tissues or cells that express and produce the protein of interest (eg, rat fibroblasts).
Is prepared. The mRNA is prepared, for example, by the guanidine thiocyanate method (Chirgwin et al., Biochemistry, Vol. 18, No. 5294).
1979), a total RNA prepared by a known method such as the hot phenol method or the AGPC method is oligo (d)
T) It can be carried out by subjecting to affinity chromatography using cellulose, poly-U-sepharose, or the like. Next, known methods such as, for example, using reverse transcriptase using the obtained mRNA as a template, such as the method of Okayama et al. (Molecular Cell Biology (Mol. Cel.
l. Biol.), Vol. 2, p. 161, 1982, and the same volume 3, p. 280, 1983) and Hoffm.
an) et al. (Gene, Vol. 25, 263)
Page, 1983) and the like.
A is converted to double-stranded cDNA. This cDNA is incorporated into a plasmid vector, phage vector or cosmid vector, and transformed into Escherichia coli, or after in vitro packaging, transfected into Escherichia coli to prepare a cDNA library.

The plasmid vector used here is not particularly limited as long as it can be replicated and maintained in the host, and any phage vector may be used as long as it can be propagated in the host. PUC19, λgt10, and cloning vectors used in a conventional manner.
λgt11 and the like are exemplified. However, when subjected to the immunological screening described below, a vector having a promoter capable of expressing the gene encoding the target protein in the host is preferable. Methods for incorporating cDNA into a plasmid include, for example, Maniatis
Method (Molecular Cloning, A Laboratory Ma
nual, second edition), Cold Spring Harbor Laboratory,
1.53, 1989).
Also, as a method of incorporating cDNA into a phage vector, the method of Hyunh et al. (DNA cloning, practical approach (DNA Cloning, a pract
ical approach), Vol. 1, p. 49, 1985). Conveniently, a commercially available cloning kit (for example, manufactured by Takara Shuzo) can also be used. Recombinant plasmids and phage vectors thus obtained can be used for prokaryotic cells (eg, E. coli: HB101, D.
H5α, Y1090, DH10B or MC1061 /
P3, etc.).

Methods for introducing a plasmid into a host include (Molecular Cloning, A Laboratory
Manual (Molecular Cloning, A Laboratory Manua
l, second edition), Cold Spring Harbor Laboratory, 1.74
1989), the calcium chloride method, the calcium chloride / rubidium chloride method, and the electroporation method. Examples of a method for introducing a phage vector into a host include a method in which phage DNA is packaged in vitro and then introduced into a grown host. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (eg, manufactured by Stratagene, Amersham). CD prepared by the above method
CDNA encoding target protein from NA library
Can be performed by combining general cDNA screening methods.

For example, after separately synthesizing an oligonucleotide which is considered to correspond to the amino acid sequence of the target protein, label it with ³² P to form a probe, and use a known colony hybridization method (Crunstein et al.). , Proceedings of National Academy of Science (Proc. Natl. Acid. Sci.
USA), 72, 3961, 1975) or plaque hybridization (Molecular Clonin).
g, A Laboratory Manual, second edition, Cold Spri
ng Harbor Laboratory, page 2.108, 1989), a method for screening a clone containing the cDNA of interest, preparing PCR primers, amplifying a specific region of the target protein by PCR, and
A method of selecting a clone having the A fragment is exemplified.

When a cDNA library prepared using a vector capable of expressing cDNA (for example, a phage vector such as λgt11) is used, an antigen-antibody reaction using an antibody reactive with the target protein is used. Thus, a desired clone can be selected. When a large number of clones are processed, it is preferable to use a screening method utilizing a PCR method. The nucleotide sequence of the DNA thus obtained is determined by the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA., No. 7).
4, 560, 1977) or phage M1
Method for terminating dideoxynucleotide synthesis using No. 3 (Sanger et al., Proc. Natl. Acad. Sci. US
A., Vol. 74, pp. 5463-5467, 1977)
Can be determined by The gene encoding the target protein can be obtained by cutting out all or a part of the clone obtained as described above with a restriction enzyme or the like.

Further, a genomic DNA derived from a cell expressing the target protein as described above can
As a preparation method by isolating NA, for example, the following method is exemplified. The cells are preferably lysed using SDS or proteinase K and the like, and the extraction with phenol is repeated to deproteinize the DNA. The RNA is digested, preferably by ribonuclease. The resulting DN
DNA obtained by partially digesting A with an appropriate restriction enzyme
The library is prepared by amplifying the fragment with an appropriate phage or cosmid. Then, a clone having the target sequence is detected by, for example, a method using a radiolabeled DNA probe, and all or a part of the gene encoding the target protein is cut out from the clone with a restriction enzyme or the like and obtained.

The DNA encoding the target protein is prepared by PCR using a known mRNA or cDNA of the target protein.
Can be prepared by a conventional method using the above as a template ("Gene amplification PCR method, basic and new development", published by Kyoritsu Shuppan Co., Ltd., 1992, etc.). The production of DNA encoding the target protein by chemical synthesis can be carried out according to a conventional method based on the base sequence of the target protein.

The rat CTGF of the present invention can be obtained by cleaving the rat CTGF-encoding DNA (cDNA or genomic DNA containing introns) prepared using the above-described methods with appropriate restriction enzymes. A DNA fragment encoding the rat CTGF is obtained, and the fragment is commonly used using a DNA obtained by ligating the fragment together with a linker DNA or a tag (Tag) as necessary using an appropriate DNA polymerase or the like. It can be prepared as a recombinant protein by a conventional method using a gene recombination technique. Specifically, it is as exemplified below. That is, the DNA prepared as described above is inserted into a vector as described in detail below to prepare an expression vector, and the expression vector is used to transform a host cell as described below to obtain a transformant. . By culturing the transformant, the target protein is produced in the culture supernatant. The target protein in the culture supernatant can be easily purified using column chromatography or the like.

The present invention also relates to an expression vector containing the DNA encoding the rat CTGF of the present invention. The expression vector of the present invention is not particularly limited as long as it can replicate and maintain or self-proliferate in various prokaryotic and / or eukaryotic host cells, and includes plasmid vectors and phage vectors (Cloning Vectors: A
Laboratory Manual, Elsview, New York, 1
985). The expression vector can be simply used as a recombination vector (plasmid DNA and bacterial phage DNA) available in the art.
It can be prepared by ligating DNA encoding F by a conventional method. Specific examples of the recombination vector used include, for example, pBR322, pBR325, pUC12, pUC13, and pUC19 as plasmids derived from Escherichia coli, pSH19 and pSH15 as plasmids derived from yeast, and pUB110, pTP5, and pC194 as plasmids derived from Bacillus subtilis. And the like. Examples of the phage include bacteriophages such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nucleopolyhedrovirus.

DN encoding rat CTGF of the present invention
For the purpose of expressing A and producing the rat CTGF, a plasmid vector is useful. The plasmid vector is not particularly limited as long as it has a function of expressing the gene encoding the rat CTGF in various prokaryotic and / or eukaryotic host cells and producing these polypeptides. For example, pMAL C2, p
cDNA3.1 (-), pEF-BOS (Nucleic Acid Research, Vol. 18, No. 5322)
PME18S (Experimental Medicine Separate Volume “Genetic Engineering Handbook”, 1992, etc.).

When bacteria, particularly Escherichia coli, are used as host cells, the expression vector generally contains at least a promoter-
It is composed of an operator region, a start codon, a DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit. When a yeast, animal cell or insect cell is used as a host, the expression vector is at least a promoter, an initiation codon, and a rat CT of the present invention.
It preferably contains DNA encoding GF and a stop codon. DN encoding a signal peptide
A, which may include an enhancer sequence, 5 'and 3' untranslated regions of the gene encoding the rat CTGF of the present invention, a splicing junction, a polyadenylation site, a selectable marker region, or a replicable unit. . Further, it may contain a gene amplification gene (marker) which is generally used depending on the purpose.

The promoter-operator region for expressing the rat CTGF of the present invention in bacteria includes a promoter, an operator and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is a bacterium belonging to the genus Escherichia, preferably a Trp promoter, lac promoter, recA promoter, λ
Those containing the PL promoter, lpp promoter, tac promoter and the like are exemplified. Promoters for expressing the rat CTGF of the present invention in yeast include PH05 promoter, PGK promoter, GA
P promoter and ADH promoter. When the host is Bacillus, SL01 promoter, SP
02 promoter, penP promoter and the like. When the host is a eukaryotic cell such as a mammalian cell, examples thereof include an SV40-derived promoter, a retrovirus promoter, and a heat shock promoter. Preferably, SV-40 is a retrovirus. However, it is not particularly limited to these. Use of an enhancer is also an effective method for expression.

[0130] A preferred initiation codon is, for example, methionine codon (ATG). Examples of the stop codon include common stop codons (eg, TAG, TAA, TGA). As the terminator region, a commonly used natural or synthetic terminator can be used. A replicable unit refers to DNA capable of replicating its entire DNA sequence in a host cell, including natural plasmids, artificially modified plasmids (DNA fragments prepared from natural plasmids) and Includes synthetic plasmids and the like. As a preferred plasmid, plasmid pBR322 or an artificially modified product thereof (a DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme) in E. coli, or yeast 2 in yeast.
μ plasmid or yeast chromosomal DNA, and in mammalian cells, plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmid pdBPV-MMTneo ATCC
37224, plasmid pSV2neo ATCC37149, plasmid pSV
2bsr and the like. As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, for example, can be used.

As the selection marker, a commonly used marker can be used by a conventional method. Examples thereof include antibiotic resistance genes such as tetracycline, ampicillin, and kanamycin. Examples of gene amplification genes include dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, and aspartate transcarbamylase gene. And the like.

The expression vector of the present invention is prepared by ligating at least the above-mentioned promoter, initiation codon, DNA encoding the protein of the present invention, termination codon and terminator region to an appropriate replicable unit in a continuous and circular manner. can do. At this time, if desired, an appropriate DNA fragment (for example, a linker or another restriction site) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase. The transformed cell of the present invention comprises
It can be prepared by introducing the above-described expression vector into a host cell.

The host cell used in the present invention is not particularly limited as long as it is compatible with the above-described expression vector and can be transformed. Natural host cells or artificial cells generally used in the technical field of the present invention are used. Various cells such as established recombinant cells (for example, bacteria (Escherichia,
Bacillus), yeast (Saccharomyces, Pichia, etc.), animal cells or insect cells, etc.).
Escherichia coli or animal cells are preferred, and specifically, Escherichia coli (DH5α, DH10B, TB1, HB101, XL-2Blue, etc.) and mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1 or N-1)
IH3T3, etc.), rat-derived cells, hamster-derived cells (BHK
And CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1
And Velo et al.) And human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells and Namalwa).
Etc.) are exemplified. Introduction (transformation (transfection)) of an expression vector into a host cell can be performed using a conventionally known method.

For example, bacteria (E. coli, Bacillus subtili)
s etc.), for example, the method of Cohen et al. (Proc. Natl. Ac
ad. Sci. USA. 69, 2110, 1972
Year), protoplast method (Mol. Gen. Genet., 168)
Vol. 111, 1979) and the competent method (Journal of Molecular Biology (J. Mol. Bio).
l.), 56, 209, 1971).
In the case of Saccharomyces cerevisiae, for example, the method of Hinnen et al. (Proc. Natl. Acad. Sci. USA.,
75, 1927, 1978) and the lithium method (J. Bacteriol., 153, 163, 1983).
Year), in the case of animal cells, for example, Graham (Gr.
aham) method (Virology, Vol. 52,
456, 1973), and in the case of insect cells, for example, by the method of Summers et al. (Mol. Cell. Biol., Vol. 3, pp. 2156-2165, 1983). Can be.

The rat CTGF of the present invention can be produced by culturing a transformed cell containing the expression vector prepared as described above (hereinafter referred to as including a transfectant) in a nutrient medium. it can. The nutrient medium is
It preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose, and examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, Examples include soybean meal and potato extract. If desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be contained. . The culturing is performed by a method known in the art. Culture conditions such as temperature, pH of the medium, and culture time are appropriately selected so that the protein of the present invention is produced in large quantities.

[0136] Specific media and culturing conditions used in accordance with the host cell are described below, but the present invention is not limited thereto. When the host is a bacterium, actinomycete, yeast, or filamentous fungus, for example, a liquid medium containing the above-mentioned nutrients is suitable. Preferably, the medium has a pH of 5 to 8. When the host is E. coli, a preferred medium is LB medium, M9 medium (Miller et al., Exp. Mol.
Genet, Cold Spring Harbor Laboratory, p. 431,
1972), YT medium and the like. In such cases,
Cultivation is usually performed with aeration and stirring, if necessary, for 14 to 4 times.
It can be performed at 3 ° C. for about 3 to 24 hours.

When the host is a bacterium belonging to the genus Bacillus, it is usually at 30 to 40 ° C. and about 16 to 96
Time can be done. When the host is yeast, for example, Burkholder minimum culture (Bostia (Bostia))
nl), Proc. Natl. Acad. Sci. USA, Vol. 77, No. 45
05, 1980), and the pH is preferably 5-8. The culture is usually performed at about 20-35 ° C for about 14-
This is performed for 144 hours, and aeration and stirring may be performed as necessary. When the host is an animal cell, for example, a MEM medium containing about 5 to 20% of fetal bovine serum (Science, vol. 122, p. 501, 1952)
Year), DMEM medium (Virology, No. 8)
Volume, 396, 1959), RPMI 1640 medium (J. Am. Med. Assoc., 199, 519, 19).
67), 199 medium (proc. Soc. Exp. Biol. Med.,
73, page 1, 1950), HamF12 medium and the like. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and stirring can be performed.

When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p. 8404, 1985) and the like can be mentioned. ~ 8 is preferred. Culture is usually about 2
The reaction is performed at 0 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and stirring can be performed. Rat CTGF of the present invention
Can be produced by culturing the above-mentioned transformed cells (especially animal cells or Escherichia coli) and secreting them into the culture supernatant. That is, the obtained culture is filtered or centrifuged to obtain a culture filtrate (supernatant), and the present invention is subjected to a conventional method generally used for purifying and isolating a natural or synthetic protein from the culture filtrate. Of rat CTGF is purified and isolated.

Examples of the method for isolation and purification include a method using specific affinity such as affinity column chromatography, a method using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and the like. Method using difference in molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, method using charge such as ion exchange chromatography and hydroxylapatite chromatography, use difference in hydrophobicity such as reverse phase high performance liquid chromatography And a method utilizing isoelectric point difference such as isoelectric focusing.

On the other hand, when the rat CTGF of the present invention is present in the periplasm or cytoplasm of the cultured transformant, the culture is subjected to a conventional method such as filtration or centrifugation to collect cells or cells. A membrane containing the rat CTGF of the present invention is suspended in an appropriate buffer, and disrupted by, for example, ultrasonic waves, lysozyme, and freeze-thaw to break the cell walls and / or cell membranes of cells, etc., and then centrifuged or filtered. Obtain fractions. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. And
The crude solution can be isolated and purified by using a conventional method as exemplified above.

[0141] The "transgenic mouse" of the present invention is a human CT which can be prepared according to the method described above.
DNA encoding GF (cDNA or genomic D
NA) is a transgenic mouse in which the endogenous locus is integrated on the endogenous locus of the mouse, and the transgenic mouse expresses and secretes human CTGF in the body.

The transgenic mouse can be prepared by a conventional method (for example, the latest manual for animal cell experiments, published by LCI, Chapter 7, Chapters 361 to 361), which is usually used in the production of transgenic animals as described above. Fortieth
8, page 1990). Specifically, for example, normal mouse blastocysts (blas
embryonic stem cells (embryonic stem cel)
l) is transformed with an expression vector into which the gene encoding human CTGF has been inserted so that it can be expressed. ES cells in which a gene encoding human CTGF has been integrated on an endogenous gene are selected by a conventional method. Next, the selected ES cells are microinjected into fertilized eggs (pulmonary blastocysts) obtained from another normal mouse (Pr
oc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7
384, 1980; U.S. Pat. No. 4,873,191). The blastocysts are implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse is obtained. By crossing the heterogeneic transgenic mice, homogenic transgenic mice can be obtained according to Mendel's law.

[0143]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to only the embodiments described in the Examples.

Example 1 Preparation of Polyclonal Antibody Against Human CTGF Amino acid sequence at positions 242 to 252 of human CTGF (Cy
The peptide corresponding to s-Glu-Ala-Asp-Leu-Glu-Glu-Asn-Ile-Lys was converted to a peptide synthesizer (Applied Biosyste
(manufactured by MS Corporation) according to a conventional method. As the immunizing antigen, a peptide emulsified with Freund's complete adjuvant was used. The peptide (0.32 mg / kg)
New Zealand White Rabbit (NZW, Simunek, In
c.) subcutaneously on day 1 (0.8 mg), day 14 (0.8 mg),
Dosing was performed at intervals and in amounts of 35 days (0.8 mg) and 49 days (0.8 mg). Using the peptide, the antibody titer in the serum was appropriately measured. Next, serum is obtained by a standard method,
A polyclonal antibody against human CTGF (IgG) was purified from the serum by affinity chromatography using agarose to which the peptide was coupled. The reactivity to human CTGF was determined by ELISA.
(Enzyme-linked immunosorbent assay) and Western blotting.

Example 2 Preparation of Recombinant Human CTGF <2-1> Transient Expression in Human Kidney-Derived Fibroblast Cell Line 293 cDNA encoding human CTGF was prepared by a conventional method using PCR. Specifically, the human chondroma cell line H
Using cDNA prepared from CS2 / 8 as a template, human CT
GF cDNA (The Journal of Cell Biology, Vol. 1
14, No. 6, p. 1287-1294, 1991). Human C containing the obtained translation region
TGF cDNA was converted to plasmid pcDNA3.1 (-) (Invi
trogen), and an expression vector was prepared. The vector was used to transform human kidney-derived fibroblast cell line 293-T (ATCC CRL-1573) by electroporation. The transformed cells were cultured for 3 days in a serum-free medium ASF104 (manufactured by Ajinomoto Co.) to transiently express recombinant human CTGF.
The expression of human CTGF was confirmed by Western blotting using the polyclonal antibody prepared in Example 1. The culture supernatant was collected, subjected to the ammonium sulfate precipitation method, and then subjected to heparin column chromatography.
After washing with 3M NaCl / PBS, elute with 0.5M NaCl / PBS,
A partially purified human CTGF fraction was obtained.

<2-2> Stable Expression in Human Epithelial-Like Cell Line Hela In the same manner as in Example <2-1>, cDNA encoding human CTGF was prepared by a conventional method using PCR. CDNA of human CTGF containing the obtained translation region
Was inserted into a plasmid pcDNA3.1 (-) (manufactured by Invitrogen) to prepare an expression vector, and the vector was subjected to electroporation and the human epithelioid cell line Hela (ATCC CCL-
2) was transformed. The transformed cells were transformed with Geneticin (0.8m
g / ml; GIBCO-BRL) and 10% fetal bovine serum (fetal ca
Geneticin-resistant transformed cell clones were selected by culturing in RPMI1640 medium containing lf serum) for about 2 weeks. The selected transformed cells were subjected to serum-free medium ASF1.
04 (manufactured by Ajinomoto Co.) to express recombinant human CTGF. The expression of human CTGF was confirmed by Western blotting using the polyclonal antibody prepared in Example 1. The culture supernatant was collected, subjected to ammonium sulfate precipitation, subjected to heparin column chromatography, washed with 0.3 M NaCl / PBS, and then 0.5 M NaCl / P
The fraction was eluted with BS to obtain a partially purified human CTGF fraction.

Example 3 Preparation of Recombinant Mouse CTGF A cDNA sequence encoding a previously reported mouse CTGF (Japanese Unexamined Patent Publication No. 5-255397, Cell Growth Differ., Vol. 2, No.
5, p.225-233, 1991; FEBS Letters, Vol.327, No.2, p.
125-130, 1993; and DNA Cell Biol., Vol. 10, No. 4,
See pages 293-300, 1991. ), A partially purified recombinant mouse CTGF was prepared in the same manner as in Example 2.

Example 4 Preparation of Anti-Human CTGF Monoclonal Antibody and Anti-Mouse CTGF Monoclonal Antibody The preparation of the monoclonal antibody in this example was carried out according to the Experimental Medicine (separate volume) Cell Engineering Handbook (edited by Toshio Kuroki et al.
It was prepared according to general methods as described in Yodosha, pp. 66-74, 1992) and an introduction to monoclonal antibody experimental procedures (written by Tamto Ando et al., Published by Kodansha, 1991). The human CTGF as the immunogen was the recombinant human CTGF prepared in Example 2 using two methods.
Or a mixture thereof was used. Further, mouse CTGF was obtained from the recombinant mouse CTG prepared in Example 3.
F was used. Normal mice (BALB
/ c mouse, female, 4-5 weeks old, Shizuoka Research Animal Center (manufactured)), normal rat (Wistar rat, female, 4-5 weeks old, Shizuoka Research Animal Center (manufactured)), normal hamster (Armenian hamster, Male, 4-5 weeks old, oriental yeast (manufactured by Oriental Yeast)) and human antibody-producing transgenic mice (Nature Genetics,
Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.
146-156, 1997; Japanese Patent Publication No. Hei 4-504365; Japanese Patent Publication No. Hei 7-50913
No. 7; Nikkei Science, June, Nos. 40-50
, 1995, etc. ) Was used. Unless otherwise specified, the same method was used to prepare monoclonal antibodies using any of the animals. The cell culture operation was performed using a multiwell microplate.

<4-1> Preparation of anti-human CTGF monoclonal antibody-producing hybridoma The partially purified recombinant human CTGF (1 μg / mouse) prepared in Example 2 was added to each of the above-mentioned normal mouse and human antibody-producing transgenic mouse. First immunization (day 0) by injection in Hoodpad with Complete Freund's Adjuvant. The recombinant human CTGF was boosted four times or more by injection into the footpad every week from the initial immunization, and the final immunization was performed in the same manner two days before the lymph node cell acquisition described below. Lymph node cells and mouse myeloma cells collected from each animal were mixed at a ratio of 5: 1, and polyethylene glycol 4000 or polyethylene glycol 1500 was used as a fusion agent.
A hybridoma was prepared by cell fusion using (manufactured by GIBCO). Cell fusion of lymph node cells from normal mice was performed using mouse myeloma PAI (JCR No. B01).
13; Res. Disclosure Vol. 217, p. 155, 1982), cell fusion of lymph node cells of a human antibody-producing transgenic mouse was performed using mouse myeloma P3 / X63-AG8.653.
(ATCC No .: CRL-1580) was used. Hybridoma selection was performed using 10% fetal calf serum (Fetal Calf Serum, FC
It was carried out by culturing in HAT-containing ASF104 medium (Ajinomoto Co.) containing S) and aminopterin. The reactivity of the culture supernatant of each hybridoma clone to the recombinant human CTGF used as an immunogen was measured by ELISA described below, whereby a large number of antibody-producing hybridomas were obtained for each animal species.

For normal mice (mouse anti-human CTGF monoclonal antibody), 8-64-6, 8-86-2, 8-97-3,
Clones named 8-149-3 and 15-38-1 were obtained (FIG. 1). Both hybridoma clones 8-86-2 and 8-64-6 were established on December 18, 1997 by the Ministry of International Trade and Industry, Ministry of Industry and Industry, Institute of Biotechnology and Industrial Technology (1-Higashi, Tsukuba, Ibaraki, Japan).
Clone 8-86-2: International Deposit No. FERM BP-6208; Clone 8-64-6: International Deposit No. FERM B
P-6209). For transgenic mice producing human antibodies (human anti-human CTGF monoclonal antibody), A
4.3, A11.1, A15.1, A29.6, B13.7, B22.2, B29.6, B3
5.1, C2.1, C26.11, C59.1, C114.4, M32.2, M33.3, M8
4.4, M107.2, M122, M124, 6, M194.2, M244, M255, M26
8.1, M288.5, M291.2, M295.2, M315, M320.2, N45.2,
Clones named N50.1 and N60.1 were obtained (FIGS. 1 and 2).

Hybridoma clone A11.1 was
It was internationally deposited on September 25, 2000 at the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry of Japan (1-3-1 Higashi, Tsukuba, Ibaraki, Japan) (International Deposit Number FERM BP-6535). In addition, hybridoma clones B22.2, M84.4 and M320.2 are all
On December 15, 1998, the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-1-1 Higashi, Tsukuba, Ibaraki, Japan)
No. 3) (Clone B22.2: International Depositary Number)
FERM BP-6598; clone M84.4: international deposit number FERM BP
-6599; clone M320.2: international deposit number FERM BP-660
0). As described above, in any of the following examples including this example, and in the drawings or tables shown as the test results in the examples, the hybridoma clone producing the human monoclonal antibody of the present invention was used. , Symbols. The alphabet and the number described before the period symbol in the symbol together indicate the name of the parent clone. In addition, the hybridoma clone subcloned from each of the above parent clones,
It was named by adding an additional number following the period symbol immediately after the parent clone name. Incidentally, in any of the following examples including this example, and in the drawings or tables shown as test results in the examples,
In some cases, the numbers by the subcloning are omitted, but all of them are the same cells as the clones shown in FIG. 1 or FIG.

<4-2> Preparation of Anti-Mouse CTGF Monoclonal Antibody-Producing Hybridoma The partially purified recombinant mouse CTGF (2 μg / animal) prepared in Example 3 was added to the above-mentioned normal rat and normal hamster.
Complete Freund's Adju
vant) together with an injection in the footpad (day 0). The recombinant mouse CTGF was boosted four times or more every week from the initial immunization by injection into the footpad, and finally immunized two days before the lymph node cell acquisition described below. Popliteal lymph node cells of each immunized animal were collected by surgery according to a conventional method. Lymph node cells collected from each animal and mouse myeloma cells PAI (JCR No. B0113; Res. Disclosure Vo
l. 217, p. 155, 1982) at a ratio of 5: 1, and cell fusion was performed using polyethylene glycol 4000 or polyethylene glycol 1500 (manufactured by GIBCO) as a fusion agent to prepare a hybridoma. Hybridoma selection was performed using 10% fetal calf serum (Fetal Calf Serum, F
CS) and an HAT-containing ASF104 medium (Ajinomoto (manufactured by Ajinomoto Co.)) containing aminopterin.

Reactivity of the culture supernatant of each hybridoma clone with the recombinant mouse CTGF used as an immunogen was measured by ELISA described later, and a large number of antibody-producing hybridomas were obtained for each animal species. For normal rats (rat anti-mouse CTGF monoclonal antibody), 13-51-2, 17-132, 23-96, 24
-53, 24-67, 25-91, 25-101, 25-256, 25-338, 25-410
And clones designated 25-463 (FIG. 1). As for normal hamster (hamster anti-mouse CTGF monoclonal antibody), a clone named 2-228-1 was obtained (FIG. 1).

<4-3> Screening of monoclonal antibody-producing hybridoma by ELISA The ELISA performed in Examples <4-1> and <4-2> is as follows. Recombinant human CT prepared in Example 2
GF (0.2 μg / well) or recombinant mouse CTGF (0.2 μg / well) prepared in Example 3 was added to each well of a 96-well microplate for ELISA (Corning) and incubated at room temperature for 2 hours. After incubation, recombinant human CTGF or recombinant mouse CTGF was adsorbed to the microplate. Then, discard the supernatant,
Add blocking reagent (200 μl, 3% BSA) to each well
(A phosphate buffer solution) was added and the mixture was incubated at room temperature for 2 hours to block a site where CTGF was not bound. Each well contains 200 μl of phosphate buffer containing 0.1% Tween 20.
Washed 3 times with l. Thus, a microplate was prepared in which each well was coated with recombinant human CTGF or recombinant mouse CTGF. The culture supernatant (100 μl) of each hybridoma was added to each well, and after reacting for 40 minutes, each well was washed three times with 200 μl of a phosphate buffer containing 0.1% Tween20.

Next, a well containing a culture supernatant of a monoclonal antibody-producing hybridoma derived from a normal mouse was treated with a biotin-labeled sheep anti-mouse immunoglobulin antibody (50 μl, manufactured by Amersham), and a monoclonal antibody-producing hybridoma derived from a normal rat. Biotin-labeled sheep anti-rat immunoglobulin antibody (50 μl, manufactured by Amersham) is added to the wells containing the culture supernatant, and biotin is added to the wells containing the culture supernatant of the monoclonal antibody-producing hybridoma derived from normal hamster. Goat anti-hamster immunoglobulin antibody (50 μl, Cedarlane
And a well containing a culture supernatant of a monoclonal antibody-producing hybridoma derived from a human antibody-producing transgenic mouse was added with a biotin-labeled goat anti-human immunoglobulin antibody (50 μl, manufactured by American Corex Corporation). Incubated for 1 hour at room temperature.

After washing the microplate with a phosphate buffer containing 0.1% Tween 20, bovine serum albumin (BS
A, 1 mg / ml) containing a mixture of 0.5 M NaCl and 20 mM HEPES (pH 7.0) diluted 1000-fold with streptavidin-β-galactosidase (Streptoavidin-β-galacto).
sidase, 50μl, manufactured by Gibco BRL))
Incubate for 30 minutes at room temperature. The microplate was then washed with a phosphate buffer containing 0.1% Tween 20, followed by 100 mM NaCl containing 1 mg / ml BSA, 1 mM MgC
l 1% 4-methyl-umbelliferyl-β-D-diluted with a solution (pH 7.0) consisting of ₂ and 10 mM phosphate buffer
Galactoside (4-Methyl-umbelliferyl-β-D-galactosi
de, 50 μl, Sigma) was added to each well,
Incubate at room temperature for 10 minutes. 1M in each well
Na ₂ CO ₃ (100 μl) was added to stop the reaction. Wavelength 460nm
(Excitation: 355nm) Fluoroscan II microplate fluorometer (Fluoroscan II micr
oplate fluorometer, Flow Laboratorie
s Inc.) (product).

<4-4> Mass Preparation of Monoclonal Antibody ICR nude mice (female, 7 to 8 weeks old, manufactured by Charles River) were cloned into each of the above hybridoma clones (10 ⁶ to 10 ⁷ cells / 0.5 ml / each). Mice) were injected intraperitoneally. Ten to twenty days later, the mice were laparotomized under anesthesia, and a large amount of each monoclonal antibody was prepared from ascites collected by a conventional method.

<4-5> Purification of Monoclonal Antibody The centrifuged supernatant obtained by centrifuging each of the monoclonal antibody ascites obtained in <4-4> was mixed with a 0.06 M acetate buffer (pH
The mixture was diluted 3-fold with 4.0), and the pH was adjusted to 4.8 by adding 1N hydrochloric acid. Next, caprylic acid (Caprylic acid, manufactured by Wako Pure Chemical Industries, Ltd.) was added little by little at room temperature with stirring so that the volume became 0.033 ml per 1 ml of ascites, and 3 ml with stirring.
The reaction was performed for 0 minutes. Then, centrifugation (10,000 rpm, 2
(0 min) to precipitate proteins other than the antibody. The centrifuged supernatant was collected and filtered with a filter (manufactured by Millipore) to remove white precipitate. The obtained filtrate was dialyzed (2 hours) against a phosphate buffer. After dialysis, ammonium sulfate (26.2g / 100ml)
Is added little by little while stirring, and 120 ° C at 4 ° C with stirring.
Allowed to react for minutes. Then, centrifugation (10,000 rpm, 2
0 minutes) and the precipitate was collected. In the collected sediment,
Add phosphate buffer and dialyze against phosphate buffer (4 ° C, 24
Time) to obtain each purified monoclonal antibody.

<4-6> Determination of isotype Using a mouse monoclonal-antibody isotype determination kit (manufactured by Amersham), the procedure was carried out according to the experimental operation protocol attached to the kit. Monoclonal antibodies (8-64-6, 8-86
-2, 8-97-3, 8-149-3 and 15-38-1) were determined. All were confirmed to be IgG1 / κ (FIG. 1). Using a human monoclonal antibody isotype determination kit (manufactured by American Corex Co., Ltd.), the operation was performed according to the experimental operation protocol attached to the kit. 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, N5
0.1 and N60.1) were determined. All were confirmed to be IgG2 / κ (FIGS. 1 and 2).

<4-7> Preparation of Affinity Column NHS-activated high trap column (HiTrap-NHS-activat)
ed Sepharose HP, 5ml, Pharmacia Biotech)
Was used to prepare an affinity column according to the attached protocol. Specifically, it is as follows. 0.
0.2 M sodium bicarbonate solution containing 5 M NaCl (pH
8.3) was dissolved in monoclonal antibody 8-86-2 (10 mg / ml Sepharose) prepared in Example <4-5>
The S-activated high trap column was injected. 45 at 20 ° C
After reacting for 2 minutes, the monoclonal antibody 8-86-2 was immobilized on NHS-activated Sepharose. Monoclonal antibody 8-86
-2 indicates human CTGF, mouse CTGF and rat CTG
Since F has high reactivity with any of them, it is possible to purify any of human CTGF, mouse CTGF and rat CTGF by preparing an affinity column using this antibody.

<4-8> Purification of Mammalian CTGF by Affinity Chromatography Transformed Hela cells expressing human CTGF (Example <2)
-2>), transformed HeLa cells expressing mouse CTGF (Example 3) and transformed Hela cells expressing rat CTGF.
The culture supernatant of each of the la cells (Example <11-2>) was collected and subjected to heparin column chromatography.
After washing with NaCl / PBS, elution was performed with 0.7 M NaCl / PBS to obtain crude fractions of human, mouse, and rat CTGF. Each crude product was added to the affinity column on which the anti-CTGF antibody 8-86-2 prepared in Example <4-7> was immobilized, washed with a phosphate buffer, and then 0.1 M glycine buffer ( pH 2.5), 0.75M Tris buffer (pH 9.0)
And neutralized. The collected eluate was dialyzed against a phosphate buffer to obtain highly purified recombinant human CTGF, mouse CTGF and rat CTGF. Each purified recombinant CTGF
With a 10-20% concentration gradient of sodium dodecyl sulfate-
Electrophoresis was performed using a polyacrylamide gel. As a result of silver staining of the separated band, a band was detected at about 38 kDa in all of human, mouse and rat CTGF (FIG. 3).

<4-9> Test of Purified CTGF for Promoting Cell Proliferation In order to confirm whether or not the various recombinant CTGFs purified in Example <4-8> retain the biological activity, each purified CTGF Was examined for its cell growth promoting activity. Rat kidney fibroblast NRK-49F (ATCC
(CRL-1570; 2 × 10 ³ / well) was cultured in DMEM medium containing 10% fetal calf serum (FCS) for 3 days. After removing the culture supernatant and washing once with DMEM medium, DME
Cultured in M medium for 1 day. Next, various concentrations (100, 50, 25, 12.5, 6.3, and 3.1 ng / ml) of purified recombinant CTGF were added to each culture system and cultured for 2 days, followed by [ ³ H] -Thym
Further, idine (3.7 kBq / well) was added and the cells were further cultured for 6 hours. The cells were collected (harvested) and the amount of [ ³ H] -Thymidine incorporated into the cells was measured using a liquid scintillation counter (Beckman). In addition, as a control, [
³ H] -Thymidine uptake was measured. FIG. 4 shows the results. Purified recombinant CT of human, mouse and rat
GF also showed a concentration-dependent cell growth promoting activity, indicating that all recombinant CTGFs retain biological functions.

<4-10> Cross Reactivity Various anti-human CTGF monoclonal antibodies (10 μg / ml) and anti-mouse CTGF monoclonal antibodies (10 μg / ml) prepared as described above, human CTGF and mouse CT
The reactivity to each of GF and rat CTGF was examined in the same manner as in the ELISA described in Example <4-3>. In this test, human, mouse and rat purified recombinant CTGF purified using the affinity column prepared in Example <4-7> were subjected to (A) 100, 30 and 10n, respectively.
Macroplates coated at g / well or (B) at concentrations of 100, 10 and 1 ng / well were used. (B)
As a negative control test, KLH (keyhole) was added to the above-mentioned human antibody-producing transgenic mouse as a negative control test.
A test was performed in the same manner as described above using an anti-KLH human monoclonal antibody prepared by immunization with limpet hemocyanin (PIERCE). The test results at the concentration (A) are shown in FIGS. 5 to 7, and the test results at the concentration (B) are shown in FIGS.
10 to FIG. Further, the results shown in FIGS. 5 to 10 are simplified and shown in the column of “cross-reactivity” in FIGS. 1 and 2. In the column of “Cross-reactivity” in FIG. 1, the coating concentrations were 100, 30, and 10 ng /
The results for wells are shown. The reactivity at each concentration is indicated by “○” when the fluorescence intensity is 1000 or more, “Δ” when the fluorescence intensity is 500 or more and less than 1000, and “×” when the fluorescence intensity is less than 500.
In the column of “Cross-reactivity” in FIG. 2, the results at the coating concentrations of 100, 10 and 1 ng / well are shown in order from the left. The reactivity at each concentration is indicated by “○” when the fluorescence intensity is 1000 or more, “Δ” when the fluorescence intensity is 500 or more and less than 1000, and “×” when the fluorescence intensity is less than 500. The monoclonal antibodies of the present invention have been shown to have various cross-reactivity characteristics.

<4-11> Inhibitory activity of binding between CTGF and various cells Recent studies have revealed that CTGF is involved in cell adhesion (Exp. Cell. Res., Vol. 233, p. 63
-77, 1997). Therefore, it was examined whether or not the various monoclonal antibodies prepared as described above had an activity to inhibit the function of CTGF (neutralizing activity), using the inhibitory effect on the cell adhesion effect mediated by CTGF as an index. The test used three types of methods described in <4-11-1> to <4-11-3> below.

<4-11-1> Human kidney-derived fibroblast cell line 293-
Inhibition of binding to T. Recombinant human CT prepared in the same manner as in Example <4-3>
GF-immobilized microplate (coating concentration: 0.5
μg / well), various monoclonal antibodies (0.5 μg / well) reactive with human CTGF prepared as described above were added. Further, each well of a recombinant mouse CTGF-immobilized microplate (coating concentration: 0.5 μg / well) prepared in the same manner as in Example <4-3> was reacted with the mouse CTGF prepared as described above. Monoclonal antibodies (0.5μg / wel
l) was added.

After removing the supernatant of each plate, the wells were added to each well.
BCECF (2 ', 7'-bis (2-carboxyethyl) -5 (6) -carboxyfluor
Human kidney-derived fibroblast cell line 293-T (A) labeled with escein tetraacetoxymethyl ester (Molecular Probes)
TCC CRL-1573) (5 × 10 ⁴ / well) was added and left at 4 ° C. for 1 hour. The suspended cells were removed, and the cells adhered to the plate were solubilized by adding a phosphate buffer (100 μl) containing 1% NP-40. The fluorescence intensity of BCECF released into the culture supernatant by cell lysis was measured using a Fluoroscan II microplate fluorometer.
fluorometer, Flow Laboratories In
c.) (manufactured)). As a control test, the test was performed in the same manner as above without adding any antibody. The results are shown in FIG. In addition, the results of FIG. 11 are simplified and shown in the column of “Binding inhibitory activity of 293 cells” in FIG. In this column of FIG. 1, "O" indicates that the activity of inhibiting cell adhesion was significantly different, and "X" indicates that the activity was not exhibited.

<4-11-2> Rat kidney-derived fibroblast cell line NR
Inhibition of binding to K-49F Recombinant human CT prepared in the same manner as in Example <4-3>
GF-immobilized microplate (coating concentration: 1μ)
g / well), various human monoclonal antibodies (final concentration: 20, 6, or 2 μg / ml) reactive with human CTGF prepared as described above were added. Next, BCECF (2 ′, 7′-bis (2-carboxyethyl) -5) was added to each well.
(6) -carboxyfluorescein tetraacetoxymethyl ester,
Rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570) (1 × 10 ⁴ / well) labeled with a molecular probe (Molecular Probes Inc.) was added, and the mixture was allowed to stand at 4 ° C. for 1 hour. Remove suspended cells, add 1% NP-40-containing phosphate buffer (100 μl),
Cells adhering to the plate were solubilized. The fluorescence intensity of BCECF released into the culture supernatant by cell lysis was measured using a fluoroscan II microplate fluorometer (Fluo
roscan II microplate fluorometer, Flow Lab (Fl
ow Laboratories Inc.) (manufactured by). As a control test, the test was performed in the same manner as above without adding any antibody. As a negative control test, KLH (k
The test was carried out in the same manner as described above using an anti-KLH human monoclonal antibody prepared by immunization with eyhole limpet hemocyanin (PIERCE). In addition, as another control test, a test was performed in the same manner as described above using microplate wells not coated with CTGF and without adding any antibody. The result is shown in FIG. The results are shown in terms of the cell binding rate (%) based on the obtained fluorescence intensity values. In addition, the results of FIG. 12 are shown in a simplified manner in the column of “Binding inhibitory activity of NRK-49F cells” in FIG. In FIG. 2, the mark “○” in this column indicates that the cell adhesion was inhibited with a significant difference, and the mark “x” indicates that the inhibitory activity was weak or did not show the inhibitory activity.

<4-11-3> Inhibition of binding to various cells Recombinant human CT prepared in the same manner as in Example <4-3>
GF-immobilized microplate (coating concentration: 1μ)
g / well), various human monoclonal antibodies (final concentration: 20 μg / ml) reactive with human CTGF prepared as described above were added. After standing for 60 minutes, the supernatant of each plate was removed, and BCECF (2 ′, 7′-b) was added to each well.
is (2-carboxyethyl) -5 (6) -carboxyfluorescein tetraac
Add the following cells (each 1 × 10 ⁴ / well) labeled with etoxymethyl ester (Molecular Probes) and add 4 ° C.
For 1 hour. The following cells were used. (1) Human lung-derived fibroblasts (NHLF2837; Clonetics)
Made). (2) Human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427). (3) Rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-
1570). The suspended cells were removed, and a 1% NP-40-containing phosphate buffer (100 μl)
l) was added to solubilize the cells adhering to the plate. The fluorescence intensity of BCECF released into the culture supernatant by cell lysis was measured using a Fluoroscan II microplate fluorometer (Flow Laboratories Inc.). As a control test, the test was performed in the same manner as above without adding any antibody. As a negative control test, KLH (keyhole limpet hemocyanin, Pierce (P
A test was performed in the same manner as described above using an anti-KLH human monoclonal antibody prepared by immunization with IERCE). In addition, as another control test, a test was performed in the same manner as described above using microplate wells not coated with CTGF and without adding any antibody. FIG. 13 shows the results. The results are shown in terms of the cell binding rate (%) based on the obtained fluorescence intensity values.

<4-12> Cross-reactivity with rabbit tissue An arteriosclerotic lesion of a hyperlipidemia model rabbit WHHL (manufactured by Oriental Yeast Co.) was collected by surgical operation, and the artery of the arteriosclerosis site was collected by a conventional method. Frozen sections were prepared. Each section was subjected to Vectastain Elite as described below.
Elite) ABC kit (manufactured by Funakoshi Co., Ltd.). The sections were fixed with acetone for 1-2 minutes, dried, and then diluted with diluted serum (PBS (10 ml) / serum (150 μl)).
Wet for a minute. After washing each section with PBS, an anti-human CTGF monoclonal antibody derived from a normal mouse (clone: 8-86-2 and 8-
149-3), an anti-mouse CTGF monoclonal antibody derived from a normal rat (clone: 13-51-2) or an anti-human CTGF monoclonal antibody derived from a human antibody-producing transgenic mouse (clone: A4.3, A11.1, A29. 6, B29.
6, B35.1, C26.11 and C114.4) (10 μg / ml or hybridoma culture supernatant, respectively) were added and allowed to stand for 40 minutes.

Next, the plate was washed with PBS, a biotinylated secondary antibody solution (100 μl) was added, and the mixture was allowed to stand for 30 minutes. In addition,
When an anti-human CTGF monoclonal antibody derived from a normal mouse is used as a primary antibody, a biotin-labeled horse anti-mouse immunoglobulin antibody is used. When a rat immunoglobulin antibody was used, and when a human antibody-producing transgenic mouse-derived anti-human CTGF monoclonal antibody was used as the primary antibody, a biotin-labeled goat anti-human immunoglobulin antibody was used. Each section was dissolved in methanol 3
After standing in a 10% hydrogen peroxide solution for 10 minutes and washing with PBS, 100 μl of avidin-peroxidase solution (PBS
(5 ml) / peroxidase-labeled avidin DH (100 μl)
l) / biotinylated hydrogen peroxide H (100 μl))
Let stand for minutes.

After washing with PBS, DAB solution (water (5 ml)
/ Buffer solution (100 µl) / DAB (diaminobenzidine tetrahydrochloride) solution (200 µl) / hydrogen peroxide solution (100 µl)) and left to stand for 2 to 10 minutes. After washing with cold water for 5 minutes, it was subjected to a Giemsa staining method to perform an encapsulation treatment. As a control, a primary antibody which had no reactivity to CTGF and was stained in the same manner as described above using a monoclonal antibody having the same isotype was used as a control. Each of the stained and sealed tissue sections was observed under a microscope at a magnification of 100 and 200 times, and the results are shown in FIG. FIG.
The results are shown in a simplified manner in the column of "Reactivity to WHHL rabbit atherosclerotic lesion tissue" in FIG. In this column of FIG. 1, "O" indicates that the tissue section was stained, and "X" indicates that the tissue section was weakly stained or was not stained. Clone A, an anti-human CTGF human monoclonal antibody from human antibody-producing transgenic mice
4.3, A11.1, A29.6, C26.11 and C114.4, and clone 13-51-2, an anti-mouse CTGF monoclonal antibody from normal rat, showed reactivity to rabbit atherosclerotic lesion tissue .

<4-13> Inhibitory Activity of Cell Proliferation by CTGF Stimulation As shown in the test of Example <4-9> above, CTG
F induces cell proliferation of various cells (for example, fibroblasts, various tumor cells, and vascular endothelial cells derived from various tissues such as kidney and lung). In this test, the inhibitory effect of the monoclonal antibody of the present invention on cell proliferation caused by such CTGF stimulation was examined as follows.

<4-13-1> Preparation of Cell Culture Solution Containing CTGF Human fetal skin-derived fibroblasts (Neonatal human dermal
fibroblast, NHDF; manufactured by Becton Dickinson) was cultured in a Petri dish, washed twice with a DMEM culture solution containing no fetal calf serum (FCS), and then subjected to human TGF-β (Transforming growth facto).
r; 1 ng / ml, manufactured by R & D Systems), and further cultured for 1 day. The culture supernatant was collected and added to a heparin column (HiTrap, manufactured by Pharmacia Biotech). After washing the column with 0.2 M NaCl / PBS, the factors trapped on the column were eluted with 0.6 M NaCl / PBS. The eluate was dialyzed against PBS and used in the following cell proliferation assay. Since CTGF is heparin-binding, CTGF can be partially purified by using a heparin column. Using the rabbit polyclonal antibody against human CTGF prepared in Example 1, Western blotting was performed according to a conventional method, and it was confirmed that CTGF was contained in the sample obtained above. In the control test, human CTGF
Was used before immunization with rabbits (same as in Example 1). The results are shown in FIG.

<4-13-3> Cell proliferation promoting activity by purified CTGF Rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570, 1 × 10 ⁴ cells / well) was placed in a 96-well microtart plate.
, And cultured for 1 day. Next, the plate was washed twice with a DMEM medium containing no FCS, and then cultured for another day.
Next, the CTGF sample (0.
20-fold dilution of the elution fraction with 6M NaCl in DMEM medium)
Was added to each well and cultured for 18 hours. Next, [ ³ H] -Thymidine (3.7 kBq / well) was added to each well, and the cells were further cultured for 6 hours. The cells were collected (harvested) and the amount of [ ³ H] -Thymidine incorporated into the cells was measured using a liquid scintillation counter (Beckman). As a positive control test, PDGF was used instead of purified CTGF.
The test was performed in the same manner as described above using (platelet-derived growth factor). In addition, as a negative control test, a test was performed in the same manner as described above without adding a CTGF sample. The results are shown in FIG. It was shown that the purified CTGF obtained above promoted the proliferation of rat kidney-derived fibroblast cell line NRK-49F in a concentration-dependent manner.

<4-13-3> Cell proliferation inhibitory activity Rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570, 1 × 10 ⁴ cells / well) was placed in a 96-well microturator plate.
, And cultured for 1 day. Next, the plate was washed twice with a DMEM medium containing no FCS, and then cultured for another day.
Next, the CTGF sample (0.
20-fold dilution of the elution fraction with 6M NaCl in DMEM medium)
A mixture prepared by reacting the above-prepared anti-human CTGF human monoclonal antibody of the present invention (final concentration: 20, 2 or 0.2 μg / ml) for 30 minutes was added to each well, and the mixture was cultured for 18 hours. Next, [ ³ H] -Thymidine (3.7
(kBq / well) was added and the cells were further cultured for 6 hours. Cells were harvested, and [ ³ H]-
The amount of Thymidine was measured with a liquid scintillation counter (Beckman). As a positive control test, the test was performed in the same manner as above without adding any antibody. In addition, as a negative control test, a test was performed in the same manner as described above without adding a CTGF sample or any antibody. The results are shown in FIGS. Further, the results of the same multiple tests as described above (including the results of this test shown in FIGS. 15 and 16) are simplified in the column of “NRK-49F cell growth inhibitory activity” in FIG. Shown. “O” in this column of FIG. 2 means that the activity of inhibiting cell proliferation was significantly different. Anti-human CTGF of the present invention
Human monoclonal antibodies have been shown to significantly suppress or inhibit the growth of human fibroblasts.

<4-14> Epitope mapping The following test was conducted in order to analyze the site (epitope) in the structure of human CTGF to which the anti-human CTGF human monoclonal antibody of the present invention specifically binds. This test was performed using the sandwich ELISA of the present invention using two types of monoclonal antibodies described in detail in Example 5 below. Specifically, the following steps were followed. (Step 1) In the same manner as in Example <5-1> described below, each of the anti-human CTGF human monoclonal antibodies (0.3
μg / 50 μl / well) was prepared.

(Step 2) Each of the following monoclonal antibodies A to D of the present invention was labeled in the same manner as in Example <5-2> described below to prepare labeled monoclonal antibodies. [Antibody A] Mouse monoclonal antibody 8-64-6 reactive to human CTGF (derived from the hybridoma identified by International Deposit No. FERM BP-6209) [Antibody B] anti-human CTGF human monoclonal antibody A11.1
(Derived from the hybridoma identified by International Deposit No. FERM BP-6535.) [Antibody C] Anti-human CTGF human monoclonal antibody C26.11
(Consists of a heavy chain having an amino acid sequence including the amino acid sequence of SEQ ID NO: 8 and a light chain having an amino acid sequence including the amino acid sequence of SEQ ID NO: 18.) [Antibody D] anti-human CTGF human Monoclonal antibody C59.1
(Consists of a heavy chain having an amino acid sequence including the amino acid sequence of SEQ ID NO: 10 and a light chain having an amino acid sequence including the amino acid sequence of SEQ ID NO: 20.) (Step 3) Examples described below. ELISA was performed in the same manner as in <5-3>. That is, each of the antibody-immobilized microplates prepared in step 1 was placed on the purified recombinant human CTGF prepared in the above example.
(15 ng / well) was added to advance the antigen-antibody reaction, and then each labeled monoclonal antibody (0.1 μl / 5
(0 μl / well). After the same operation described in Example <5-3> described below, the reaction was stopped by adding a reaction stopping solution to each well, and the fluorescence intensity at a wavelength of 460 nm (excitation: 355 nm) was measured with a fluorometer.

[0178] The value of the required fluorescence intensity is determined by the human CT to which the monoclonal antibody immobilized on the microplate binds.
Depending on the difference between the site (epitope) on GF and the site (epitope) on human CTGF to which the labeled monoclonal antibody binds, it is thought that the following results (1) to (3) are obtained. . (1) If the site (epitope) on human CTGF to which the monoclonal antibody immobilized on the microplate binds is the same as the site (epitope) on human CTGF to which the labeled monoclonal antibody binds, immobilize on the microplate Since the labeled monoclonal antibody added later cannot bind to the antigen-antibody complex formed between the converted monoclonal antibody and human CTGF, the value of the fluorescence intensity measured in step 3 is extremely close to zero. . (2) If the site (epitope) on human CTGF to which the monoclonal antibody immobilized on the microplate binds and the site (epitope) on human CTGF to which the labeled monoclonal antibody binds are located close to each other, the microplate Since the binding of the subsequently added labeled monoclonal antibody to the antigen-antibody complex formed between the monoclonal antibody immobilized on and the human CTGF undergoes some steric hindrance, the value of the fluorescence intensity measured in step 3 is Will be lower. (3) If the site (epitope) on human CTGF to which the monoclonal antibody immobilized on the microplate binds was different from the site (epitope) on human CTGF to which the labeled monoclonal antibody binds, the antibody was immobilized on the microplate. Since the labeled monoclonal antibody added later can bind to the antigen-antibody complex formed between the monoclonal antibody and human CTGF, the value of the fluorescence intensity measured in step 3 is significantly higher.

The results of this test were consistent with the above estimates. The results are shown in the column of "Epitope mapping" in FIG. In addition, the meaning of each alphabet shown in the column in FIG. 2 is exemplarily shown below. “(A)” means “Antibody A” used as the labeled antibody above. "(B)": means "antibody B" used as the labeled antibody above. "(C)": means "antibody C" used as the labeled antibody above. "(D)": means "Antibody D" used as a labeled antibody above. “A”: It means that the epitope is the same or almost the same as the epitope of “Antibody A”. “B”: means the same or almost the same epitope as the epitope of “antibody B”. "C": means the same or almost the same epitope as that of "antibody C". "D": means the same or almost the same epitope as the epitope of "antibody D". "-": Means that the epitope is different from the epitope of any of the "antibody A", "antibody B", "antibody C" or "antibody D". “B / C”: means the same or almost the same epitope as the epitope of “antibody B” and / or the epitope of “antibody C”. "A- / B": means an epitope at a position close to the epitope of "antibody A" and is the same or almost identical to the epitope of "antibody B". The description methods other than the above have the same meaning as described above.

<4-15> Therapeutic Effect on Kidney Disease and Tissue Fibrosis The therapeutic effect of the anti-human CTGF human monoclonal antibody of the present invention on various diseases was examined using a mouse model of the disease. The mouse model used in this study is a disease model that exhibits some of the pathological features or clinical findings found in any of the following diseases or pathological conditions. The effect is representative of the therapeutic effect of any of the following diseases or pathological conditions. Synovial tissue associated with rheumatoid arthritis, such as kidney disease (renal failure, nephritis, renal fibrosis, etc.), various tissue fibrosis (renal fibrosis, pulmonary fibrosis, fibrosis in liver tissue, fibrosis in skin tissue, etc.) Fibrosis, fibrosis associated with various cancers), skin disease (scleroderma, psoriasis, atopy, etc.), liver disease (cirrhosis, fibrosis in liver tissue, hepatitis, etc.), lung disease (pulmonary fibrosis, pneumonia, etc.) ),
Rheumatoid arthritis, arteriosclerosis, etc.

<4-15-1> Preparation of disease mouse model B6C3F1 mice (male, 7 weeks old, 6 mice per group, manufactured by SLC) were surgically anesthetized with pentobarbital (Pentobarbital, 50 mg / kg). The left flank was opened. Next, two places of the ureter extending from the left kidney were ligated with a suture, and the ureter between the two places of ligature was cut (UUO, Unilateral urine).
eteral obstruction). After the procedure, the laparotomy was sutured. By this operation, the left kidney loses the most important function of a normal kidney, that is, the filtration function of body fluids such as blood, and develops various pathological symptoms seen in various kidney diseases.

<4-15-2> Therapeutic Effect of Anti-CTGF Monoclonal Antibody Each of the model mice prepared above was treated with anti-human CTGF human monoclonal antibody M84 or M dissolved in phosphate buffer.
320 (prepared in the above example, 5 mg / kg each) was administered intraperitoneally. The antibody administration is administered for the first time immediately after the completion of the operation,
Further, the test was performed four times in total every three days from the first administration. After the final administration (14 days after the completion of the operation), the left kidney was removed from each mouse by surgery. The excised kidney was defatted and dehydrated with acetone, and then the protein was hydrolyzed with 6N hydrochloric acid. Next, the sample was dried by applying nitrogen gas under warm conditions, and then dissolved in purified water to obtain a sample for quantification. The concentration of hydroxyproline (OH-Proline) in the obtained kidney tissue sample was measured according to a previously reported method (Analytical Biochemistry, Vol.
288-291, 1973; Kidney Int., Vol. 54, No. 1, p.
99-109, 1998). An increase in the production of hydroxyproline is an indicator of the onset of nephritis and renal fibrosis caused by renal dysfunction, and a decrease in hydroxyproline concentration indicates that the monoclonal antibody is effective for treating the renal disease. It is shown.

The following control experiment was carried out in the same manner as described above. (1) Mice that had been subjected to the above-mentioned UUO (ureter ligation treatment) were intraperitoneally administered with only a phosphate buffer (not containing any antibodies) in the same manner as described above. (2) The case of a normal mouse in which only the laparotomy was performed and the laparotomy was sutured without performing UUO. (3) In the case of a normal mouse not subjected to UUO. (4) Pirfenidone (Kidney Int., Vol. 5) in clinical trials as a therapeutic agent for fibrosis such as renal fibrosis instead of the above antibody
4, No. 1, p. 99-109, 1998) (approximately 500 mg / kg) mixed in the feed (positive control test). The results are shown in FIG. As a result, it was shown that the monoclonal antibody of the present invention has a significant inhibitory and therapeutic effect on kidney disease and tissue fibrosis. Also surprisingly, the efficacy of the monoclonal antibodies of the present invention is demonstrated by the very high dose of the positive control (eg,
Equivalent to about 100 g) when converted to four doses in a 50 kg patient.

<4-16> Determination and Analysis of Gene Sequence and Amino Acid Sequence of Anti-human CTGF Human Monoclonal Antibody Variable heavy chain (Heavy Chain) constituting human monoclonal antibodies against various human CTGFs prepared in the above Examples CDNA sequence encoding the region, as well as the light chain (Light Cha
The cDNA sequences encoding the variable region and constant region of in) were determined as described below, and the structural characteristics of the gene were analyzed. FIG. 18 schematically shows the sequence of the sequence analysis in this example. Human CTGF prepared in the above example
Hybridomas (clones: A29, C26, C59, C114 and M295; each about 5 × 10 ⁷ cells) were cultured, centrifuged, the precipitate was collected, and the extraction of PolyA ⁺ RNA described later Stored at -80 ° C until time.

For extraction and purification of PolyA ⁺ RNA from each hybridoma, a commercially available FastTrack 2.0 kit (manufactured by INVITROGEN)
Was used as follows. Said frozen cells,
The cells were lysed in a cell lysis buffer (Lysis Buffer), disrupted by POLYTRON, and solubilized. After incubating the lysate at 45 ° C., Oligo (dT) cellulose was added, and the mixture was gently shaken for about 1 hour. Next, Oligo (dT) cellu
After washing the lose, PolyA ⁺ RNA was eluted with Ellution Buffer. The eluted PolyA ⁺ RNA is ethanol precipitated and 20 μl
It was dissolved in Tris-EDTA buffer. The concentration of the resulting PolyA ⁺ RNA was determined by measuring the absorbance at a wavelength of 260 nm.

Using the obtained PolyA ⁺ RNA as a template, commercially available MarA
Using athon cDNA Amplification Kit (CLONTECH)
CDNA was synthesized by the RACE-PCR method according to a conventional method ("Gene amplification PCR method, basics and new development", second edition, 1992, published by Kyoritsu Shuppan Co., Ltd., p.13-15). That is, 1st strand cDNA and 2nd strand cDNA were sequentially synthesized using PolyA ⁺ RNA (1 to 5 μg) purified from each hybridoma as a template. The cDNA was subjected to extraction once each using phenol / chloroform / isoamino alcohol and chloroform. Next, the cDNA was precipitated with ethanol and ligated to the adapter DNA (SEQ ID NO: 25). Obtained DNA
Using the reaction product diluted 1/250 as a template, PCR was performed by a conventional method using synthetic primers to prepare cDNAs encoding the antibody heavy chain and the antibody light chain, respectively. P related to antibody heavy chain
The primer described in SEQ ID NO: 26 was used for CR. The primer according to SEQ ID NO: 27 was used for PCR relating to the antibody light chain.

[0187] Each PCR product was fractionated by agarose gel electrophoresis, and DNA was recovered. Determination of the nucleotide sequence of each of the obtained cDNAs was performed using commercially available DyeTerminator Cycle Sequencing.
FS Kit (PE-Applied Biosystems) and PRISM377 DNA
This was performed using a Sequencer (manufactured by PE-Applied Biosystems). Sequencing Primer for this sequencing is
The primers used in the aforementioned PCR were used. Further, an appropriate sequencing primer was prepared from the obtained sequence, and the reaction was further performed. The cDNA sequence encoding the variable region of the heavy chain of the human monoclonal antibody against human CTGF produced by each of the above hybridomas,
The amino acid sequences deduced from the cDNA sequences encoding the variable regions of the (Chain) and the respective cDNA sequences are shown in the sequence listing below.

<Clone A29> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 5 (signal sequence: base number 1)
To 57, V region: base numbers 58 to 363) Amino acid sequence: SEQ ID NO: 6 (signal sequence: amino acids 1 to 19, variable region: including amino acids 21 to 120) (light chain variable region) DNA sequence: sequence No. 15 (signal sequence: base number 1
To 60, V region: base numbers 61 to 365) Amino acid sequence: SEQ ID NO: 16 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 120)

<Clone C26> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 7 (signal sequence: base number 1)
To 57, V region: base numbers 58 to 357) Amino acid sequence: SEQ ID NO: 8 (signal sequence: amino acids 1 to 19, variable region: including amino acids 21 to 118) (light chain variable region) DNA sequence: sequence No. 17 (signal sequence: base No. 1
To 60, V region: base numbers 61 to 364) Amino acid sequence: SEQ ID NO: 18 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 121)

<Clone C59> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 9 (signal sequence: base number 1)
To 57, V region: base numbers 58 to 350) Amino acid sequence: SEQ ID NO: 10 (signal sequence: amino acids 1 to 19, variable region: including amino acids 21 to 116) (light chain variable region) DNA sequence: sequence No. 19 (signal sequence: base No. 1
To 66, V region: base numbers 67 to 353) Amino acid sequence: SEQ ID NO: 20 (signal sequence: amino acids 1 to 22, variable region: including amino acids 23 to 117)

<Clone C114> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 11 (signal sequence: base number 1)
To 57, V region: base numbers 58 to 350) Amino acid sequence: SEQ ID NO: 12 (signal sequence: amino acids 1 to 19, variable region: including amino acids 21 to 116) (light chain variable region) DNA sequence: sequence No. 21 (signal sequence: base number 1
V region: containing nucleotide numbers 48 to 335) Amino acid sequence: SEQ ID NO: 22 (signal sequence: variable region containing amino acid numbers 1 to 16, amino acid numbers 17 to 111)
including)

<Clone M295> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 13 (signal sequence: base number 1)
To 58, V region: base numbers 59 to 353) Amino acid sequence: SEQ ID NO: 14 (signal sequence: amino acids 1 to 19, variable region: including amino acids 21 to 117) (light chain variable region) DNA sequence: sequence No. 23 (signal sequence: base No. 1
To 66, V region: base numbers 67 to 356) Amino acid sequence: SEQ ID NO: 24 (signal sequence: amino acids 1 to 22, variable region: including amino acids 23 to 118)

Based on the determined DNA sequences, using a gene sequence analysis software, a library of human immunoglobulin variable region genes V BASE Sequence (Immunol. Today, Vol. 1) prepared by Tomlinson et al.
6, No. 5, p. 237-242, 1995). As a result, it was found that each V region gene of the heavy chain and light chain of the human monoclonal antibody was composed of the following segments. <Heavy chain V region gene> Clone A29: DP-38 Clone C26: DP-75 Clone C59: DP-5 Clone C114: DP-5 Clone M295: DP-65 <Light chain V region gene> Clone A29: DPK24 Clone C26 : DPK12 clone C59: DPK1 clone C114: DPK1 clone M295: DPK9, which encodes the heavy chain of the above human monoclonal antibody
It was considered that the cDNA sequence had an N region (N-addition) between the V region and the downstream D region, and between the D region and the downstream J region.

Example 5 Human CTGF and mouse CTG
Establishment of sandwich ELISA system for quantification of F <5-1> Preparation of antibody-immobilized microplate In this example, the monoclonal antibody immobilized on the microplate was the monoclonal antibody derived from a normal mouse prepared as described above. -64-6 (International deposit number FERM B
(Derived from the hybridoma identified by P-6209). This monoclonal antibody has high reactivity with human CTGF and cross-reactivity with mouse CTGF. The monoclonal antibody 8-64-6 was diluted with a phosphate buffer, added to each well of a 96-well ELISA microplate (manufactured by Corning) at a concentration of 1 μg / 50 μl / well, and incubated at room temperature for 1 hour. Then, the monoclonal antibody 8-64-6 was adsorbed on the microplate.
Then, after the plate was washed with a phosphate buffer, 3% of bovine serum albumin (BSA) was added to each well.
bumin) (200 μl / well) and incubated at room temperature for 2 hours to block unbound antibody sites. The plate was then washed three times with phosphate buffer.

<5-2> Preparation of Labeled Monoclonal Antibody The monoclonal antibody to be labeled in this example is a normal mouse-derived monoclonal antibody 8-86-2 (International Deposit No. FERM BP-6208) prepared as described above. (Derived from the hybridoma identified by). This monoclonal antibody has high reactivity with human CTGF, mouse CTGF and rat CTGF. Monoclonal antibody 8-86-2 (1 ml of 20 mg / ml) was added to 0.1 M NaHCO
₃ (pH 8.2-8.3) solution was dialyzed (4 ° C., 24 hours). Next, NHS-biotin (100 μl of 2 mg / ml, Pierce) was added, and the mixture was stirred vigorously and incubated at room temperature for 30 minutes. Then, dialyzed against phosphate buffer (4
° C, 24 hours).

<5-3> Establishment of Quantitative Method by Sandwich ELISA The sandwich ELISA system established in the present invention for quantifying human CTGF and mouse CTGF is as follows. A measurement sample (50 μl / well) was added to 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 a phosphate buffer containing 0.1% Tween20, and then prepared in Example <5-2> in which each well was diluted with a phosphate buffer containing 1% BSA and 0.1% Tween20. The biotin-labeled monoclonal antibody (0.3 μl / 50 μl / well) was added and incubated at room temperature for 1 hour. The microplate is washed three times with a phosphate buffer containing 0.1% Tween20, and then diluted 1000-fold with a solution (containing BSA (1 mg / ml), pH 7.0) containing 0.5 M NaCl and 20 mM HEPES. Streptavidin-β-galactosidase (Streptoavi
din-β-galactosidase, 50 μl, Gibco BR
L) was added to each well and incubated at room temperature for 30 minutes.

After the microplate was washed three times with a phosphate buffer containing 0.1% Tween 20, 100 mM NaCl, 1 mM Mg were used.
1% 4-methyl-umbelliferyl-β-diluted with a solution (BSA (1 mg / ml), pH 7.0) consisting of Cl ₂ and 10 mM phosphate buffer (containing Na and K) D-galactoside (4-methyl-umbelliferyl-β-D-galactoside, 50 μl
1, Sigma) was added to each well and incubated at room temperature for 10 minutes. 1 M Na ₂ C in each well
O ₃ (100 μl) was added to stop the reaction. The fluorescence intensity at a wavelength of 460 nm (excitation: 355 nm) was measured using a Fluoroscan II microplate fluorometer.
te fluorometer, Flow Laboratories In
c.) (manufactured)). The amount of human CTGF or mouse CTGF in the measurement sample was determined from a calibration curve created in the following example.

<5-4> Preparation of Calibration Curve The affinity-purified recombinant human CTGF or recombinant mouse CTGF prepared in Example <4-8> was prepared using CTG.
Example <5- used as F standard (standard)
A calibration curve was prepared using the sandwich ELISA established in 3>. The results are shown in FIG. For human CTGF, a calibration curve having a significant difference was obtained in the extremely low concentration range of 3 ng / ml to 1000 ng / ml. For mouse CTGF, a calibration curve having a significant difference was obtained in the concentration range of 30 ng / ml to 1000 ng / ml. However, Example <5-3
In the sandwich ELISA system established in>, rat CTG
F could not be quantified.

Example 6 Mouse CTGF and Rat CT
Establishment of sandwich ELISA system for quantification of GF <6-1> Preparation of antibody-immobilized microplate In this example, the monoclonal antibody immobilized on the microplate was the normal rat-derived monoclonal antibody 13 prepared as described above. -51-2 was used. This monoclonal antibody has high reactivity with mouse CTGF and cross-reactivity with rat CTGF. Monoclonal antibody 13-51-2 was diluted with phosphate buffer (PBS), and was diluted to a concentration of 1 μg / 50 μl / well with 96 μl for ELISA.
The solution was added to each well of a well microplate (manufactured by Corning) and incubated at room temperature for 1 hour to adsorb the monoclonal antibody 13-51-2 to the microplate. Then, after the plate was washed with a phosphate buffer, 3% of bovine serum albumin (BSA; Bovine seru) was added to each well.
Phosphate buffer (200 μl / well) containing m albumin) was added, and incubation was performed at room temperature for 2 hours to block the site where no antibody was bound. The plate was then washed three times with phosphate buffer.

<6-2> Preparation of Labeled Monoclonal Antibody The biotin-labeled monoclonal antibody prepared in Example <5-2>, that is, a monoclonal antibody having high reactivity with any of human CTGF, mouse CTGF and rat CTGF 8 -86-2 (derived from the hybridoma identified by International Accession No. FERM BP-6208) was labeled with biotin, and a labeled monoclonal antibody was used.

<6-3> Establishment of quantitative method by sandwich ELISA Mouse CTGF and rat CTGF established in the present invention
The sandwich ELISA system for quantification of is as follows. In each well of the antibody-immobilized microplate prepared in Example <6-1>, a measurement sample (50 μl / well) was added.
Was added and incubated at room temperature for 1 hour. The microplate was washed three times with a phosphate buffer containing 0.1% Tween20, and then prepared in Example <6-2> in which each well was diluted with a phosphate buffer containing 1% BSA and 0.1% Tween20. The biotin-labeled monoclonal antibody (0.3 μl / 50 μl / well) was added and incubated at room temperature for 1 hour. The microplate is washed three times with a phosphate buffer containing 0.1% Tween20, and then diluted 1000-fold with a solution (containing BSA (1 mg / ml), pH 7.0) containing 0.5 M NaCl and 20 mM HEPES. Streptavidin-β-galactosidase (Streptoavi
din-β-galactosidase, 50 μl, Gibco BR
L) was added to each well and incubated at room temperature for 30 minutes.

After the microplate was washed three times with a phosphate buffer containing 0.1% Tween 20, 100 mM NaCl, 1 mM Mg were used.
1% 4-methyl-umbelliferyl-β-diluted with a solution (BSA (1 mg / ml), pH 7.0) consisting of Cl ₂ and 10 mM phosphate buffer (containing Na and K) D-galactoside (4-methyl-umbelliferyl-β-D-galactoside, 50 μl
1, Sigma) was added to each well and incubated at room temperature for 10 minutes. 1 M Na ₂ C in each well
O ₃ (100 μl) was added to stop the reaction. The fluorescence intensity at a wavelength of 460 nm (excitation: 355 nm) was measured using a Fluoroscan II microplate fluorometer.
te fluorometer, Flow Laboratories In
c.) (manufactured)). Mouse CTGF in the measurement sample
Alternatively, the amount of rat CTGF was determined from a calibration curve created in the following example.

<6-4> Preparation of Calibration Curve The affinity-purified recombinant mouse CTGF or recombinant rat CTGF prepared in Example <4-8> was subjected to CT.
Example <6 used as GF standard (standard)
Calibration curve was prepared using the sandwich ELISA established in -3>. The results are shown in FIG. For mouse CTGF, a calibration curve having a significant difference was obtained in the extremely low concentration range of 1 ng / ml to 1000 ng / ml. Rat CTGF
For, a calibration curve having a significant difference was obtained in the concentration range of 10 ng / ml to 1000 ng / ml. However, Example <6-
In the sandwich ELISA system established in 3>, human CTG
F could not be quantified.

Example 7 Quantification of CTGF in Serum of Patients Suffering from Various Diseases The sandwich ELISA established in Example <5-3>
Was used to quantify CTGF in the serum of various patients.

<7-1> Biliary atresia, rheumatic vasculitis, malignant rheumatoid arthritis, psoriasis and atopic dermatitis The human serum used in this test was healthy (33 samples) and suffered from biliary atresia. , Postoperative samples of patients who underwent surgery (<Group 1> patients with normal clinical findings (17 samples), <Group 2> patients with ongoing symptoms (14 samples), and <Group 3>
Severe patients requiring liver transplantation (8 specimens), patients suffering from rheumatoid vasculitis (10 specimens), patients suffering from malignant rheumatoid arthritis (MRA) (17 specimens) , Serum collected from each of patients suffering from psoriasis (24 samples) and patients suffering from atopic dermatitis (34 samples). The results are shown in FIG. 21 (biliary atresia) and FIG. 22 (rheumatic vasculitis, malignant rheumatoid arthritis, psoriasis and atopic dermatitis). In patients with biliary atresia, CTGF is second
It was revealed that the expression was significant in the group (the advanced stage of the symptoms). In addition, it was revealed that significantly more CTGF was expressed in rheumatic vasculitis and malignant rheumatoid arthritis than in healthy subjects.

<7-2> Rheumatoid arthritis and osteoarthritis The human serum used in this test was rheumatoid arthritis (Rheuma arthritis).
toid arthritis (RA) (36 patients)
1) and synovial fluid collected from each of patients (19) suffering from osteoarthritis (Osteoarthritis, OA). The results are shown in FIG. CTGF concentration in synovial fluid of patients with rheumatoid arthritis was found to be significantly higher than that of osteoarthritis. These results indicate that the assay system of the present invention can be used for CTG not only in healthy subjects but also in various disease patients.
The expression state of F can be quantified with high sensitivity, and it can be said that it has utility as a clinical diagnostic agent for accurately grasping the progress of the disease state.

Example 8 Antibody Fragment F (ab ′)_TwoPreparation of Fab and Fab
Body fragment F (ab ') _TwoAnd Fab are adjusted as follows:
To make. Monoclonal antibody (5 mg / ml) was converted to 20 mM acetic acid
Add to sodium buffer (pH 3.5) and 37 ° C for 30 minutes
Incubate. Next, insolubilized pepsin (1 m
l, manufactured by Pierce Corporation) and rotate it with a rotator.
And incubated at 37 ° C for 12 hours. Collect reaction solution
Centrifuged (3000 rpm, 10 minutes) and collect the supernatant
You. Protein A affinity chromatography
Of the protein A column kit (Amersham)
Perform the following according to the protocol. Centrifugal sediment
Add binding buffer and centrifuge (3000 rpm, 10 minutes)
And collect the supernatant. Supernatant collected by two centrifugations
Collect, add an equal volume of binding buffer, and add 1N
Adjust to pH 8.9 by adding thorium. The mixed solution is
Add to the Protein A column equilibrated with binding buffer
After washing with the binding buffer (5 ml) twice, the eluted fraction was washed.
to recover. The eluted fraction thus obtained was added to a 5 mM phosphate buffer solution.
(2 L, pH 6.8) for dialysis (4 ° C., 24 hours).

For further purification, high performance liquid chromatography (HPLC) is performed using a hydroxyapatite column (manufactured by Bio-Rad). The solution obtained by dialysis is added to the hydroxyapatite column, a 5 mM phosphate buffer is allowed to flow for 15 minutes, and then a linear gradient elution is performed with a 5 mM to 0.4 M phosphate buffer. The eluate was collected with a fraction collector, the absorbance at 280 nm was measured, and F (a
b ') Collect the fraction containing ₂ The obtained fraction is dialyzed (4 ° C., 24 hours) against a phosphate buffer (2 L) to obtain a purified F (ab ′) ₂ of a monoclonal antibody.

Example 9 Preparation of Transgenic Mouse Expressing Human CTGF cDNA encoding human CTGF obtained in Example 2
Into the expression vector pCAGGS (Gene, Vol. 108, p. 193-200, 1991) having the chicken β actin promoter.
It was inserted using a DNA end blunting kit (manufactured by Takara) to obtain a plasmid phCTGF. By electroporation, human kidney-derived fibroblast cell line 293-T (A
TCC CRL-1573). By the sandwich ELISA established in Example 5, it was confirmed that the obtained transformed cells expressed and secreted human CTGF in the culture supernatant. For transgenic mouse production, ph
CTGF was linearized by restriction enzyme treatment.

The foster mother mice were white ICR mice (female,
Female ICR mice having a plug (or vaginal plug) obtained by mating a white ICR mouse (male, Nippon SLC) ligated with vas deferens were used. Moreover, the mouse for egg collection for obtaining a fertilized egg for introducing the human CTGF gene was superovulated by administering PEAMEX (5 units, manufactured by Sankyo Zoki) and pregnyl (5 units, manufactured by Organon). BDF-1
A mouse (female, manufactured by Japan SLC) was bred with a BDF-1 mouse (male, manufactured by Nippon SLC).
After mating, the oviduct was excised from a BDF-1 mouse (female), and only a fertilized egg was obtained by treatment with hyaluronidase and stored in a medium.

The introduction of the human CTGF gene into fertilized eggs
This was performed by a conventional method using a manipulator under a microscope. The fertilized egg is fixed with a retaining needle, and tris E
A solution containing the above-mentioned linear gene of human CTGF diluted with DTA buffer was injected into the male pronucleus of a fertilized egg using a DNA introduction needle. After the gene transfer, only fertilized eggs that maintained a normal state were selected, and the human CTGF gene-transferred fertilized eggs were inserted into the fallopian tubes in the ovaries of a foster parent mouse (white ICR mouse). The tail of a child mouse (chimeric mouse) born from the foster mother was cut off, the genomic gene was recovered, and it was confirmed by PCR that the human CTGF gene had been introduced into the mouse genome. In addition, by the sandwich ELISA established in Example 5, human CT was added to the serum of the mouse.
It was confirmed that GF was expressed and secreted. Then
This chimeric mouse was crossed with a normal mouse to produce a human CTGF-highly expressing heterotransgenic mouse. A homo mouse was prepared by crossing the hetero mice.

Example 11 Preparation of Rat CTGF <11-1> Cloning of cDNA (1) Preparation of rat cDNA library and probe Rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-1570,
About 1 × 10 ⁶ / ml) was centrifuged (2,000 × g, 5 minutes, 4 ° C.), and the precipitated cells were suspended using ISOGEN (Nippon Gene), followed by shaking extraction with chloroform. The supernatant was collected. Isopropanol was added to the obtained supernatant, left at room temperature for 10 minutes, and then centrifuged (12,000 × g, 10 minutes, 4 ° C.) to precipitate RNA. The precipitated RNA was washed with ethanol and then dissolved in a TE buffer. All obtained
Poly (A) ⁺ RNA was purified from the RNA using the mRNA Purification Kit (Pharmacia).

Using poly (A) ⁺ RNA (5 μg) as a template,
script l system for cDNA Synthesis Kit (GIBCO-BRL
CDNA). In order to increase the efficiency of the screening, an oligo dT primer having a NotI cleavage site (GIBCO-BRL) was used. After addition of the SalI adapter, NotI digestion was performed to obtain cDNA having unidirectionality.
Furthermore, a cDNA size fraction column (cDNA size fraction)
ation column, manufactured by GIBCO-BRL). The cDNA base sequences encoding human and mouse CTGF obtained in Example 2 were compared, and the 5 'primer (SEQ ID NO: 3) and 3'
A primer (SEQ ID NO: 4) was designed and synthesized.

Using the cDNA library prepared as described above as a template, PCR (Polymerase Cha) was performed using both primers and Ex Taq DNA polymerase (Takara Shuzo).
in Reaction). The reaction was performed using a DNA thermal cycler (manufactured by Perkin Elmer Cetus) under the conditions of a final primer concentration of 0.4 μM and a Mg ²⁺ concentration of 1.5 mM for 1 minute at 94 ° C., 1 minute at 55 ° C., and 72 ° C. For 1 cycle, and a total of 35 cycles were performed. After the amplified DNA is subjected to agarose gel electrophoresis, the QUIAEX DNA extraction kit (QUIAEX
EX DNA Extraction Kit, manufactured by QUIAGEN).

[0215] The recovered DNA fragment was subjected to vector pCRII (Invitroge) using a TA cloning kit (Invitrogen).
Auto read sequencing kit (Pharmacia)
And the nucleotide sequence was determined by the dideoxy method using an ALF DNA sequencer (Pharmacia). Obtained c
As a result of comparing the base sequence of the DNA fragment with the base sequences of human and mouse CTGF obtained in Examples 2 and 3, respectively, the cDNA fragment was found to be human and mouse CTGF.
Contains a region encoding the rat homolog (rat CTGF). The cDNA fragment (about 0.
8kb) using the ECL random priming labeling kit (ECLr
FITC labeling was performed using an andom prime labeling system (Amersham) and used as a probe for plaque hybridization.

(2) Incorporation and packaging of cDNA library into vector The cDNA fragment obtained in (1) above is
x NotI-SalI arm (GIBCO-BRL). DNA ligation kit (DNA ligation Kit,
Takara Shuzo) was used. Next, GIGA PACK II GOLD (S
After in vitro packaging using tratagene, E. coli Y1090 (G
Using IBCO-BRL) as a host, a cDNA library comprising plaques containing the recombinant phage was prepared.

(3) Screening of cDNA library Rapid hybrid buffer (Rapid hybrid buffer)
plaque hybridization method using an amplification buffer (manufactured by Amersham) (Maniatis et al.,
Molecular Cloning: A Labolatory Manual, Cold Sprin
g Harbor Laboratory, Cold Spring Harbor, New Yor
According to k), the cDNA library prepared in the above (2) was screened as follows. The library (1 × 10 ⁴ plaques) obtained in the above (2) was sown on an agar plate, and a replica was prepared using Hybond-N nylon membrane (Amersham). Using the replica and the FITC-labeled probe prepared in the above (1), a rapid hybridization buffer (Rapid hybridization buffer, Am
(manufactured by ersham). Primary screening and secondary screening were performed, and 13 positive clones were obtained. After isolation of each clone in a single plaque, in vivo excision was performed according to the manual of GIBCO-BRL.
and 13 clones were recovered as plasmid DNA.

(4) Determination of base sequence Autoread sequencing kit (Auto ReadSequencing Kit, manufactured by Pharmacia) for 13 clones
And the ALF DNA sequencer (Pharmacia) to determine the nucleotide sequence by the dideoxy method. All 13 clones contained the same nucleotide sequence. As a result of comparison with the cDNA sequences of human and mouse CTGF, the obtained clone r311 contained cD encoding the full length of rat CTGF.
It was confirmed that an NA region was included. The obtained full-length cDNA sequence of rat CTGF (including the 5 ′ and 3 ′ terminal nucleotide sequences) is shown in SEQ ID NO: 1, and the amino acid sequence deduced from the sequence is shown in SEQ ID NO: 2.

<11-2> Preparation of Recombinant Rat CTGF The clone r311 containing the cDNA encoding the rat CTGF obtained in Example <11-1> was digested with SalI-DraI to obtain the cDNA encoding the rat CTGF. Including D
The NA fragment was cut out. The DNA fragment was converted to plasmid pc.
It was inserted into DNA3.1 (-) (manufactured by Invitrogen) to prepare an expression vector. The vector was used to transform the human epithelioid cell line Hela (ATCC CCL-2) by electroporation. The transformed cells were transformed with Geneticin (0.8 mg / ml; GIBCO-BRL).
) And RPMI1640 medium containing 10% fetal calf serum for about 2 weeks.
Geneticin resistant transformed cell clones were selected. The selected transformed cells were cultured in a serum-free medium ASF104 (manufactured by Ajinomoto Co.) to express recombinant rat CTGF. The expression of rat CTGF was confirmed by Western blotting using a monoclonal antibody cross-reactive with rat CTGF prepared in Example 4 above. After collecting the culture supernatant and subjecting it to the ammonium sulfide precipitation method,
Subject to heparin column chromatography, 0.3 M NaCl
After washing with / PBS, elution was carried out with 0.5M NaCl / PBS to obtain a partially purified rat CTGF fraction.

[0220]

Industrial Applicability The present invention has not been provided yet.
For TGF, antigen specificity, antigen affinity, neutralizing activity,
It is intended to provide various monoclonal antibodies derived from various mammals having different characteristics in properties such as cross-reactivity and the like. In particular, the present invention provides, for the first time in the world, various human monoclonal antibodies against human CTGF by using, as an immunized animal, a transgenic mouse prepared to produce a human antibody using a genetic recombination technique.

Among the monoclonal antibodies of the present invention, human C
Monoclonal antibodies against TGF and pharmaceutical compositions thereof may be used for various disease symptoms, such as renal diseases (renal fibrosis, nephritis, renal failure, etc.), whose onset may be predicted to be due to CTGF. Lung disease (eg, pulmonary fibrosis, pneumonia, etc.), liver disease (eg, liver tissue fibrosis, cirrhosis, hepatitis, etc.), skin disease (eg, wound, scleroderma, psoriasis, keloids, etc.), arthritis (eg, , Rheumatoid arthritis, osteoarthritis, etc.), vascular diseases (eg, rheumatoid vasculitis, etc.), tissue fibrosis complicated by various cancers, and arteriosclerosis (especially, complicated tissue fibrosis)
It is useful as a medicament for suppressing or preventing the onset and / or progression of the disease and treating or preventing the disease. In particular, human monoclonal antibodies and pharmaceutical compositions thereof have no antigenicity against humans, which has been a major problem (side effect) in the treatment of antibody pharmaceuticals composed of non-human mammal-derived antibodies such as mouse-derived antibodies. Thus, it dramatically increases the value as a drug.

Further, by using various monoclonal antibodies of the present invention, various mammals (human, mouse,
CTGF in body fluids (such as serum) of rats and rabbits
And various immunoassay systems (methods and kits) that can be simply and highly sensitively quantified in an intact state. In addition, by preparing an affinity column in which the monoclonal antibody is immobilized on an insoluble carrier, it becomes possible to easily purify various mammalian CTGFs with high purity.

Further, the transgenic non-human mammal (human transgenic mouse or the like) expressing the human CTGF of the present invention is useful not only as a model animal for elucidating the physiological function of human CTGF but also as a human CGF.
Controls the function of TGF (inhibition, suppression, activation, stimulation, etc.)
It is extremely useful as a tool for screening various drugs (low molecular compounds, antibodies, antisense, polypeptides other than human CTGF, etc.). That is, such a drug is administered to the transgenic non-human mammal, and the degree of expression of human CTGF in the animal is determined using the assay system (sandwich ELISA) of the present invention.
), It is possible to evaluate the effect of the administered drug on human CTGF.

[0224]

[Sequence List] SEQUENCE LISTING <110> Japan Tobacco, Inc. <120> Monoclonal Antibody For Connective Tissue Growth Factor and Pharmaceutical Use Thereof <130> J98-0215 <140> <141> <150> JP P1997-367699 <151> 1997-12-25 <150> JP P1998-356183 <151> 1998-12-15 <160> 27 <170> PatentIn Ver. 2.0 <210> 1 <211> 2338 <212> DNA <213> Rat <220> <221> 5'UTR <222> (1) .. (212) <220> <221> CDS <222> (213) .. (1256) <220> <221> 3'UTR <222> (1257) .. (2338) <220> <221> polyA_signal <222> (2297) .. (2302) <400> 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 Aru Pro Ala Thr 10 15 20 ggc cag gac tgc agc gcg cag tgt cag tgc gca cgt gaa gcg g cg 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 Val Val Ser 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 cag 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 gtcttc 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 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 atcgg accaaa 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 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 aacgt 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 tgctg cttct ttgactatga ctggtttggg aggcagttta tttgttgaga 2256 gtgtgaccaa aagttacatg tttgcacctt tctagttgaa aataaagtat atatattttt 2316 tatatgaaaa aaaaaaaaaa aa 2338 <210> 2 <211> 347 <212> PRT <La> La <A> La <A> La <A> La <A> La <A> La <A> La <A> La <A> 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 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221 > primer_bind <222> (1) .. (20) <400> 3 tgcggctgct gccgcgtctg 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221> primer_bind <222> (1) .. (21) <400> 4 gcacaggtct tgatgaacat c 21 <210> 5 <211> 444 <212> DNA <213> Homo sapiens <220> <221 > CDS <222> (1) .. (444) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (363) < 400> 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 gg 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 <210> 6 <211 > 148 <212> PRT <213> Homo sapiens <400> 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 <210> 7 <211> 447 <212> DNA <213> Homo sapiens <220 > <221> CDS <222> (1) .. (447) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. 357) <400> 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 S er 145 <210> 8 <211> 149 <212> PRT <213> Homo sapiens <400> 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 Val Thr Ser Ser Ala Ser Ser 130 135 140 Thr Lys Gly Pro Ser 145 <210> 9 <211> 438 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (438) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (350) <400> 9 atg gac tgc acc tgg agg atc ctc ttc ttg gtg gca gca gct aca ggc 48 Met Asp Cy s 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 T hr 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 <210> 10 <211> 146 <212> PRT <213> Homo sapiens <400> 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 <210> 11 <211> 438 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (438) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58 ) .. (350) <400> 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 g ag 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 <210> 12 <211> 146 <212> PRT <213> Homo sapiens <400> 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 Va l 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 <210> 13 < 211> 450 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (450) <220> <221> sig_peptide <222> (1) .. (58) <220 > <221> V_region <222> (59) .. (353) <400> 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 Gl y 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 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 <210> 14 <211> 150 <212> PRT <213> Homo sapiens <400> 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 <210> 15 <211> 423 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (423) < 220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (365) <400> 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 <210> 16 <211> 141 <212> PRT <213> Homo sapiens <400> 16 Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser 1 5 10 1 5 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 <210> 17 <211> 420 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (420) <220> <221> sig_peptide <222> (1). . (60) <220> <221> V_region <222> (61) .. (364) <400> 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 G ln 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 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 1 40 <210> 18 <211> 140 <212> PRT <213> Homo sapiens <400> 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 <210> 19 <211> 405 < 212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (405) <220> <221> sig_peptide <222> (1) .. (66) <220> <221> V_region <222> (67) .. (353) <400> 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 Le u 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 I le Lys 115 120 125 cga act gtg gct gca cca tct 405 Arg Thr Val Ala Ala Pro Ser 130 135 <210> 20 <211> 135 <212> PRT <213> Homo sapiens <400> 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 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 <210> 21 <211> 387 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (387) <220> <221> sig_peptide <222 > (1) .. (47) <223> Initiation codon and a portion of a signal sequence are lacked. <220> <221> V_region <222> (48) .. (3 35) <400> 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 <210> 22 <211> 129 <212 > PRT <213> Homo sapiens <400> 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 <210> 23 <211> 411 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (411 ) <220> <221> sig_peptide <222> (1) .. (66) <220> <221> V_region <222> (67) .. (356) <400> 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 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 <210> 24 <211> 137 <212> PRT <213> Homo sapiens <400> 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 <210> 25 <211> 27 <212> DNA < 213> Artificial Sequence <220> <223> Description of Artificial Sequence : Artificially synthesized adaptor sequence <220> <221> misc_difference <222> (1) .. (27) <400> 25 ccatcctaat acgactcact atagggc 27 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221> primer_bind <222> (1) .. (25) <400> 26 ccagggccgc tgtgctctcg gaggt 25 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221> primer_bind <222> (1) .. (23) <400> 27 gggggtcagg ctggaactga gga 23

“Sequence List Free Text” SEQ ID NO: 3 Other information: Description of artificial sequence: Primer sequence synthesized artificially. SEQ ID NO: 4 Other information: Description of artificial sequence: Primer sequence artificially synthesized. SEQ ID NO: 21 Other information: Initiation codon and part of signal sequence are missing. SEQ ID NO: 25 Other information: Description of artificial sequence: Artificially synthesized adapter sequence. SEQ ID NO: 26 Other information: Description of artificial sequence: Primer sequence artificially synthesized. SEQ ID NO: 27 Other information: Description of artificial sequence: Primer sequence artificially synthesized.

[0226]

[Brief description of the drawings]

FIG. 1 is a view showing characteristics of various monoclonal antibodies prepared by immunizing various mammals with human CTGF or mouse CTGF.

FIG. 2 is a view showing characteristics of various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF.

FIG. 3: Recombinant human C purified using an affinity column adsorbed with monoclonal antibody 8-86-2 reactive to CTGF of human, mouse and rat.
TGF, recombinant mouse CTGF and recombinant rat CTG
The figure which shows the state of electrophoresis in SDS polyacrylamide gel of F.

FIG. 4: Recombinant human C purified using an affinity column adsorbed with monoclonal antibody 8-86-2 reactive to CTGF of human, mouse and rat.
TGF, recombinant mouse CTGF and recombinant rat CTG
The figure which shows the cell growth promotion activity of rat kidney-derived fibroblast NRK-49F of F. The vertical axis shows the amount of [ ³ H] thymidine taken up into cells as an indicator of the strength of the cell growth promoting activity, and the horizontal axis shows the concentration of each recombinant CTGF.

FIG. 5 shows the reactivity of various monoclonal antibodies prepared by immunizing various mammals with human CTGF or mouse CTGF to human CTGF. The vertical axis indicates the fluorescence intensity as an index of the reactivity of the antibody, and the horizontal axis indicates the clone name of the monoclonal antibody tested by ELISA at various concentrations of human CTGF.

FIG. 6 shows the reactivity of various monoclonal antibodies prepared by immunizing various mammals with human CTGF or mouse CTGF to mouse CTGF. The vertical axis indicates the fluorescence intensity as an index of the reactivity of the antibody, and the horizontal axis indicates the clone name of the monoclonal antibody tested by ELISA with various concentrations of mouse CTGF.

FIG. 7 shows the reactivity of various monoclonal antibodies prepared by immunizing various mammals with human CTGF or mouse CTGF to rat CTGF. The vertical axis shows the fluorescence intensity as an index of the reactivity of the antibody, and the horizontal axis shows the clone name of the monoclonal antibody tested by ELISA with various concentrations of rat CTGF.

FIG. 8 is a diagram showing the reactivity of human CTGF with various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF. The vertical axis indicates the fluorescence intensity as an index of antibody reactivity, and the horizontal axis indicates the clone name of the human monoclonal antibody tested by ELISA at various concentrations of human CTGF.

FIG. 9 is a diagram showing the reactivity of various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF to mouse CTGF. The vertical axis shows the fluorescence intensity as an index of antibody reactivity, and the horizontal axis shows the clone names of human monoclonal antibodies tested by ELISA with various concentrations of mouse CTGF.

FIG. 10 is a diagram showing the reactivity of rat CTGF with various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF. The vertical axis shows the fluorescence intensity as an index of antibody reactivity, and the horizontal axis shows the clone names of human monoclonal antibodies tested by ELISA with various concentrations of rat CTGF.

FIG. 11: Human kidney-derived fibroblast cell line 293-T and human CT
Human CT for adhesion to GF or mouse CTGF
The figure which shows the inhibitory activity of various monoclonal antibodies prepared by immunizing various mammals with GF or mouse CTGF.
The vertical axis shows the fluorescence intensity as an index of the inhibitory activity, and the horizontal axis shows the clone names of the various monoclonal antibodies tested.

FIG. 12 shows the inhibitory activity of various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF on the adhesion between rat kidney-derived fibroblast cell line NRK-49F and human CTGF. The vertical axis shows the cell binding rate (%), and the horizontal axis shows the clone names of the various monoclonal antibodies tested. The total number of cells added
100%.

FIG. 13 shows that human CTGF was applied to human antibody-producing transgenic mice for adhesion of human CTGF to each of rat kidney-derived fibroblast cell line NRK-49F, human osteosarcoma-derived cell line MG-63 or human lung-derived fibroblast cells. The figure which shows the inhibitory activity of various human monoclonal antibodies prepared by immunization.
The vertical axis shows the cell binding rate (%), and the horizontal axis shows the clone names of the various monoclonal antibodies tested. The total number of added cells was set to 100%.

FIG. 14 is a view showing the staining state of various types of monoclonal antibodies prepared by immunizing various mammals with human CTGF or mouse CTGF to show the reactivity of atherosclerosis model rabbit WHHL with atherosclerotic lesion tissue sections. . (A) shows the stained state in the control test, (b) shows the stained state in the test with the monoclonal antibody B35.1, and (c) shows the stained state in the test with the monoclonal antibody B29.6. (D) shows the staining state in the test with the monoclonal antibody 13-51-2, and (e) shows the staining state in the test with the monoclonal antibody A4.3. , (F) shows the staining state in the test with the monoclonal antibody C114.4, (g) shows the staining state in the test with the monoclonal antibody A11.1, and (h) shows the monoclonal state. Antibody A2
The staining condition in the test at 9.6 is shown, and the subdivision (i) shows the staining condition in the test with monoclonal antibody C26.11.

FIG. 15 shows human CTGF against cell proliferation of rat kidney-derived fibroblast cell line NRK-49F stimulated by purified human CTGF.
FIG. 4 shows the inhibitory activity of various human monoclonal antibodies prepared by immunizing a human antibody-producing transgenic mouse with. The vertical axis represents [ ^3] as an index of the level of cell growth promoting activity.
[H] shows the amount of thymidine taken up into cells, and the horizontal axis shows the clone name of a human monoclonal antibody tested using various concentrations of purified human CTGF.

FIG. 16 shows human CTGF against cell proliferation of rat kidney-derived fibroblast cell line NRK-49F stimulated by purified human CTGF.
FIG. 4 shows the inhibitory activity of various human monoclonal antibodies prepared by immunizing a human antibody-producing transgenic mouse with. The vertical axis represents [ ^3] as an index of the level of cell growth promoting activity.
[H] shows the amount of thymidine taken up into cells, and the horizontal axis shows the clone name of a human monoclonal antibody tested using various concentrations of purified human CTGF.

FIG. 17 is a view showing the therapeutic effects of various human monoclonal antibodies prepared by immunizing human antibody-producing transgenic mice with human CTGF on kidney disease and tissue fibrosis. The vertical axis shows the concentration of hydroxyproline as an indicator of the progress of disease symptoms, and the horizontal axis shows the clone name of the administered human monoclonal antibody. The term Sham group in the footnote refers to normal mice (only laparotomy group) and Ve
The term hicle group refers to the PBS administration group.

FIG. 18 is a diagram schematically showing a procedure for determining a DNA sequence encoding each of a heavy chain and a light chain of an anti-human CTGF human monoclonal antibody.

FIG. 19: Standard human CTG quantified by sandwich ELISA using monoclonal antibodies 8-64-6 and 8-86-2
F, Calibration curves of standard mouse CTGF and standard rat CTGF. The vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the standard CTG.
Shows the concentration of F.

FIG. 20: Standard human CTG quantified by sandwich ELISA using monoclonal antibodies 13-51-2 and 8-86-2
F, Calibration curves of standard mouse CTGF and standard rat CTGF. The vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the standard CTG.
Shows the concentration of F.

FIG. 21 shows CTGF concentrations in various serum samples of biliary atresia patients determined by sandwich ELISA using monoclonal antibodies 8-64-6 and 8-86-2. The vertical axis indicates the quantitative value (CTGF content), and the horizontal axis indicates the type of the tested sample group. The first group (I) was a specimen of a patient with normal clinical findings, the second group (II) was a specimen of a patient whose symptoms were progressing, and the third group (III) required a liver transplant. This is a sample of a severely ill patient.

FIG. 22 is a view showing the concentration of CTGF contained in serum samples of various patients quantified by sandwich ELISA using monoclonal antibodies 8-64-6 and 8-86-2. The vertical axis indicates the quantitative value (CTGF content), and the horizontal axis indicates the name of the disease affected by the patient from whom the tested sample was collected.

FIG. 23: C contained in synovial fluid samples of rheumatoid arthritis patients and osteoarthritis patients quantified by sandwich ELISA using monoclonal antibodies 8-64-6 and 8-86-2
The figure which shows a TGF density | concentration. The vertical axis indicates the quantitative value (CTGF content), and the horizontal axis indicates the name of the disease affected by the patient from whom the tested sample was collected.

FIG. 24 is a view showing the state of electrophoresis of human CTGF purified from human fetal skin-derived fibroblasts by SDS polyacrylamide gel by Western blotting test. Lanes 1, 2, and 3 show the test results when the eluted fractions with 0.2, 0.6, and 2.0 M NaCl, respectively, were used with a pre-mune antibody (normal rabbit serum before immunization with human CTGF). Nos. 4, 5 and 6 show the test results when the fractions eluted with 0.2, 0.6 and 2.0 M NaCl, respectively, were used with a rabbit anti-human CTGF polyclonal antibody.

FIG. 25 is a graph showing the activity of various concentrations of purified human CTGF to promote cell proliferation of rat kidney-derived fibroblast cell line NRK-49F. The vertical axis indicates the amount of [ ³ H] thymidine taken up into cells as an indicator of the level of cell growth promoting activity,
The horizontal axis shows the concentration (dilution factor) of purified human CTGF. N
C shows the result of a negative control (negative control) test when no purified CTGF was added. PDGF shows the results of a positive control test using PDGF (platelet-derived growth factor) instead of purified CTGF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 13/12 A61K 31/00 613G 17/00 617 19/02 619A 29/00 629A 35/00 635 A61K 39 / 395 39/395 N C07K 1/22 C07K 1/22 14/52 14/52 16/22 16/22 17/00 17/00 C12N 5/10 C12P 21/08 15/02 G01N 33/53 D C12P 21 / 08 33/543 501A G01N 33/53 33/545 33/543 501 33/577 B 33/545 C12N 5/00 B 33/577 15/00 C // (C12N 15/09 ZNA C12R 1:91) ( C12N 5/10 C12R 1:91) (C12P 21/08 C12R 1:91) (72) Inventor Shinji Sakamoto 1-13-2 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Japan Pharmaceutical Research Laboratory (72) Inventor Masaharu Takikawa Okayama, Okayama Prefecture Higashiyama 2-chome 17-22-304 F-term (reference) 4B024 AA01 AA11 BA43 BA44 GA03 HA01 HA04 HA15 4B064 AG27 CA10 CA20 CE13 DA01 DA13 4B065 AA90X AA91X AA91Y AA93Y AB04 BA08 CA25 CA44 CA46 4C085 AA14 BB17 CC CA40 DA76 EA28 EA50 FA72

Claims (82)

[Claims] 1. A monoclonal antibody or a part thereof having the following characteristics (a) to (g): (a) human, mouse and rat connective tissue growth factor (C
(B) reactive with both human and mouse CTGF, and not reactive with rat CTGF; (c) both mouse and rat CTGF And (d) a human kidney-derived fibroblast cell line 293-T (ATCC CRL-157).
Inhibits the binding of 3) to human CTGF or the binding of the cell line 293-T to mouse CTGF; (e) rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-
1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427)
Or any of human lung-derived fibroblasts and human CTG
Inhibiting binding to F; (f) inhibiting cell proliferation of rat kidney-derived fibroblast NRK-49F (ATCC CRL-1570) upon stimulation of human CTGF or mouse CTGF; or (g) hydroxproline Suppresses the elevation of the hydroxyproline in the kidney, whose production shows an upward trend. 2. The monoclonal antibody or a part thereof according to claim 1, wherein the monoclonal antibody has the property described in any one of the following (a) to (c): A monoclonal antibody or a part thereof obtained by immunizing a mouse with CTGF or a part thereof, and having reactivity with any of human, mouse and rat CTGF; (b) mouse CTGF or a part thereof A monoclonal antibody or a part thereof obtained by immunizing a hamster, which is reactive with any of human, mouse and rat CTGF; or (c) immunizing a rat with a mouse CTGF or a part thereof The obtained monoclonal antibody or a part thereof, which has reactivity with any of human, mouse and rat CTGF. 3. The monoclonal antibody or a part thereof according to claim 1, wherein the monoclonal antibody has the property described in any one of the following (a) to (c): A monoclonal antibody obtained by immunizing a mouse with CTGF or a part thereof, which is reactive with any of human, mouse and rat CTGF, and which is a human kidney-derived fibroblast cell line 293-T (ATCC CRL (B) a monoclonal antibody obtained by immunizing a rat with mouse CTGF or a part thereof and reacting with any of human, mouse and rat CTGF. Inhibits the binding between human kidney-derived fibroblast cell line 293-T (ATCC CRL-1573) and mouse CTGF; or (c) transforming mouse CTGF or a part thereof into hamster A monoclonal antibody obtained by immunization,
It is reactive with both mouse and rat CTGF and has a human kidney-derived fibroblast cell line 293-T (ATCC CRL-15).
73) inhibits binding to mouse CTGF 4. The method according to claim 1, wherein the monoclonal antibody is an international deposit number.
The monoclonal antibody or a part thereof according to claim 1, which is a monoclonal antibody produced from a fusion cell identified by FERM BP-6208. 5. The method according to claim 5, wherein the monoclonal antibody is an international deposit number.
The monoclonal antibody or a part thereof according to claim 1, which is a monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fusion cell identified by FERM BP-6208. 6. The method according to claim 6, wherein the monoclonal antibody is an international deposit number.
The monoclonal antibody or a part thereof according to claim 1, which is a monoclonal antibody produced from a fused cell identified by FERM BP-6209. 7. The method according to claim 7, wherein the monoclonal antibody is an international deposit number.
2. The monoclonal antibody or a part thereof according to claim 1, which is a monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fused cell identified by FERM BP-6209. 8. A human, mouse, or rat CTGF.
A human monoclonal antibody or a part thereof reactive with any of the above. 9. The method according to claim 9, wherein the human monoclonal antibody is a human CT.
9. The human monoclonal antibody or a part thereof according to claim 8, which is a monoclonal antibody reactive with GF. 10. A human monoclonal antibody reactive to human CTGF having any of the following characteristics (a) to (d) or a part thereof: (a) human kidney-derived fibroblast cell line 293 -T (ATCC CRL-157
3) inhibits the binding of human CTGF; (b) rat kidney-derived fibroblast cell line NRK-49F (ATCC CRL-
1570), human osteosarcoma-derived cell line MG-63 (ATCC CRL-1427)
Or any of human lung-derived fibroblasts and human CTG
Inhibiting binding to F; (c) inhibiting cell proliferation of rat kidney-derived fibroblast NRK-49F (ATCC CRL-1570) upon stimulation with human CTGF or mouse CTGF; or Suppresses the elevation of the hydroxyproline in the kidney, whose production shows an upward trend. 11. The human according to claim 8, wherein the human monoclonal antibody is a monoclonal antibody derived from a transgenic non-human mammal capable of producing a human antibody. Monoclonal antibodies or parts thereof. 12. The method according to claim 12, wherein the human monoclonal antibody is human C.
12. A monoclonal antibody obtained by immunizing a transgenic non-human mammal capable of producing a human antibody with TGF.
Or the human monoclonal antibody thereof. 13. The human monoclonal antibody or a part thereof according to claim 8, wherein the transgenic non-human mammal is a transgenic mouse. 14. The DNA of the V region encoding the variable region of the heavy chain of the human monoclonal antibody may be selected from the group consisting of DP-5, DP-38 and DP-
14. The human monoclonal antibody or a part thereof according to any one of claims 8 to 13, wherein the human monoclonal antibody is derived from any gene segment selected from the group consisting of 65 and DP-75. 15. The DNA of the V region encoding the variable region of the light chain of the human monoclonal antibody comprises DPK1, DPK9, and DPK1.
The human monoclonal antibody or a part thereof according to any one of claims 8 to 13, wherein the human monoclonal antibody is derived from any gene segment selected from the group consisting of 2 and DPK24. 16. The DNA of the V region encoding the variable region of the heavy chain of the human monoclonal antibody may be selected from the group consisting of DP-5, DP-38, and DP-38.
The V region DNA derived from any of the gene segments selected from the group consisting of 65 and DP-75 and encoding the light chain variable region of the human monoclonal antibody is DPK1, DPK
9. It is derived from any gene segment selected from the group consisting of K9, DPK12 and DPK24.
A human monoclonal antibody according to any one of claims 15 to 15, or a part thereof. 17. The human monoclonal antibody or the human monoclonal antibody thereof according to claim 9, wherein the variable region of the heavy chain of the human monoclonal has an amino acid having any one of the following amino acid sequences (a) to (j). Partially: (a) the amino acid sequence of amino acids 21 to 120 of the amino acid sequence described in SEQ ID NO: 6; (b) the amino acid sequence of amino acids 21 to 120 of the amino acid sequence described in SEQ ID NO: 6, An amino acid sequence in which one or several amino acids have been deleted, substituted, inserted or added; (c) an amino acid sequence of amino acids 21 to 118 of the amino acid sequence described in SEQ ID NO: 8; In the amino acid sequence of amino acids 21 to 118 of the described amino acid sequence, one or several amino acids are deleted, (E) an amino acid sequence of amino acids 21 to 116 of the amino acid sequence of SEQ ID NO: 10; (f) an amino acid sequence of amino acids 21 to 116 of the amino acid sequence of SEQ ID NO: 10 In the amino acid sequence at position 116, 1
Or an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added; (g) an amino acid sequence of amino acids 21 to 116 of the amino acid sequence described in SEQ ID NO: 12; In the amino acid sequence of amino acid Nos. 21 to 116 of the amino acid sequence to be
Or an amino acid sequence in which several amino acids have been deleted, substituted, inserted, or added; (i) an amino acid sequence of amino acids 21 to 117 of the amino acid sequence described in SEQ ID NO: 14; or (j) an amino acid sequence of SEQ ID NO: 14 In the amino acid sequence of amino acids 21 to 117 of the amino acid sequence described, 1
Or, an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added. 18. The human monoclonal antibody according to claim 9, wherein the human monoclonal light chain variable region has an amino acid comprising any one of the following amino acid sequences (a) to (j): Partially: (a) an amino acid sequence of amino acid numbers 21 to 120 of the amino acid sequence described in SEQ ID NO: 16; 1
Or an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added; (c) an amino acid sequence of amino acids 21 to 121 of the amino acid sequence described in SEQ ID NO: 18; (d) an amino acid sequence described in SEQ ID NO: 18 In the amino acid sequence of amino acids 21 to 121 of the amino acid sequence to be
Or an amino acid sequence in which several amino acids are deleted, substituted, inserted or added; (e) an amino acid sequence of amino acids 23 to 117 of the amino acid sequence described in SEQ ID NO: 20; (f) an amino acid sequence described in SEQ ID NO: 20 In the amino acid sequence of amino acids 23 to 117 of the amino acid sequence to be
Or an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added; (g) an amino acid sequence of amino acids 17 to 111 of the amino acid sequence described in SEQ ID NO: 22; (h) an amino acid sequence described in SEQ ID NO: 22 In the amino acid sequence of amino acids 17 to 111 of the amino acid sequence to be
Or an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added; (i) the amino acid sequence of amino acids 23 to 118 of the amino acid sequence described in SEQ ID NO: 24; or (j) the amino acid sequence of SEQ ID NO: 24 In the amino acid sequence of amino acids 23 to 118 of the amino acid sequence described, 1
Alternatively, an amino acid sequence in which several amino acids have been deleted, substituted, inserted or added. 19. A monoclonal antibody reactive with human CTGF, which is produced from a fused cell identified by International Accession No. FERM BP-6535, or a part thereof. 20. A monoclonal antibody reactive with human CTGF, the monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fused cell identified by International Accession No. FERM BP-6535, or Part of that. 21. A monoclonal antibody reactive with human CTGF, which is produced from a fusion cell identified by International Accession No. FERM BP-6598, or a part thereof. 22. A monoclonal antibody reactive with human CTGF, the monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fusion cell identified by International Accession No. FERM BP-6598, or Part of that. 23. A monoclonal antibody reactive with human CTGF, which is produced from a fused cell identified by International Accession No. FERM BP-6599, or a part thereof. 24. A monoclonal antibody reactive with human CTGF, the monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fusion cell identified by International Accession No. FERM BP-6599, or Part of that. 25. A monoclonal antibody reactive with human CTGF, which is produced from a fused cell identified by International Accession No. FERM BP-6600 or a part thereof. 26. A monoclonal antibody reactive with human CTGF, the monoclonal antibody having substantially the same properties as a monoclonal antibody produced from a fusion cell identified by International Accession No. FERM BP-6600, or Part of that. 27. The monoclonal antibody according to claim 17 or 18, which is a monoclonal antibody reactive with human CTGF or a part thereof, wherein said monoclonal antibody has reactivity with human CTGF. A monoclonal antibody or a part thereof, which has no reactivity with the antigen-antibody complex when reacted with an antigen-antibody complex comprising any one of the monoclonal antibodies and human CTGF. 28. The monoclonal antibody or a part thereof according to claim 27, wherein the monoclonal antibody is a human monoclonal antibody. 29. A monoclonal antibody reactive with rat CTGF or a part thereof. 30. The variable region is a variable region derived from the monoclonal antibody according to any one of claims 2 to 7, 27 or 29, and the constant region is a constant region derived from human immunoglobulin. A recombinant chimeric monoclonal antibody reactive with human CTGF. 31. A part or all of the complementarity determining region of the hypervariable region is a complementarity determining region derived from the monoclonal antibody according to any one of claims 2 to 7, 27 or 29. A recombinant human monoclonal antibody having reactivity to human CTGF, wherein the framework region of the hypervariable region is a framework region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin. . 32. A cell that produces the monoclonal antibody according to any one of claims 1 to 29. 33. A cell that produces the recombinant monoclonal antibody according to claim 30 or 31. 34. The cell according to claim 32, wherein the cell is a fused cell obtained by fusing a mammal-derived B cell with a mammalian-derived myeloma cell for the ability to produce the monoclonal antibody. A cell as described. 35. The cell, wherein the cell is a DNA encoding the heavy chain of the monoclonal antibody or D encoding the light chain thereof.
34. The cell according to claim 32 or 33, wherein the cell is a transgenic cell transformed by introducing one or both DNAs of NA into the cell. 36. The fusion cell as described in International Deposit No. FERM BP-
35. The fusion cell according to claim 34, which is a fusion cell identified by 6535. 37. The fusion cell according to International Accession No. FERM BP-
35. The fusion cell of claim 34, which is a fusion cell identified by 6598. 38. The fusion cell according to International Accession No. FERM BP-
35. The fusion cell of claim 34, wherein the fusion cell is identified as 6599. 39. The fusion cell according to International Accession No. FERM BP-
35. The fusion cell of claim 34, wherein the fusion cell is identified as 6600. 40. The fusion cell as described in International Deposit No. FERM BP-
35. The fusion cell of claim 34, wherein the fusion cell is identified as 6208. 41. The fusion cell according to International Accession No. FERM BP-
35. The fusion cell of claim 34, wherein the fusion cell is identified as 6209. 42. An 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 claim 42, wherein the insoluble carrier is an insoluble carrier selected from the group consisting of a plate, a test tube, a tube, a bead, a ball, a filter, and a membrane. 44. The antibody-immobilized insoluble carrier according to claim 42, wherein the insoluble carrier is a filter, a membrane, or an insoluble carrier used for affinity column chromatography. 45. A label obtained by labeling the monoclonal antibody according to any one of claims 1 to 31 alone or with a labeling substance capable of producing a detectable signal by reacting with another substance. antibody. 46. The labeled antibody according to claim 45, wherein the labeling substance is an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin, or a radioisotope. 47. The monoclonal antibody according to any one of claims 1 to 31, the insoluble carrier immobilized on the antibody according to claim 42 or 43, and the labeled antibody according to claim 45 or 46. A kit for detecting or quantifying a mammalian CTGF, comprising at least one monoclonal antibody selected from the group consisting of: an insoluble carrier immobilized on an antibody, or a labeled antibody. 48. The mammal 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. A kit used for detecting or quantifying CTGF in animals. 49. The monoclonal antibody according to any one of claims 1 to 31, the insoluble carrier immobilized on the antibody according to claim 42 or 43, and the labeled antibody according to claim 45 or 46. A method for detecting or quantifying a mammalian CTGF by an immunoassay, which comprises using at least one monoclonal antibody selected from the group consisting of: an insoluble carrier immobilized on an antibody, and a labeled antibody. 50. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following steps (a) and (b): (a) the method according to claim 42 or 43; Reacting the sample with the antibody-immobilized insoluble carrier; and (b) forming an antigen-antibody complex formed by the binding of the antibody-immobilized insoluble carrier to the mammalian CTGF contained in the sample. Or a step of reacting the labeled antibody according to claim 46. 51. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following steps (a) and (b): (a) the method according to claim 45 or 46; Reacting the sample with a labeled antibody; and (b) mammalian CTG contained in the sample with the labeled antibody.
A step of reacting the antibody-immobilized insoluble carrier according to claim 42 or 43 with an antigen-antibody complex formed by binding to F. 52. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following step (a): (a) the antibody immobilized insoluble according to claim 42 or 43; A step of reacting a mixture comprising a carrier, the labeled antibody according to claim 45 or 46, and a sample. 53. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following step (a): (a) the antibody immobilized insoluble according to claim 42 or 43; Reacting the carrier with a sample and a standard substance of mammalian CTGF labeled with a labeling substance capable of producing a detectable signal either alone or by reacting with another substance. 54. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following steps (a) and (b): (a) a sample, and alone or other substances; 32. A step of reacting the monoclonal antibody according to any one of claims 1 to 31 with a mixture of a mammalian CTGF standard substance labeled with a label substance capable of producing a detectable signal upon reaction. And (b) reacting with the monoclonal antibody in response to an antigen-antibody complex formed by binding the mammalian CTGF or the labeled standard substance of mammalian CTGF contained in the sample to the monoclonal antibody. Reacting an antiserum derived from a mammal having sex properties. 55. The method according to claim 49, wherein the CTGF of the mammal is detected or quantified by an immunoassay comprising at least the following steps (a) to (c): And (b) a labeling substance capable of producing a detectable signal by reacting the reaction system, which has been subjected to the step (a), alone or with another substance. CT of mammals labeled with
Reacting a GF standard substance; and (c) an antigen-antibody complex formed by binding of the mammalian CTGF or the labeled mammalian CTGF standard substance contained in the sample to the monoclonal antibody. A step of allowing the body to react with a mammal-derived antiserum reactive with the monoclonal antibody. 56. A mammalian CTGF comprising the antibody-immobilized insoluble carrier according to claim 42 or 44.
A kit used for the separation or purification of DNA. 57. A mammalian CTG using affinity chromatography using the antibody-immobilized insoluble carrier according to claim 42 or 44.
A method for separating or purifying F. 58. The affinity chromatography according to claim 5, wherein the affinity chromatography is an affinity column chromatography.
8. The method for purifying mammalian CTGF according to 7. 59. DNA encoding human CTGF
Is integrated into an endogenous locus. 60. A rat CTGF having the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence substantially identical to the amino acid sequence. 61. A DNA encoding rat CTGF having the amino acid sequence set forth in SEQ ID NO: 2. 62. The DNA according to claim 61, wherein the DNA comprises a base sequence from base numbers 213 to 1256 in the base sequence set forth in SEQ ID NO: 1. 63. A pharmaceutical composition comprising the monoclonal antibody according to any one of claims 2 to 31 or a part thereof, and a pharmaceutically acceptable carrier. 64. A pharmaceutical composition comprising the human monoclonal antibody or a part 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 according to any one of claims 14 to 18 or 28 or a part thereof. 66. The pharmaceutical composition according to any one of claims 63 to 65, wherein the pharmaceutical composition is used for suppressing the proliferation of cells capable of proliferating by stimulation of CTGF. object. 67. The pharmaceutical composition according to any one of claims 63 to 65, wherein the pharmaceutical composition is for treating or preventing a disease accompanied by proliferation of cells capable of proliferating by stimulation of CTGF. 68. The proliferation of the cells is brain, neck, lung, heart, liver, pancreas, kidney, stomach, large intestine, small intestine, duodenum, bone marrow, uterus, ovary, testis, prostate, skin, oral cavity, tongue, and 68. The pharmaceutical composition according to claim 66 or claim 67, wherein the composition is cell proliferation in a tissue selected from the group consisting of 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 a kidney. 71. The pharmaceutical composition according to claim 67, wherein the disease is further accompanied by tissue fibrosis. 72. The pharmaceutical composition according to claim 71, wherein the fibrosis of the tissue is fibrosis in lung, liver, kidney or skin. 73. The pharmaceutical composition according to claim 72, wherein the fibrosis of the tissue is fibrosis in the kidney. 74. A pharmaceutical composition for treating or preventing a disease in the kidney, comprising a CTGF inhibitor or a CTGF production inhibitor, and a pharmaceutically acceptable carrier. 75. The method according to claim 7, wherein the inhibitor is a monoclonal antibody reactive with CTGF.
5. The pharmaceutical composition according to 4. 76. The inhibitor according to any one of claims 9 to 31.
The pharmaceutical composition according to claim 74, which is the monoclonal antibody according to any one of the above. 77. The inhibitor according to any one of claims 14 to 1
A pharmaceutical composition according to claim 76, which is the human monoclonal antibody according to any of claims 8 or 28. 78. The pharmaceutical composition according to any one of claims 74 to 77, wherein the disease is a disease associated with tissue fibrosis. 79. A substance capable of suppressing the growth of cells capable of proliferating by stimulation of CTGF by the stimulation of CTGF, and a pharmaceutically acceptable carrier. Pharmaceutical composition for inhibiting. 80. The substance according to claim 79, wherein the substance is a monoclonal antibody reactive to CTGF.
A pharmaceutical composition according to claim 1. 81. The inhibitor according to claims 9 to 31.
80. The pharmaceutical composition according to claim 79, which is the monoclonal antibody according to any one of the above. 82. The inhibitor according to claim 14, wherein the inhibitor is
A pharmaceutical composition according to claim 81, which is the human monoclonal antibody according to any of claims 8 or 28.