U.S. patent application number 15/164615 was filed with the patent office on 2016-09-08 for connective tissue growth factor antibodies.
This patent application is currently assigned to FibroGen, Inc.. The applicant listed for this patent is FibroGen, Inc.. Invention is credited to Al Y. Lin, Thomas B. Neff, Noelynn A. Oliver, William R. Usinger, Qingjian Wang, David A. Yeowell.
Application Number | 20160257740 15/164615 |
Document ID | / |
Family ID | 33493433 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160257740 |
Kind Code |
A1 |
Lin; Al Y. ; et al. |
September 8, 2016 |
Connective Tissue Growth Factor Antibodies
Abstract
The present invention relates to antibodies that bind to CTGF.
The antibodies are particularly directed to regions of CTGF
involved in biological activities associated with fibrosis. The
invention also relates to methods of using the antibodies to treat
disorders associated with CTGF including localized and systemic
fibrotic disorders including those of the lung, liver, heart, skin,
and kidney.
Inventors: |
Lin; Al Y.; (Castro Valley,
CA) ; Neff; Thomas B.; (Atherton, CA) ;
Oliver; Noelynn A.; (Ridgefield, CT) ; Usinger;
William R.; (Lafayette, CA) ; Wang; Qingjian;
(Belmont, CA) ; Yeowell; David A.; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FibroGen, Inc. |
San Francisco |
CA |
US |
|
|
Assignee: |
FibroGen, Inc.
San Francisco
CA
|
Family ID: |
33493433 |
Appl. No.: |
15/164615 |
Filed: |
May 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14690727 |
Apr 20, 2015 |
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15164615 |
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13691356 |
Nov 30, 2012 |
9034643 |
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14690727 |
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12927243 |
Nov 10, 2010 |
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13691356 |
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12157262 |
Jun 9, 2008 |
7871617 |
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12927243 |
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10858186 |
Jun 1, 2004 |
7405274 |
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12157262 |
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60475598 |
Jun 4, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/02 20180101;
C07K 2317/21 20130101; A61P 9/00 20180101; C07K 2317/76 20130101;
C07K 2317/54 20130101; A61P 27/02 20180101; A61P 3/10 20180101;
C07K 16/22 20130101; A61P 1/16 20180101; A61P 9/12 20180101; A61P
13/12 20180101; A61P 19/04 20180101; A61P 35/04 20180101; C07K
2317/35 20130101; A61P 9/10 20180101; A61P 27/06 20180101; C07K
2317/55 20130101; C07K 2317/24 20130101; A61P 43/00 20180101; A61K
2039/505 20130101; A61P 17/00 20180101; C07K 2317/92 20130101; C07K
2317/41 20130101; A61P 35/02 20180101; A61P 29/00 20180101; A61P
35/00 20180101; A61P 11/00 20180101; C07K 2317/31 20130101; C07K
2317/622 20130101; A61P 27/00 20180101; A61P 9/04 20180101 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Claims
1-5. (canceled)
6. A method for treating a connective tissue growth factor
(CTGF)-associated disorder or condition comprising administering to
a subject in need thereof an effective amount of an antibody or
fragment thereof, wherein the antibody, or fragment binds to a
region of human connective tissue growth factor (CTGF) set forth as
amino acids 134 to 158 of SEQ ID NO:2, thereby treating the
CTGF-associated disorder or condition.
7. The method of claim 6, wherein the antibody is a monoclonal
antibody.
8. The method of claim 6, wherein the affinity of the antibody for
CTGF is at least about 10.sup.-9 M.
9. The method of claim 6, wherein the antibody is a single chain
antibody.
10. The method of claim 6, wherein the antibody is a humanized
antibody.
11. The method of claim 6, wherein the antibody is a human
antibody.
12. The method of claim 6, wherein the antibody is a chimeric
antibody.
13. The method of claim 6, wherein the antibody is a multivalent
antibody.
14. The method of claim 6, wherein the antibody is
glycosylated.
15. The method of claim 6, wherein the antibody is
non-glycosylated.
16. The method of claim 6, wherein the antibody fragment is
selected from the group consisting of a Fab, a F(ab).sub.2, and a
F.sub.v fragment.
17. The method of claim 6, wherein the CTGF-associated disorder or
condition is arthritis, atherosclerosis, retinopathy, nephropathy,
transplant rejection, hypertrophic scarring, Crohn's disease,
inflammatory bowel disease, keloids or macular degeneration.
18. The method of claim 17, wherein the transplant rejection is the
rejection of an organ selected from the group consisting of eye,
heart, kidney, liver, lung and skin.
19. The method of claim 6, wherein the CTGF-associated disorder is
cancer.
20. The method of claim 19, wherein the cancer is selected from the
group consisting of acute lymphoblastic leukemia, angioleimyoma,
angiolipoma, breast cancer, colorectal cancer, dermatofibromas,
desmoplastic cancers, fibrosarcoma, gastrointestinal cancer,
glioma, glioblastoma, liver cancer, ovarian cancer, pancreatic
cancer, prostate cancer and rhabdomyosarcoma.
21. The method of claim 6, wherein the CTGF-associated disorder or
condition is a fibrotic disorder.
22. The method of claim 21, wherein the fibrotic disorder is
selected from the group consisting of cardiac fibrosis,
interstitial fibrosis, liver fibrosis, kidney fibrosis, lung
fibrosis, ocular fibrosis, osteoarthritis, scleroderma, skin
fibrosis, systemic fibrosis, peridural fibrosis or peritoneal
fibrosis.
23. The method of claim 22, wherein the lung fibrosis is idiopathic
pulmonary fibrosis.
24. The method of claim 21, wherein the fibrotic disorder results
from surgery, chemotherapy or radiation therapy.
25. A pharmaceutical composition comprising an antibody, or
fragment thereof, that binds to a region of human connective tissue
growth factor (CTGF) set forth as amino acids 134 to 158 of SEQ ID
NO:2.
26. The pharmaceutical composition of claim 25, further comprising
a carrier or excipient.
27. A method of treating a CTGF-associated disorder or condition
selected from the group consisting of arthritis, atherosclerosis,
retinopathy, nephropathy, transplant rejection, hypertrophic
scarring, Crohn's disease, inflammatory bowel disease, keloids or
macular degeneration comprising administering the pharmaceutical
composition of claim 25.
28. A method of treating a CTGF-associated disorder or condition
selected from the group consisting of cancer and fibrotic disorders
comprising administering the pharmaceutical composition of claim
25.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 13/691,356, filed 30 Nov. 2012, which is a continuation of U.S.
application Ser. No. 12/927,243, filed 10 Nov. 2010, which is a
continuation of U.S. application Ser. No. 12/157,262, filed 9 Jun.
2008, which is a continuation of U.S. application Ser. No.
10/858,186, filed 1 Jun. 2004, which claims the benefit of U.S.
Provisional Application Ser. No. 60/475,598, filed on 4 Jun. 2003,
incorporated in their entireties by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to antibodies that bind to
connective tissue growth factor (CTGF). The antibodies are
particularly directed to regions of CTGF involved in biological
activities associated with various disorders.
BACKGROUND
Connective Tissue Growth Factor (CTGF)
[0003] CTGF is a 36 kD, cysteine-rich, heparin binding, secreted
glycoprotein originally isolated from the culture media of human
umbilical vein endothelial cells. (See e.g., Bradham et al. (1991)
J Cell Biol 114:1285-1294; Grotendorst and Bradham, U.S. Pat. No.
5,408,040.) CTGF belongs to the CCN (CTGF, Cyr61, Nov) family of
proteins (secreted glycoproteins), which includes the serum-induced
immediate early gene product Cyr61, the putative oncogene Nov, the
ECM-associated protein FISP-12, the src-inducible gene CEF-10, the
Wnt-inducible secreted protein WISP-3, and the anti-proliferative
protein HICP/rCOP (Brigstock (1999) Endocr Rev 20:189-206; O'Brian
et al. (1990) Mol Cell Biol 10:3569-3577; Joliot et al. (1992) Mol
Cell Biol 12:10-21; Ryseck et al. (1990) Cell Growth and Diff
2:225-233; Simmons et al. (1989) Proc Natl Acad Sci USA
86:1178-1182; Pennica et al. (1998) Proc Natl Acad Sci USA,
95:14717-14722; and Zhang et al. (1998) Mol Cell Biol
18:6131-6141.) CCN proteins are characterized by conservation of 38
cysteine residues that constitute over 10% of the total amino acid
content and give rise to a modular structure with N- and C-terminal
domains. The modular structure of CTGF includes conserved motifs
for insulin-like growth factor binding protein (IGF-BP) and von
Willebrand's factor (VWC) in the N-terminal domain, and
thrombospondin (TSP1) and a cysteine-knot motif in the C-terminal
domain.
[0004] CTGF expression is induced by members of the Transforming
Growth Factor beta (TGF.beta.) superfamily, which includes
TGF.beta.-1, -2, and -3, bone morphogenetic protein (BMP)-2, and
activin, as well as a variety of other regulatory modulators
including dexamethasone, thrombin, vascular endothelial growth
factor (VEGF), and angiotensin II; and environmental stimuli
including hyperglycemia and hypertension. (See, e.g., Franklin
(1997) Int J Biochem Cell Biol 29:79-89; Wunderlich (2000) Graefes
Arch Clin Exp Ophthalmol 238:910-915; Denton and Abraham (2001)
Curr Opin Rheumatol 13:505-511; and Riewald (2001) Blood
97:3109-3116; Riser et al. (2000) J Am Soc Nephrol 11:25-38; and
International Publication No. WO 00/13706). TGF.beta. stimulation
of CTGF expression is rapid and prolonged, and does not require
persistent application. (Igarashi et al. (1993) Mol Biol Cell 4:
637-645.) Enhanced expression of CTGF by TGF.beta. involves
transcriptional activation via DNA regulatory elements present in
the CTGF promoter. (Grotendorst et al. (1996) Cell Growth Differ 7:
469-480; Grotendorst and Bradham, U.S. Pat. No. 6,069,006; Holmes
et al. (2001) J Biol Chem 276:10594-10601.)
[0005] CTGF has been shown to increase steady-state transcription
of .alpha.1(I) collagen, .alpha.5 integrin, and fibronectin mRNAs,
as well as to promote cellular processes including proliferation
and chemotaxis of various cell types in culture. (See e.g., Frazier
et al. (1996) J Invest Dermatol 107:406-411; Shi-wen et al. (2000)
Exp Cell Res 259:213-224; Klagsburn (1977) Exp Cell Res 105:99-108;
Gupta et al. (2000) Kidney Int 58:1389-1399; Wahab et al. (2001)
Biochem J 359(Pt 1):77-87; Uzel et al. (2001) J Periodontol
72:921-931; and Riser and Cortes (2001) Ren Fail 23:459-470.)
Subcutaneous injection of CTGF in neonatal mice results in the
local deposition of granulation tissue. Similarly, subcutaneous
injection of TGF.beta. generates granulation tissue formation and
induces high levels of CTGF mRNA in local fibroblasts. Moreover,
combination or sequential treatment with TGF.beta. and CTGF results
in the development of a more persistent granuloma. (Mori et al.
(1999) J Cell Physiol 181:153-159.) Thus, CTGF appears to mediate a
subset of the effects elicited by TGF.beta., in particular, the
production and deposition of extracellular matrix (ECM). Further,
the ability to respond to CTGF, or the extent of the CTGF response,
may rely upon a priming stimulus provided by TGF.beta. treatment
that enables cellular "competence." (International Publication No.
WO 96/08140.)
[0006] Although a plethora of interacting factors have been
characterized that modulate tissue organization, a consensus is now
emerging for the role of CTGF in regulating skeletal development,
wound healing and extracellular matrix (ECM) remodeling, fibrosis,
tumorigenesis, and angiogenesis. For example, elevated CTGF
expression has been observed in cirrhotic liver, pulmonary
fibrosis, inflammatory bowel disease, sclerotic skin and keloids,
desmoplasia, and atherosclerotic plaques. (Abraham et al. (2000) J
Biol Chem 275:15220-15225; Dammeier et al. (1998) Int J Biochem
Cell Biol 30:909-922; diMola et al. (1999) Ann Surg 230(1):63-71;
Igarashi et al. (1996) J Invest Dermatol 106:729-733; Ito et al.
(1998) Kidney Int 53:853-861; Williams et al. (2000) J Hepatol
32:754-761; Clarkson et al. (1999) Curr Opin Nephrol Hypertens
8:543-548; Hinton et al. (2002) Eye 16:422-428; Gupta et al. (2000)
Kidney Int 58:1389-1399; Riser et al. (2000) J Am Soc Nephrol
11:25-38.)
[0007] CTGF is also upregulated in glomerulonephritis, IgA
nephropathy, focal and segmental glomerulosclerosis and diabetic
nephropathy. (See, e.g., Riser et al. (2000) J Am Soc Nephrol
11:25-38.) An increase in the number of cells expressing CTGF is
also observed at sites of chronic tubulointerstitial damage, and
CTGF levels correlate with the degree of damage. (Ito et al. (1998)
Kidney Int 53:853-861.) Further, CTGF expression is increased in
the glomeruli and tubulointerstium in a variety of renal diseases
in association with scarring and sclerosis of renal parenchyma.
Elevated levels of CTGF have also been associated with liver
fibrosis, myocardial infarction, and pulmonary fibrosis. For
example, in patients with idiopathic pulmonary fibrosis (IPF), CTGF
is strongly upregulated in biopsies and bronchoalveolar lavage
fluid cells. (Ujike et al. (2000) Biochem Biophys Res Commun
277:448-454; Abou-Shady et al. (2000) Liver 20:296-304; Williams et
al. (2000) J Hepatol 32:754-761; Ohnishi et al. (1998) J Mol Cell
Cardiol 30:2411-22; Lasky et al. (1998) Am J Physiol 275: L365-371;
Pan et al. (2001) Eur Respir J 17:1220-1227; and Allen et al.
(1999) Am J Respir Cell Mol Biol 21:693-700.) Thus, CTGF represents
a valid therapeutic target in disorders, such as those described
above.
[0008] The association of CTGF with various aspects of these
disorders has been established; and methods for treating disorders
through modulation of CTGF have been described. (See, e.g.,
Grotendorst and Bradham, U.S. Pat. No. 5,783,187; International
Publication No. WO 00/13706; and International Publication No. WO
03/049773.) Modulation of growth factors, cytokines, and cell
surface receptors can be accomplished using monoclonal antibodies,
and several therapeutic monoclonal antibodies have been approved or
are underdevelopment. (See, e.g., Infliximab (Remicade; Maini et
al. (1998) Arthritis Rheum 41:1552-1563; Targan et al. (1997) N
Engl J Med 337:1029-1035); Basiliximab (Simulect) and Daclizumab
(Zenapax) (Bumgardner et al. (2001) Transplantation 72:839-845;
Kovarik et al. (1999) Transplantation 68:1288-1294); and
Trastuzumab (Herceptin; Baselga (2001) Ann Oncol 12 Suppl
1:S49-55.))
[0009] Antibodies have been generated against CTGF, and have proven
efficacious in vivo at, e.g., inhibiting angiogenesis. (See, e.g.,
Grotendorst and Bradham, U.S. Pat. No. 5,408,040; International
Publication No. WO 99/07407; and Shimo et al. (2001) Oncology
61:315-322). Further, the modular nature of CTGF appears to
distinguish domains involved in specific biological activities. For
example, the N-terminal half of CTGF has been shown to stimulate
cell differentiation and ECM production, whereas the C-terminal
half stimulates cell proliferation. (See, e.g., International
Publication Nos. WO 00/35936 and WO 00/35939; and Brigstock and
Harding, U.S. Pat. No. 587,670.) This demonstrates that antibodies
directed to different regions of the CTGF molecule exhibit
different effects with respect to modulating biological activities
of CTGF. (See, e.g., International Publication Nos. WO 00/35936 and
WO 00/35939). Currently, no clear distinction has been made between
anti-CTGF antibodies that produce a desired effect, and those which
either produce multiple effects or are non-neutralizing. (See,
e.g., International Publication No. WO 99/33878.)
[0010] There is clearly a need in the art for agents that
effectively neutralize the activity of CTGF in disease. Antibodies,
particularly monoclonal antibodies, provide the specificity and
pharmacokinetic profiles appropriate for a therapeutic agent, and
neutralizing antibodies targeted to specific activities of CTGF
would fulfill a need in the art and would find use in therapeutic
treatment of CTGF-associated disorders including pulmonary
disorders such as idiopathic pulmonary fibrosis (IPF), etc.; renal
disorders such as diabetic nephropathy, glomerulosclerosis, etc.;
and ocular disorders such as retinopathy, macular degeneration,
etc.
SUMMARY OF THE INVENTION
[0011] The present invention provides antibodies, particularly
monoclonal antibodies, and portions thereof that specifically bind
to a region on the N-terminal fragment of a CTGF polypeptide.
[0012] In one aspect, an antibody of the invention specifically
binds to a region on human CTGF (SEQ ID NO:2) as set forth from
about amino acid 103 to amino acid 164 (SEQ ID NO:21), more
specifically from about amino acid 135 to about amino acid 157 (SEQ
ID NO:22); and even more specifically from about amino acid 142 to
about amino acid 154 (SEQ ID NO:25); or an orthologous region on
CTGF derived from another species. In particular embodiments, the
antibody has the same specificity as an antibody produced by the
cell line identified by ATCC Accession No. PTA-6006. (Deposited
with the ATCC on 20 May 2004.) In specific embodiments, the
antibody is substantially identical to mAb1, as described infra.
More preferably, the antibody is substantially similar to CLN1, as
described infra. In yet another embodiment, an antibody of the
invention competitively binds with any of the foregoing antibodies
to a CTGF polypeptide.
[0013] In one embodiment, the present invention provides a
monoclonal antibody or portion thereof comprising at least one
member of the group consisting of an immunoglobulin heavy chain
sequence comprising SEQ ID NO:14, an immunoglobulin heavy chain
sequence comprising the variable domain of SEQ ID NO:14, an
immunoglobulin light chain sequence comprising SEQ ID NO:20, an
immunoglobulin light chain sequence comprising the variable domain
of SEQ ID NO:20, or conservative variants thereof. In a specific
embodiment, the antibody comprises the immunoglobulin heavy chain
variable domain from amino acid residue 1 through amino acid
residue 167 of SEQ ID NO:14. In another specific embodiment, the
antibody comprises the immunoglobulin light chain variable domain
from amino acid residue 1 through amino acid residue 136 of SEQ ID
NO:20. In a particular embodiment, the antibody comprises the
immunoglobulin heavy chain sequence of SEQ ID NO:14 and the
immunoglobulin light chain sequence of SEQ ID NO:20. Within this
embodiment, the present invention specifically provides the
antibody of CLN1 or a portion thereof comprising at least the
antigen binding region residues of CLN1.
[0014] In certain aspects, the antibody of the invention is a
polyclonal antibody. In other aspects, the antibody is a monoclonal
antibody. In certain embodiments, the antibody is a humanized
monoclonal antibody; more preferably a human monoclonal antibody.
Any of the aforementioned antibodies may additionally contain
various amounts of glycosylation, incorporated by the cell
producing the antibody or applied and/or modified synthetically; or
the antibody may be free of glycosylation. The antibody may be
optionally pegylated and/or similarly modified to increase plasma
half-life, etc. In various embodiments, the invention provides
fragments of the antibody, particularly wherein the fragment is a
Fab, F(ab).sub.2, or Fv fragment.
[0015] In certain aspects, the antibody or portion thereof is
produced by a cloned cell line. The cell line may be derived from
any animal model used for monoclonal antibody production including,
but not limited to, mice, goat, chicken, etc. In particular, the
cell line may be derived from mice. The mice may be standard mice
used for antibody production, e.g., BALB/C, or a modified, e.g.,
transgenic, mouse strain optimized or developed for production of
specific isotype, idiotype, or species-specific monoclonal
antibodies. In one embodiment, the cell line is a hybridoma cell
line that produces and secretes mAb1. In other embodiments, the
cell line produces and secretes an antibody or portion thereof that
has a property substantially equivalent to mAb1. In still other
embodiments, the cell line produces and secretes an antibody or
portion thereof that has a property substantially equivalent to
CLN1. In a particular embodiment, the invention provides a cell
line identified by ATCC Accession No. PTA-6006. (Deposited 20 May
2004.)
[0016] In another aspect, the antibody or portion thereof is
derived from a non-human transgenic animal, particularly a
non-human transgenic mammal, capable of producing a human antibody.
The animal may be of any species including, but not limited to,
mouse, chicken, cow, goat, etc. In particular, the animal may be
mouse. Such antibodies may be obtainable by immunizing a non-human
transgenic mammal with a fragment of human CTGF, e.g., SEQ ID
NO:21, or, more specifically, SEQ ID NO:22, or to an orthologous
region on CTGF derived from a non-human species. In certain
embodiments, the antibodies are obtained by immunizing the
non-human transgenic mammal with a fragment of CTGF selected from
the group consisting of SEQ ID NOs:23 thru 26 or an orthologous
region on CTGF derived from a non-human species. In specific
embodiments, the antibodies are obtained by immunizing a transgenic
mouse with any of the aforementioned CTGF fragments. In other
embodiments, the antibodies are obtained by immunizing a transgenic
mouse with functional equivalents of any of the aforementioned CTGF
fragments.
[0017] By "specifically binds to a region on CTGF", it is meant
that the antibodies have binding specificity for a particular
region on CTGF, which may be defined by a linear amino acid
sequence, or by a tertiary, i.e., three-dimensional, conformation
on part of the CTGF polypeptide. Binding specificity means that the
antibodies affinity for the portion of CTGF is substantially
greater than their affinity for other related polypeptides. By
"substantially greater affinity" we mean that there is a measurable
increase in the affinity for the portion of CTGF as compared with
the affinity for other related polypeptides. Preferably, the
affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold,
10.sup.3-fold, 10.sup.4-fold, 10.sup.5-fold, 10.sup.6-fold or
greater for the particular portion of CTGF than for other proteins.
Preferably, the binding specificity is determined by affinity
chromatography, immunoprecipitation or by an in vitro binding
assay, such as radioimmunoassay (RIA) or enzyme-linked
immunoabsorbent assay (ELISA), or by fluorescence-activated cell
sorting (FACS) analysis. More preferably, the binding specificity
is obtained by RIA or affinity chromatography, as described
infra.
[0018] In preferred embodiments of the invention, the antibodies
have an affinity that is equal to or greater than that of mAb1,
described infra, as determined, for example, by the Scatchard
analysis of Munson and Pollard (1980, Anal Biochem 107:220).
Antibody affinity is defined as the strength of the total
noncovalent interactions between a single antigen-binding site on
an antibody and a single epitope on an antigen. Affinity is
calculated by measuring the association constant (KO, such that
Affinity = K a = [ Ab Ag ] [ Ab ] [ Ag ] = 1 K d ##EQU00001##
where [Ab] is the concentration of free antigen binding site on the
antibody, [Ag] is the concentration of free antigen, [AbAg] is the
concentration of antigen binding site on the antibody occupied by
antigen, and K.sub.d is the dissociation constant of the
antibody-antigen complex. Preferably, antibodies of the invention
have an affinity for CTGF that is greater than Kd=10.sup.-8,
preferably greater than 10.sup.-9, preferably greater than
10.sup.-10, particularly for therapeutic use. Advantageously, an
antibody according to the invention has an affinity similar to or
greater than that of mAb1 (that is, a Kd.ltoreq.10.sup.-9).
However, antibodies sharing epitope binding with mAb1, but having
lower affinity (i.e., higher Kd) than mAb1, are also embodied
within the present invention and are potentially useful in various
assays and diagnostic applications as described herein. Such
antibodies may additionally be useful in therapeutic applications,
especially if they have a high avidity for antigen, as described
below.
[0019] Antibodies according to the invention may be monovalent,
bivalent or they may be multi-valent. In certain embodiments of the
invention, it is preferred that the antibodies of the invention are
bivalent or multivalent. Any of the antibodies of the invention may
be manipulated to improve avidity, e.g., by combining
epitope-binding sites into a single antibody construct, e.g., a
tribody, etc. Antibodies according to the invention may be single
chain antibodies.
[0020] It may be useful in some circumstances for antibodies of the
invention to show suitable affinity for CTGF from other species,
for example, for treatment and prevention of disorders in those
species. For example, an antibody of the invention that shows a
suitable K.sub.d for canine CTGF could be used to treat a
CTGF-associated disorder in dogs. Antibodies of the invention that
show cross-species affinity, such as mAb1, are also useful as
research tools, to study CTGF-associated disorders in various
animal models. In another aspect, the antibody or portion thereof
is encoded by genetic material originally derived from a human. The
antibody may be generated by cells in culture, e.g., using phage
display techniques, or may be produced within an animal, e.g., a
non-human transgenic animal containing immunoglobulin genes derived
from a human.
[0021] Additionally, the invention provides recombinant constructs
comprising portions of any of the antibodies of the invention, as
described above, and a protein derived from another source.
Specifically contemplated are embodiments encompassing chimeric
antibodies comprising a variable region derived from a monoclonal
antibody that specifically binds to a region on an N-terminal
fragment of CTGF and a constant region derived from another source.
The variable region can be derived from any antibody defined by the
invention, and specifically encompasses antibodies that bind to a
region on human CTGF from about amino acid 97 to about amino acid
180 of SEQ ID NO:2, or, more specifically, from about amino acid
103 to about amino acid 164 of SEQ ID NO:2, or, more specifically,
from about amino acid 134 to about amino acid 158 of SEQ ID NO:2,
or, even more specifically, from about amino acid 143 to about
amino acid 154 of SEQ ID NO:2, or to an orthologous region on CTGF
derived from another species. The constant region can be derived
from any source. In some embodiments, the constant region is
derived from a constant region of a human immunoglobulin.
[0022] The present invention also provides any of the antibodies
described above wherein the antibody additionally comprises a
labeling agent capable of providing a detectable signal by itself
or together with other substances. Such labeling agents can be
selected from, but are not limited to, the group consisting of an
enzyme, fluorescent substance, chemiluminescent substance, biotin,
avidin, and radioisotope. The present invention also provides any
of the antibodies described above wherein the antibody additionally
comprises a cytotoxic agent or enzyme.
[0023] In other embodiments, the antibodies of the invention, as
described above, additionally neutralize at least one activity
associated with CTGF. Such activities associated with CTGF include,
but are not limited to, stimulation of cell migration, production
of extracellular matrix by a cell in vivo or ex vivo, and/or
reduction in fibrosis in a subject. In particular embodiments, the
biological activity is selected from the group consisting of cell
growth, differentiation of fibroblasts and/or endothelial cells,
and induction of expression of proteins involved in extracellular
matrix formation and remodeling including, e.g., collagens
including, but not limited to, types I, II, III, and IV; and
fibronectin.
[0024] In certain embodiments, the antibodies specifically inhibit
cell migration in ex vivo assays. Preferably, the antibodies
inhibit CTGF-stimulated chemotactic migration of smooth muscle
cells in a Boyden chamber assay. For example, in a cell migration
assay described infra, antibodies of the invention repeatedly and
reproducibly inhibit CTGF-induced migration. In various
embodiments, the antibodies specifically reduce fibrosis in animal
models. Preferably, the antibodies inhibit development of fibrosis
in animal models of lung and kidney fibrosis. For example, the
antibodies attenuate bleomycin-induced lung fibrosis in mice by
60-70%, as determined by inhibition of pulmonary hydroxyproline
(collagen) accumulation and/or histological examination of tissue
preparations, described infra. Further, the antibodies reduce the
accumulation of collagen in a rat remnant kidney (i.e.,
nephrectomy) model, and in mice following unilateral ureter
obstruction (UUO), as described infra.
[0025] In other embodiments, antibodies of the invention modulate
the interaction between a CTGF polypeptide and a cell receptor,
and/or between a CTGF polypeptide and a secreted or
membrane-associated cofactor, thereby neutralizing a biological
activity of CTGF. The cofactor may be any protein, carbohydrate,
and/or lipid; in particular embodiments, the cofactor is a member
of the TGF-.beta. family of growth factors, e.g., TGF-.beta.,
BMP-4, etc.
[0026] In another aspect, the antibody reduces fibrosis in a
subject. In various embodiments, the subject is a tissue or organ.
In other embodiments, the subject is an animal, preferably a
mammal, most preferably a human. When the subject is a tissue, the
invention specifically contemplates both endogenous tissues and ex
vivo tissues, e.g., transplant tissues, tissues grown in culture,
etc. In various embodiments, the tissue is selected from the group
consisting of epithelial, endothelial, and connective tissue. When
the subject is an organ, the invention specifically contemplates
organs selected from the group consisting of kidney, lung, liver,
eye, heart, and skin. In preferred embodiments, the subject is an
animal, particularly, an animal of mammalian species including rat,
rabbit, bovine, ovine, porcine, murine, equine, and primate
species. In a most preferred embodiment, the subject is human.
[0027] In specific embodiments, the antibody is used to treat or
prevent a CTGF-associated disorder in a subject having or at risk
for having a CTGF-associated disorder. Such disorders include, but
are not limited to, various cancers including acute lymphoblastic
leukemia, dermatofibromas, breast cancer, breast carcinoma, glioma
and glioblastoma, rhabdomyosarcoma and fibrosarcoma, desmoplasia,
angiolipoma, angioleiomyoma, desmoplastic cancers, and prostate,
ovarian, colorectal, pancreatic, gastrointestinal, and liver cancer
and other tumor growth and metastases. CTGF-associated disorders
also include various fibrotic disorders including, but not limited
to, idiopathic pulmonary fibrosis, kidney fibrosis,
glomerulosclerosis, ocular fibrosis, osteoarthritis, scleroderma,
cardiac fibrosis, and liver fibrosis. Fibrosis can occur in any
organ or tissue including an organ selected from, but not limited
to, kidney, lung, liver, heart, and skin; or a tissue selected
from, but not limited to, epithelial, endothelial, and connective
tissue. In other embodiments, the CTGF-associated disorder may be
caused by any initiating factor including, but not limited to,
exposure to chemicals or biological agents, inflammatory response,
autoimmune reaction, trauma, surgical procedures, etc.
CTGF-associated disorders also include, but are not limited to,
disorders due to hyperglycemia and hypertension. Such disorders may
occur, e.g., due to diabetes, obesity, etc., and include diabetic
nephropathy, retinopathy, and cardiovascular disease.
[0028] Therefore, in various embodiments, the invention provides
antibodies that can be used to treat or prevent CTGF-associated
disorders in a subject. The present invention also provides the use
of such antibodies in the manufacture of a medicament for the
treatment of CTGF-associated disorders.
[0029] In another aspect, the invention provides a method of
neutralizing an activity associated with CTGF comprising contacting
an antibody of the invention and a CTGF polypeptide, thereby
neutralizing a biological activity of CTGF, such as those described
above. The biological activity can be any activity of CTGF
including, but not limited to, stimulation of cell migration and
production of extracellular matrix. In various embodiments, the
neutralizing occurs in vitro. In other embodiments, the
neutralizing occurs in a subject in vivo.
[0030] In yet another aspect, the invention provides methods of
using an antibody as described above to treat a CTGF-associated
disorder in a patient in need, the method comprising administering
the antibody or a pharmaceutical formulation thereof to the
patient, thereby treating the disorder. The subject can be a
patient diagnosed with or suspected of having a CTGF-associated
disorder, including, e.g., a disorder resulting in excess
production of extracellular matrix. In particular aspects,
CTGF-associated disorder is selected from a cancer or fibrotic
disorder. Cancers include, but are not limited to, acute
lymphoblastic leukemia, dermatofibromas, breast cancer, breast
carcinoma, glioma and glioblastoma, rhabdomyosarcoma and
fibrosarcoma, desmoplasia, angiolipoma, angioleiomyoma,
desmoplastic cancers, and prostate, ovarian, colorectal,
pancreatic, gastrointestinal, and liver cancer, and fibrotic
disorders include, but are not limited to, idiopathic pulmonary
fibrosis, kidney fibrosis, glomerulosclerosis, ocular fibrosis,
macular degeneration, osteoarthritis, scleroderma, chronic heart
failure, cardiac fibrosis, and liver fibrosis. In other
embodiments, the CTGF-associated disorder may be caused by any
initiating factor including, but not limited to, exposure to
chemicals or biological agents, inflammatory response, autoimmune
reaction, trauma, surgical procedures, etc. CTGF-associated
disorders also include, but are not limited to, disorders due to
hyperglycemia and hypertension. Such disorders may occur, e.g., due
to diabetes, obesity, etc., and include diabetic nephropathy,
retinopathy, and cardiovascular disease.
[0031] In another aspect, the present invention provides a
composition comprising an antibody as described above and at least
one other component. Components may include any compound, molecule,
or agent, including, e.g., proteins, nucleic acids, carbohydrates,
lipids, etc. Additionally, components may include various solvents,
salts, and other carriers and/or excipients. In some embodiments,
the composition is a pharmaceutical composition comprising an
antibody as described above and at least one additional component
selected from a solvent, a stabilizer, or an excipient. In a
particular embodiment, the pharmaceutical composition comprises an
antibody in admixture with a pharmaceutically acceptable carrier.
The pharmaceutical composition may additionally contain a second
therapeutic agent, e.g., an angiotensin converting enzyme (ACE)
inhibitor, an advanced glycation endproduct cleavage or inhibitory
agent, etc. The invention additionally provides medicaments
comprising an antibody as defined above for treating a subject
having a CTGF-associated disorder. Such disorders include, but are
not limited to, various cancers and fibrotic disorders; disorders
resulting from conditions such as myocardial infarction, arthritis,
and inflammation; and disorders due to diabetes, obesity, and the
like, which may include diabetic nephropathy, retinopathy, and
cardiovascular disease.
[0032] In another embodiment, the invention provides a polypeptide
sequence selected from the group consisting of SEQ ID NO:14, amino
acid 1 through amino acid 167 of SEQ ID NO:14, SEQ ID NO:20, and
amino acid 1 through amino acid 136 of SEQ ID NO:20. The invention
also encompasses conservative variants of the polypeptides. In
another embodiment, the invention provides specific fragments of
human CTGF selected from the group consisting of SEQ ID NOs:21
through 26, and orthologous CTGF fragments obtained from a
non-human species.
[0033] The polypeptides referred to above may be "altered"
polypeptides, as defined infra.
[0034] In another embodiment, the invention provides a
polynucleotide sequence encoding an antibody of the invention or a
portion thereof. In particular embodiments, the polynucleotide
sequence is selected from the group consisting of a polynucleotide
sequence encoding SEQ ID NO:14, a polynucleotide sequence encoding
from amino acid 1 through amino acid 167 of SEQ ID NO:14, the
polynucleotide sequence of SEQ ID NO:13, and a polynucleotide
comprising nucleotide 1 through nucleotide 501 of SEQ ID NO:13. In
other embodiments, the polynucleotide sequence is selected from the
group consisting of a polynucleotide sequence encoding SEQ ID
NO:20, a polynucleotide sequence encoding from amino acid 1 through
amino acid 136 of SEQ ID NO:20, the polynucleotide of SEQ ID NO:19,
and a polynucleotide comprising nucleotide 1 through nucleotide 408
of SEQ ID NO:19.
[0035] The polynucleotides referred to above may be "altered"
polynucleotides, as defined infra.
[0036] The invention additionally provides recombinant
polynucleotides comprising any of the polynucleotide sequences
described above operably linked to a vector sequence that contains
replication and transcriptional control sequences. In one aspect,
the recombinant polynucleotide encodes the amino acid sequence of
SEQ ID NO:14 or the variable domain therein. In another aspect, the
recombinant polynucleotide comprises SEQ ID NO:13. In yet another
aspect, the recombinant polynucleotide encodes the amino acid
sequence of SEQ ID NO:20 or the variable domain therein. In still
another aspect, the recombinant polynucleotide comprises SEQ ID
NO:19.
[0037] The invention also provides host cells transfected with at
least one of the recombinant polynucleotides described above. Host
cells include any prokaryotic and eukaryotic host cell, including,
e.g., cloned cell lines maintained by culture methods known to
those of skill in the art. Host cells also include transgenic
plants and animals derived from transformed cells, e.g., stem
cells. In one embodiment, the host cell comprises a cell
co-transfected with a polynucleotide encoding SEQ ID NO:14 and a
polynucleotide encoding SEQ ID NO:20, and which produces a
functional antibody with characteristics substantially the same as
mAb1. In particular embodiments, the antibody is CLN1. In another
particular embodiment, the host cell is identified by ATCC
Accession No. PTA-6006. (Deposited: 20 May 2004.)
[0038] These and other embodiments of the subject invention will
readily occur to those of skill in the art in light of the
disclosure herein, and all such embodiments are specifically
contemplated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIGS. 1A and 1B show the structure and sequence conservation
of Connective Tissue Growth Factor. FIG. 1A shows the modular
domain structure of CTGF, which includes conserved motifs for
insulin-like growth factor binding protein (IGF-BP) and Von
Willebrand's factor (VWC) in the N-terminal fragment, and
thrombospondin (TSP1) and the cysteine-knot motif (CT) in the
C-terminal fragment. FIG. 1B shows a multiple sequence alignment
between N-terminal fragments of human (hCTGF; SEQ ID NO:27), bovine
(bCTGF; SEQ ID NO:29), porcine (pCTGF; SEQ ID NO:30), rat (rCTGF;
SEQ ID NO:31), and murine (FISP12; SEQ ID NO:32) CTGF orthologs.
The alignment was created using the CLUSTAL W program (v. 1.74;
Thompson et al. (1994) Nucleic Acids Res 22:4673-4680) using
default parameters. In the figure, an asterisk (*) indicates
complete conservation of the amino acid residue among the species
represented.
[0040] FIGS. 2A and 2B show Scatchard plots of competitive binding
between labeled and unlabeled human CTGF to anti-CTGF antibodies,
mAb2 and mAb1, respectively. mAb1 is an exemplary antibody of the
present invention.
[0041] FIG. 3A shows Fab antibody fragment (M, 45 kD) obtained
following papain digestion of the corresponding IgG antibody mAb1
and subsequent protein A-Sepharose affinity chromatography (Lane
2), as demonstrated by SDS-PAGE. FIG. 3B shows binding of a Fab
fragment and corresponding IgG to CTGF over increasing
concentration of chaotropic agent (thiocyanate).
[0042] FIGS. 4A and 4B show Scatchard plots of competitive binding
between labeled recombinant human CTGF and unlabeled rat CTGF to
anti-CTGF antibodies, mAb2 and mAb1, respectively.
[0043] FIGS. 5A, 5B, and 5C show the therapeutic benefits of an
antibody of the invention in a model of interstitial fibrosis in
the lung. FIG. 5A shows the effect of antibody treatment on
bleomycin-induced increase in hydroxyproline content of mouse
lungs. The number of animals in each group is shown in parentheses
below each bar and treatment groups are indicated along the x-axis.
SA: Saline; BL: Bleomycin; AbsJ: pool of 3 monoclonal antibodies of
the invention; mAb1, an exemplary antibody of the invention. Values
are expressed as mean.+-.SE. FIGS. 5B and 5C show hematoxylin- and
eosin-stained paraffin sections of pulmonary proximal acini from
mice exposed to bleomycin by intratracheal injection and
subsequently treated with saline or antibodies of the invention,
respectively. In FIG. 5B, the thin interalveolar septa acinus have
an abnormal appearance, and inflammatory cells and fibrosis are
present. In FIG. 5C, the parenchyma is largely normal and there is
only moderate thickening of interalveolar septa.
[0044] FIGS. 6A, 6B, and 6C show the therapeutic benefits of an
antibody of the invention in a model of tubulointerstitial fibrosis
in the kidney. FIG. 6A shows the reduction in fibrosis due to
unilateral ureter obstruction (UUO) following treatment with an
antibody of the invention, mAb1, or an antibody directed to the
C-terminus of CTGF, mAb3. The extent of fibrosis is expressed as
the ratio of hydroxyproline to proline in the obstructed kidney
compared with the contralateral unobstructed kidney (mean.+-.SE).
FIGS. 6B and 6C show trichome-stained paraffin sections of
obstructed kidneys receiving saline or antibody therapy,
respectively.
[0045] FIGS. 7A and 7B show the therapeutic benefit of an antibody
of the invention in a model of glomerular fibrosis in the kidney.
FIGS. 7A and 7B show photomicrographs of trichrome-stained remnant
kidney tissue after receiving saline or antibody therapy,
respectively.
[0046] FIGS. 8A, 8B, 8C, 8D, 8E, 8F, and 8G show the induction of
localized subcutaneous granulomas in newborn mice. On the left,
FIGS. 8A and 8B show the formation of granuloma at the site of
subcutaneous injection of TGF.beta. alone or TGF.beta. and CTGF,
respectively. On the right, FIGS. 8C through 8G show a histological
panel representing the scoring system (from 0 [normal] to 4
[fibrotic]) used to evaluate therapeutic benefit of antibody.
[0047] FIG. 9 shows degree of fibrosis in a localized subcutaneous
granuloma model with and without treatment with anti-CTGF
antibodies. mAb1 is an exemplary antibody of the invention, whereas
mAb3 is an anti-CTGF antibody that specifically binds to a
C-terminal CTGF epitope.
[0048] FIGS. 10A, 10B, 10C, and 10D show the therapeutic benefit of
an antibody of the invention in organ fibrosis using a model of
systemic sclerosis. Each of the panels shows changes in collagen
accumulation in respective organs following treatment with saline
(control), TGF.beta. and CTGF, or TGF.beta. and CTGF treatment
concomitant with antibody therapy.
[0049] FIGS. 11A and 11B show a diagramatic representation of the
cloning of heavy and light immunoglobulin chains of an exemplary
antibody of the invention, mAb1. FIG. 11A shows the alignment of
heavy chain PCR fragments used to determine the mAb1 heavy chain
coding sequence (CDS). FIG. 11B shows the alignment of light chain
PCR fragments used to determine the mAb1 light chain coding
sequence (CDS).
[0050] FIGS. 12A and 12B shows binding studies between CTGF and
TGF.beta.. FIG. 12A shows the degree of binding between TGF.beta.
and either CTGF, a fragment of CTGF encoded by exon 3 (Exon 3), or
a fragment of CTGF encoded by exon 5 (Exon 5) in the presence or
absence of anti-CTGF antibody. FIG. 12B shows the degree to which
anti-CTGF antibodies inhibit TGF.beta. and CTGF interaction. In the
figure, antibodies include exemplary antibodies of the invention,
mAb1 and mAb4, and an antibody that specifically binds to a
C-terminal CTGF epitope, mAb3.
DESCRIPTION OF THE INVENTION
[0051] Before the present compositions and methods are described,
it is to be understood that the invention is not limited to the
particular methodologies, protocols, cell lines, assays, and
reagents described, as these may vary. It is also to be understood
that the terminology used herein is intended to describe particular
embodiments of the present invention, and is in no way intended to
limit the scope of the present invention as set forth in the
appended claims.
[0052] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
references unless context clearly dictates otherwise. Thus, for
example, a reference to "a fragment" includes a plurality of such
fragments, a reference to an "antibody" is a reference to one or
more antibodies and to equivalents thereof known to those skilled
in the art, and so forth.
[0053] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications cited herein are incorporated herein by
reference in their entirety for the purpose of describing and
disclosing the methodologies, reagents, and tools reported in the
publications, which might be used in connection with the invention.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of
prior invention.
[0054] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, cell biology, genetics, immunology
and pharmacology, within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Gennaro, A. R., ed.
(1990) Remington's Pharmaceutical Sciences, 18th ed., Mack
Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology,
Academic Press, Inc.; Handbook of Experimental Immunology, Vols.
I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell
Scientific Publications); Maniatis, T. et al., eds. (1989)
Molecular Cloning: A Laboratory Manual, 2nd edition, Vols. I-III,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds.
(1999) Short Protocols in Molecular Biology, 4th edition, John
Wiley & Sons; Ream et al., eds. (1998) Molecular Biology
Techniques: An Intensive Laboratory Course, Academic Press); PCR
(Introduction to Biotechniques Series), 2nd ed. (Newton &
Graham eds., 1997, Springer Verlag).
DEFINITIONS
[0055] "Connective tissue growth factor" or "CTGF" refers to the
amino acid sequences of substantially purified CTGF derived from
any species, particularly a mammalian species, including rat,
rabbit, bovine, ovine, porcine, murine, equine, and hominid,
preferably the human species, and from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0056] The term "N-terminal fragment" of CTGF refers to any
polypeptide comprising sequences derived from the amino-terminal
portion of a CTGF polypeptide, or to any variants, or fragments
thereof. N-terminal fragments can include all, none, or portions of
CTGF from the initial methionine residue through the cysteine-free
"hinge" region as shown in FIGS. 1A and 1B. Further, N-terminal
fragments can include all, none, or portions of the insulin growth
factor-binding protein motif and/or the von Willebrand type C
domain (SEQ ID NO:21) as shown in FIG. 1B. N-terminal fragments of
CTGF can also include all, none, or portions of the cysteine-free
region. Further, N-terminal fragments of CTGF can be any fifteen or
more contiguous amino acids contained within any preceding
N-terminal fragment defined above.
[0057] In one aspect, "N-terminal fragment" of CTGF refers to
polypeptide sequences derived from the amino-terminal portion of
human CTGF. Such fragments can encompass the entire region from
amino acid residue 1 to about amino acid residue 198 of SEQ ID
NO:2, or from about amino acid 23 to about amino acid 198 of SEQ ID
NO:2. The boundary of the N-terminal fragment within the hinge
region may be optionally defined by one of several protease
cleavage sites defined in SEQ ID NO:2, such as chymotrypsin
cleavage sites between residues 179 and 180, between residues 182
and 183, and between residues 188 and 189; plasmin cleavage sites
between residues 183 and 184, and between residues 196 and 197; and
a bone morphogenetic protein-1 cleavage site between residues 169
and 170. Additionally, N-terminal fragments of human CTGF can
include all, none, or portions of the region from amino acid 27 to
amino acid 97 of SEQ ID NO:2, amino acid 103 to amino acid 166 of
SEQ ID NO:2, or amino acid 167 to amino acid 198 of SEQ ID NO:2.
Further, N-terminal fragments of human CTGF can be any fifteen or
more contiguous amino acids contained within any preceding
N-terminal fragment defined above.
[0058] In specific embodiments, the CTGF N-terminal fragments of
the present invention comprise sequences selected from the
following regions of human CTGF (SEQ ID NO:2) and orthologous
fragments thereof derived from a different species, particularly a
mammalian species including rat, rabbit, bovine, ovine, porcine,
murine, and equine: amino acid residue 23 to amino acid residue 96
(encoded by exon 2); amino acid residue 27 to amino acid residue 97
(IGF-BP motif); amino acid residue 97 to amino acid residue 180
(encoded by exon 3); amino acid residue 103 to amino acid residue
166 (VWC domain); amino acid residue 167 to amino acid residue 198
(cysteine-free hinge); amino acid residue 23 to amino acid residue
180 (encoded by exons 2 and 3); amino acid residue 27 to amino acid
residue 166 (IGF-BP and VWC); and amino acid residue 23 to amino
acid residue 198. (See FIG. 1B.)
[0059] The term "C-terminal fragment" of CTGF refers to any
polypeptide comprising sequences derived from the carboxy-terminal
portion of a CTGF amino acid polypeptide sequence, or to any
variants, or fragments thereof. C-terminal fragments of CTGF can
include all, none, or portions of the cysteine-free region of CTGF
polypeptide (amino acid 167 to amino acid 198 of SEQ ID NO:2).
[0060] The C-terminal fragments can include all, none, or portions
of CTGF from the cysteine-free hinge region to the end of the
protein. Further, C-terminal fragments can include all, none, or
portions of the thrombospondin motif and/or the cysteine-knot
motif. Further, C-terminal fragments of CTGF can be any fifteen or
more contiguous amino acids contained within any preceding
C-terminal fragment defined above.
[0061] In some aspects, C-terminal fragments can encompass the
entire region from amino acid residue 181 to about amino acid
residue 349 of SEQ ID NO:2. The boundary of the C-terminal fragment
within the hinge region may be optionally defined by one of several
protease cleavage sites defined in SEQ ID NO:2, such as
chymotrypsin, plasmin, and bone morphogenetic protein-1 cleavage
sites defined above. Additionally, C-terminal fragments comprise
sequences selected from the following regions of human CTGF (SEQ ID
NO:2) and orthologous fragments thereof derived from a different
species, particularly a mammalian species including rat, rabbit,
bovine, ovine, porcine, murine, and equine: amino acid 201 to amino
acid 242 of SEQ ID NO:2, amino acid 247 to amino acid 349 of SEQ ID
NO:2, amino acid 248 to amino acid 349 of SEQ ID NO:2, or amino
acid 249 to amino acid 346 of SEQ ID NO:2. Further, C-terminal
fragments of human CTGF can be any fifteen or more contiguous amino
acids contained within any preceding C-terminal fragment defined
above.
[0062] The terms "cysteine-free region" or "hinge region" of CTGF
refer to any polypeptide derived from about amino acid residue 167
to about amino acid residue 198 of human CTGF (SEQ ID NO:2) and
orthologous fragments thereof derived from a different species,
particularly a mammalian species including rat, rabbit, bovine,
ovine, porcine, murine, and equine.
[0063] The terms "amino acid sequence" or "polypeptide" or
"polypeptides" as used herein refer to oligopeptide, peptide,
polypeptide, or protein sequences, and fragments thereof, and to
naturally occurring or synthetic molecules. A polypeptide or amino
acid fragment is any portion of a polypeptide that retains at least
one structural and/or functional characteristic of the polypeptide.
CTGF fragments include any portion of a CTGF polypeptide sequence
that retains at least one structural or functional characteristic
of CTGF. Where "amino acid sequence" is recited to refer to the
polypeptide sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native sequence associated with
the recited protein molecule.
[0064] The term "immunogenicity" relates to the ability of a
substance, when introduced into the body, to stimulate the immune
response and the production of an antibody. An agent displaying the
property of immunogenicity is referred to as being immunogenic.
Immunogenic agents can include, but are not limited to, a variety
of macromolecules such as, for example, proteins, lipoproteins,
polysaccharides, nucleic acids, bacteria and bacterial components,
and viruses and viral components. Immunogenic agents often have a
molecular weight greater than 10 kDa. Antigenic fragments refer to
fragments of CTGF polypeptide, preferably, fragments of about five
to fifteen amino acids in length, that retain at least one
biological or immunological aspect of CTGF polypeptide
activity.
[0065] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab')2, and Fv fragments, which
are capable of binding the epitopic determinant, and include
polyclonal and monoclonal antibodies. Antibodies that bind CTGF or
fragments of CTGF can be prepared using intact polypeptides or
using fragments containing small peptides of interest as the
immunizing antigen. The polypeptide or oligopeptide used to
immunize an animal (e.g., a mouse, rat, rabbit, chicken, turkey,
goat, etc.) can be derived from the translation of RNA, or
synthesized chemically, and can be conjugated to a carrier protein
if desired. Commonly used carriers chemically coupled to peptides
include, for example, bovine serum albumin, thyroglobulin, and
keyhole limpet hemocyanin (KLH).
[0066] The term "monoclonal antibody" as used herein refers to a
substantially homogeneous population of antibodies, i.e., the
individual antibodies comprising the population are identical in
specificity and affinity except for possible naturally occurring
mutations that may be present in minor amounts. Note that a
monoclonal antibody composition may contain more than one
monoclonal antibody.
[0067] The monoclonal antibodies included within the scope of the
invention include hybrid and recombinant antibodies (e.g.,
"humanized" antibodies) regardless of species of origin or
immunoglobulin class or subclass designation, as well as antibody
fragments (e.g., Fab, F(ab').sub.2, and Fv), having at least one of
the distinct characteristics of the antibodies described herein.
Preferred embodiments include antibodies capable of binding to
substantially the same epitope as that recognized by monoclonal
antibody mAb1 and/or have affinity for that epitope that is greater
than or equal to the affinity of mAb1.
[0068] The term "monoclonal" indicates the character of the
antibody as a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies of
the invention may be made using the hybridoma method first
described by Kohler and Milstein (1975, Nature 256:495-497), or may
be made by recombinant DNA methods. For example, see Celltech
Therapeutics Ltd., European Patent No. EP-0 120 694; Cabilly et
al., U.S. Pat. No. 4,816,567; or Mage and Lamoyi (1987; In:
Monoclonal Antibody Production Techniques and Applications, Marcel
Dekker, Inc., New York, pp. 79-97).
[0069] The term "neutralizing antibody" as used herein refers to an
antibody, preferably a monoclonal antibody, that is capable of
substantially inhibiting or eliminating a biological activity of
CTGF. Typically, a neutralizing antibody will inhibit binding of
CTGF to a cofactor such as TGF.beta., to a CTGF-specific receptor
associated with a target cell, or to another biological target. In
a particular embodiment, a neutralizing antibody will inhibit a
biological activity of CTGF to a degree approximately equal to or
greater than mAb1. Preferably, a neutralizing antibody will inhibit
a biological activity of CTGF to a degree approximately equal to or
greater than CLN1.
[0070] The phrase "CTGF-associated disorders" as used herein refers
to conditions and diseases associated with abnormal or altered
expression or activity of CTGF. Abnormal expression of CTGF has
been associated with cell proliferative disorders, such as those
caused by endothelial cell proliferation; cell migration;
tumor-like growths; general tissue scarring; and various diseases
characterized by inappropriate deposition of extracellular
matrix.
[0071] CTGF-associated disorders include, but are not limited to,
disorders involving angiogenesis and other processes which play a
central role in conditions such as proliferative vitreoretinopathy;
cancer, including acute lymphoblastic leukemia, dermatofibromas,
breast cancer, breast carcinoma, glioma and glioblastoma,
rhabdomyosarcoma and fibrosarcoma, desmoplasia, angiolipoma,
angioleiomyoma, desmoplastic cancers, and prostate, ovarian,
colorectal, pancreatic, gastrointestinal, and liver cancer; other
tumor growth and metastases; etc.
[0072] CTGF-associated disorders also include fibrotic disorders
and related conditions, such as excessive scarring resulting from
localized or systemic fibrosis, chronic or acute fibrosis of organs
such as the kidney, lungs, liver, eyes, heart, skin, etc.; or a
tissue selected from, but not limited to, epithelial, endothelial,
and connective tissue. Fibrosis can also occur in the eye and
joints. Such CTGF-associated disorders include, for example,
cardiac fibrosis, including cardiac reactive fibrosis or cardiac
remodeling following myocardial infarction or congestive heart
failure; pulmonary disorders, including interstitial pulmonary
fibrosis, etc.; fibrosis associated with dialysis including
peritoneal dialysis, e.g., continuous ambulatory peritoneal
dialysis (CAPD); peridural fibrosis; kidney fibrosis; pulmonary
fibrosis; interstitial fibrosis; skin fibrosis; and fibrosis
resulting from acute or repetitive traumas, including surgery,
chemotherapy, radiation treatment, allograft rejection, chronic and
acute transplant rejection (e.g., kidney, liver, or other organ);
bronchiolitis obliterans, e.g., following lung transplant; and
inflammation and infection, e.g., due to disease or injury.
[0073] Additionally, CTGF-associated disorders include, but are not
limited to, sclerotic conditions, including systemic sclerosis,
scleroderma, keloids, hypertrophic scarring, and other
dermatological diseases and conditions; atherosclerosis, such as
conditions involving atherosclerotic plaques and atherosclerosis
associated with diabetes, peritoneal dialysis, etc.; arthritis,
including rheumatoid arthritis, osteoarthritis, and other joint
inflammatory conditions, etc.; interstitial diseases, including
interstitial fibrosis; Crohn's disease; inflammatory bowel disease;
retinopathies, including, for example, proliferative
vitreoretinopathy, non-proliferative diabetic retinopathy,
proliferative diabetic retinopathy, and macular degeneration
(including age-related and juvenile (Stargardt's) disease, and
pigment epithelial detachment); nephropathies, including diabetic
nephropathy, IgA-associated nephropathy, nephropathy due to
toxicity, lupus kidney disease, etc.; and conditions associated
with chemical toxicity tubule destruction.
[0074] CTGF-associated disorders also include, but are not limited
to, disorders due to hyperglycemia, hypertension, advanced
glycation endproduct (AGE) formation, etc. Such disorders may
occur, e.g., due to diabetes, obesity, etc., and include diabetic
nephropathy, retinopathy, and cardiovascular disease. Further,
CTGF-associated disorders may be caused by any initiating factor
including, but not limited to, exposure to chemicals or biological
agents, inflammatory response, autoimmune reaction, trauma,
surgical procedures, etc. In some embodiments, the methods are used
to treat a patient predisposed to a CTGF-associated disorder due to
a condition including, but not limited to, myocardial infarction,
arthritis, and local or systemic inflammation.
[0075] The "proliferative" processes and disorders referred to
herein include pathological states characterized by the continual
multiplication of cells resulting in an overgrowth of a cell
population within a tissue. The cell populations are not
necessarily transformed, tumorigenic or malignant cells, but can
include normal cells as well. For example, CTGF may be involved
pathologically by inducing a proliferative lesion in the intimal
layer of an arterial wall, resulting in atherosclerosis, or by
stimulating neovascularization.
[0076] "Cancer" refers to any autonomous growth of tissue,
including uncontrolled, abnormal growth of cells, or to any
malignant tumor of potentially unlimited growth that expands
locally by invasion and systemically by metastasis. Cancer also
refers to any abnormal state marked by a cancer.
[0077] The term "fibrosis" refers to abnormal processing of fibrous
tissue, or fibroid or fibrous degeneration. Fibrosis can result
from various injuries or diseases, and can often result from
chronic transplant rejection relating to the transplantation of
various organs. Fibrosis typically involves the abnormal
production, accumulation, or deposition of extracellular matrix
components, including overproduction and increased deposition of,
for example, collagen and fibronectin. "Fibrosis" is used herein in
its broadest sense referring to any excess production or deposition
of extracellular matrix proteins. There are numerous examples of
fibrosis, including the formation of scar tissue following a heart
attack, which impairs the ability of the heart to pump. Diabetes
frequently causes damage/scarring in the kidneys, which leads to a
progressive loss of kidney function; and in the eyes, which causes
loss of vision. After surgery, scar tissue can form between
internal organs causing contracture, pain, and in some cases,
infertility. Major organs such as the heart, kidney, liver, eye,
and skin are prone to chronic scarring, commonly associated with
other diseases. Hypertrophic scars (non-malignant tissue bulk) are
a common form of fibrosis caused by burns and other trauma. In
addition, there are a number of other fibroproliferative disorders,
including scleroderma, keloids, and atherosclerosis, which are
associated respectively with general tissue scarring, tumor-like
growths in the skin, or sustained scarring of blood vessels which
impairs blood carrying ability.
[0078] The terms "nucleic acid" or "polynucleotide" or
"polynucleotides" refer to oligonucleotides, nucleotide sequences,
or polynucleotides, or any fragments thereof, and to DNA or RNA of
natural or synthetic origin which may be single- or double-stranded
and may represent the sense or antisense strand, to peptide nucleic
acid (PNA), or to any DNA-like or RNA-like material, natural or
synthetic in origin. Polynucleotide fragments are any portion of a
polynucleotide sequence that retains at least one structural or
functional characteristic of the polynucleotide. Polynucleotide
fragments can be of variable length, for example, greater than 60
nucleotides in length, at least 100 nucleotides in length, at least
1000 nucleotides in length, or at least 10,000 nucleotides in
length.
[0079] "Altered" polynucleotides include those with deletions,
insertions, or substitutions of different nucleotides resulting in
a polynucleotide that encodes the same or a functionally equivalent
polypeptide. Included within this definition are sequences
displaying polymorphisms that may or may not be readily detectable
using particular oligonucleotide probes or through deletion of
improper or unexpected hybridization to alleles, with a locus other
than the normal chromosomal locus for the subject polynucleotide
sequence.
[0080] "Altered" polypeptides may contain deletions, insertions, or
substitutions of amino acid residues, which produce a silent change
and result in a functionally equivalent polypeptide. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological or immunological activity of the encoded polypeptide is
retained. For example, negatively charged amino acids may include
aspartic acid and glutamic acid; positively charged amino acids may
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values may include
leucine, isoleucine, and valine, glycine and alanine, asparagine
and glutamine, serine and threonine, and phenylalanine and
tyrosine.
[0081] A polypeptide or amino acid "variant" is an amino acid
sequence that is altered by one or more amino acids from a
particular amino acid sequence. A polypeptide variant may have
conservative changes, wherein a substituted amino acid has similar
structural or chemical properties to the amino acid replaced, e.g.,
replacement of leucine with isoleucine. A variant may also have
non-conservative changes, in which the substituted amino acid has
physical properties different from those of the replaced amino
acid, e.g., replacement of a glycine with a tryptophan. Analogous
minor variations may also include amino acid deletions or
insertions, or both. Preferably, amino acid variants retain certain
structural or functional characteristics of a particular
polypeptide. Guidance in determining which amino acid residues may
be substituted, inserted, or deleted may be found, for example,
using computer programs well known in the art, such as LASERGENE
software (DNASTAR Inc., Madison, Wis.).
[0082] A polynucleotide variant is a variant of a particular
polynucleotide sequence that preferably has at least about 80%,
more preferably at least about 90%, and most preferably at least
about 95% polynucleotide sequence similarity to the particular
polynucleotide sequence. It will be appreciated by those skilled in
the art that as a result of the degeneracy of the genetic code, a
multitude of variant polynucleotide sequences encoding a particular
protein, some bearing minimal homology to the polynucleotide
sequences of any known and naturally occurring gene, may be
produced. Thus, the invention contemplates each and every possible
variation of polynucleotide sequence that could be made by
selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard codon triplet
genetic code, and all such variations are to be considered as being
specifically disclosed.
[0083] A "deletion" is a change in an amino acid or nucleotide
sequence that results in the absence of one or more amino acid
residues or nucleotides.
[0084] The terms "insertion" or "addition" refer to a change in a
polypeptide or polynucleotide sequence resulting in the addition of
one or more amino acid residues or nucleotides, respectively, as
compared to the naturally occurring molecule.
[0085] The term "functional equivalent" as it is used herein refers
to a polypeptide or polynucleotide that possesses at least one
functional and/or structural characteristic of a particular
polypeptide or polynucleotide. A functional equivalent may contain
modifications that enable the performance of a specific function.
The term "functional equivalent" is intended to include fragments,
mutants, hybrids, variants, analogs, or chemical derivatives of a
molecule.
[0086] The term "microarray" refers to any arrangement of nucleic
acids, amino acids, antibodies, etc., on a substrate. The substrate
can be any suitable support, e.g., beads, glass, paper,
nitrocellulose, nylon, or any appropriate membrane, etc. A
substrate can be any rigid or semi-rigid support including, but not
limited to, membranes, filters, wafers, chips, slides, fibers,
beads, including magnetic or nonmagnetic beads, gels, tubing,
plates, polymers, microparticles, capillaries, etc. The substrate
can provide a surface for coating and/or can have a variety of
surface forms, such as wells, pins, trenches, channels, and pores,
to which the nucleic acids, amino acids, etc., may be bound.
[0087] The term "sample" is used herein in its broadest sense.
Samples may be derived from any source, for example, from bodily
fluids, secretions, tissues, cells, or cells in culture including,
but not limited to, saliva, blood, urine, serum, plasma, vitreous,
synovial fluid, cerebral spinal fluid, amniotic fluid, and organ
tissue (e.g., biopsied tissue); from chromosomes, organelles, or
other membranes isolated from a cell; from genomic DNA, cDNA, RNA,
mRNA, etc.; and from cleared cells or tissues, or blots or imprints
from such cells or tissues. Samples may be derived from any source,
such as, for example, a human subject, or a non-human mammalian
subject, etc. Also contemplated are samples derived from any animal
model of disease. A sample can be in solution or can be, for
example, fixed or bound to a substrate. A sample can refer to any
material suitable for testing for the presence of CTGF or of
fragments of CTGF or suitable for screening for molecules that bind
to CTGF or to fragments thereof. Methods for obtaining such samples
are within the level of skill in the art.
[0088] The term "hybridization" refers to the process by which a
nucleic acid sequence binds to a complementary sequence through
base pairing. Hybridization conditions can be defined by, for
example, the concentrations of salt or formamide in the
prehybridization and hybridization solutions, or by the
hybridization temperature, and are well known in the art.
Hybridization can occur under conditions of various stringency.
[0089] In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature. For example, for purposes
of the present invention, hybridization under high stringency
conditions might occur in about 50% formamide at about 37.degree.
C. to 42.degree. C., and under reduced stringency conditions in
about 35% to 25% formamide at about 30.degree. C. to 35.degree. C.
In particular, hybridization generally occurs in conditions of
highest stringency at 42.degree. C. in 50% formamide, 5.times.SSPE,
0.3% SDS, and 200 .mu.g/ml sheared and denatured salmon sperm
DNA.
[0090] The temperature range corresponding to a particular level of
stringency can be further narrowed by methods known in the art, for
example, by calculating the purine to pyrimidine ratio of the
nucleic acid of interest and adjusting the temperature accordingly.
To remove nonspecific signals, blots can be sequentially washed,
for example, at room temperature or up to and including 60.degree.
C., under increasingly stringent conditions of up to 0.1.times.SSC
and 0.5% SDS. Variations on the above ranges and conditions are
well known in the art.
Invention
[0091] The present invention provides antibodies that specifically
bind to Connective Tissue Growth Factor (CTGF). The antibodies are
polyclonal or monoclonal antibodies, preferably monoclonal
antibodies, and more preferably human monoclonal antibodies. The
antibodies are directed toward the N-terminal fragment of CTGF,
shown in FIG. 1. More specifically, the antibodies are directed
toward a fragment of CTGF extending from about residue 97 to about
residue 180 of SEQ ID NO:2. In particular embodiments, the
antibodies are directed toward a fragment of CTGF extending from
about residue 103 to about residue 164, and more particularly a
fragment from about residue 134 to about residue 158 of SEQ ID
NO:2. More specifically, the antibodies are directed toward a
fragment of CTGF extending from about residue 143 to about residue
154 of SEQ ID NO:2.
[0092] In particular embodiments, the antibodies neutralize a
biological activity of CTGF. Biological activities of CTGF include
cell proliferation, differentiation, gene expression, etc. In
particular embodiments, the biological activity is selected from
the group consisting of cellular differentiation, e.g.,
differentiation or transdifferentiation of fibroblasts,
myofibroblasts, endothelial cells, etc., from various precursor
cells; induction of expression of proteins involved in
extracellular matrix formation and remodeling including, e.g., type
I collagen, fibronectin, etc.; cooperative induction of signaling
cascades associated with various factors including, but not limited
to, TGF-.beta., IGF, VEGF, angiotensin II, endothelin, etc.; and
cellular response to various environmental stimuli including, but
not limited to, increased glucose (hyperglycemia), increased
mechanical stress (hypertension), etc.
[0093] Although the invention is not to be limited by the mechanism
by which the antibodies neutralize CTGF activity, the antibodies
may bind to and prevent CTGF from interacting with specific cell
receptors. The receptors may have high binding affinity for CTGF
and, by binding to CTGF, stimulate an intracellular signal that
leads to proliferation, differentiation, induction of gene
expression, and/or change in cellular morphology or function. The
particular biological response of a cell to CTGF depends on the
cell and the current state of the surrounding milieu.
Alternatively, the receptors may have low binding affinity for CTGF
and, by binding to CTGF, may, e.g., position CTGF relative to high
affinity receptors to facilitate recognition for and response to
CTGF. Alternatively, the antibodies may bind CTGF within tissues or
organs and facilitate titration or elimination of CTGF from the
body.
[0094] Alternatively or in conjunction with the mechanisms
described above, the antibodies may bind to and prevent CTGF from
interacting with secreted or membrane-bound cofactors. Such
cofactors specifically include members of the TGF.beta. superfamily
including, e.g., TGF.beta.-1, -2, and -3; activin-A, -B, -C, and
-E; BMP-2, -3, -4, -5, -6, -7, -8a, -8b, -9, -10, -11, and -15; and
GDF-3, -5, -6, -7, -9, and -10. For example, CTGF has been shown to
bind to TGF.beta.-1 and BMP-4 and modulate their activity. (Abreu
et al. (2002) Nat Cell Biol 4: 599-604.) The present invention
provides evidence that the region of CTGF that binds to TGF.beta.
is encoded by exon 3 (FIG. 1B; nucleotide 418 to nucleotide 669 of
SEQ ID NO:1) and antibodies that bind within this region prevent
interaction between CTGF and TGF.beta.. (Example 12, infra.)
Further, antibodies that bind within this region of CTGF have been
shown to neutralize specific CTGF-associated processes in animal
models. For example, antibodies that bind within this region of
CTGF have been shown to specifically inhibit cell migration in ex
vivo assays, and reduce fibrosis in animal models. Exemplary
antibodies of the invention are mAb1 and CLN1; antibody CLN1 is
produced by the cell line defined by ATCC Accession No. PTA-6006,
deposited with the American Type Culture Collection (ATCC, 10801
University Boulevard, Manassas Va. 20110-2209) on 20 May 2004. The
Chinese Hamster Ovary (CHO) cell line identified as ATCC Accession
No. PTA-6006, which produces the human monoclonal antibody CLN-1,
was deposited with the ATCC under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures at ATCC for at least 30 years from the date of
deposit, or for the enforceable life of the patent, or for a period
of 5 years after the date of the most recent request for furnishing
a sample of the deposited material, whichever is longest. The
organisms will be made available by ATCC under the terms of the
Budapest Treaty and the cell line will be irrevocably and without
restriction or condition released to the public upon issuance of a
patent. Also, if the culture deposit should die or be lost or
destroyed when cultivated under suitable conditions, it will be
promptly replaced on notification with a viable specimen of the
same culture. Availability of a deposited strain is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0095] Regardless of the mechanism of action, the present invention
provides methods of using the antibodies to treat various diseases
and disorders associated with CTGF. Diseases and disorders
associated with CTGF include, but are not limited to,
nephropathies, pulmonary fibroses, retinopathies, scleroderma,
liver fibroses, heart failure, arthritis, and atherosclerosis.
Additionally, disorders associated with CTGF occur due to various
factors including, but not limited to, hyperglycemia, hypertension,
diabetes, obesity, etc; and include diabetic nephropathy,
retinopathy, cardiovascular disease, and the like. As CTGF is
overexpressed in a wide variety of diseases including those listed
above, the invention contemplates treating patients having a
CTGF-associated disorder with a CTGF antibody to improve or
stabilize the pathology, retain or restore organ function, improve
the quality of life, and prolong survival.
[0096] For example, the antibodies are particularly directed to
regions of CTGF involved in biological activities associated with
both fibrotic and non-fibrotic aspects of various disorders
including, e.g., interstitial pulmonary fibrosis, diabetic
nephropathy and retinopathy, macular degeneration, etc. The
invention also relates to methods of using the antibodies to treat
disorders associated with CTGF including localized and systemic
fibrotic disorders, such as those of the lung, liver, heart, skin,
and kidney, etc.; and localized scar formation due to, e.g.,
trauma, surgical procedures, etc.
[0097] The antibodies of the invention can also be used in any
method that involves binding to CTGF. Such methods include
purification of CTGF or fragments of CTGF, e.g., by affinity
chromatography; detection of CTGF or fragments of CTGF in a sample,
e.g., using ELISA or immunohistochemical techniques; diagnosing a
CTGF-associated disorder by using the method of detecting CTGF to
measure CTGF levels in a patient sample and comparing the level of
CTGF in the sample to a standard.
Antibodies Directed to CTGF
[0098] Modulation of the amount and/or activity of secreted
cellular factors using, e.g., monoclonal antibodies, has been
demonstrated, and several therapeutic antibodies have been approved
or are under development. (See, e.g., Abciximab (Reopro; Centocor,
Inc., Malvern Pa.), Infliximab (Remicade; Maini et al. (1998)
Arthritis Rheum 41:1552-1563; Targan et al. (1997) N Engl J Med
337:1029-1035); Basiliximab (Simulect) and Daclizumab (Zenapax)
(Bumgardner et al. (2001) Transplantation 72:839-845; Kovarik et
al. (1999) Transplantation 68:1288-1294); and Trastuzumab
(Herceptin; Baselga (2001) Ann Oncol 12 Suppl 1:S49-55.)) Numerous
methods of producing antibodies, including production in animals,
plants, fungi, and bacteria; synthetic construction; and ex vivo
culture; are known and available to those of skill in the art.
[0099] The antibodies of the invention may be prepared using any
technique that provides for the production of antibody molecules.
Techniques for in vivo and in vitro production of either monoclonal
or polyclonal antibodies are well known in the art. (See, e.g.,
Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York; Goding (1986) Monoclonal Antibodies:
Principles and Practice, 2.sup.nd Edition, Academic Press; Schook
(1987) Monoclonal Antibody Production Techniques and Applications,
Marcel Dekker, Inc.) The production of chimeric antibodies is also
well known in the art, as is the production of single-chain
antibodies. (See, e.g., Morrison et al. (1984) Proc Natl Acad Sci
USA 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;
Takeda et al. (1985) Nature 314:452-454.) Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by a variety of available means, for example, by chain
shuffling from random combinatorial immunoglobin libraries. (See,
e.g., Burton (1991) Proc Natl Acad Sci USA 88:11120-11123.)
[0100] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents. (See, e.g., Orlandi et al. (1989) Proc Natl Acad Sci USA
86:3833-3837; Winter and Milstein (1991) Nature 349:293-299.)
Antibody fragments that contain specific binding sites for the
target polypeptide may also be generated. Such antibody fragments
include, but are not limited to, F(ab').sub.2 fragments, which can
be produced by pepsin digestion of the antibody molecule, and Fab
fragments, which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed to allow rapid and easy identification
of monoclonal Fab fragments with the desired specificity. (See,
e.g., Huse et al. (1989) Science 254:1275-1281.)
[0101] Monoclonal antibodies of the invention may also be prepared
using the hybridoma method (see, e.g., Kohler and Milstein (1975)
Nature 256:495-497) or by recombinant DNA methods (see, e.g.,
Celitech Therapeutics Ltd., European Patent No. EP 0 120 694;
Cabilly et al., U.S. Pat. No. 4,816,567; and Mage and Lamoyi (1987)
In: Monoclonal Antibody Production Techniques and Applications,
Marcel Dekker, Inc., New York, pp. 79-97).
[0102] In the hybridoma method, a mouse or other appropriate host
animal is immunized with CTGF or a fragment thereof by
subcutaneous, intraperitoneal, or intramuscular routes to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the polypeptide used for
immunization. Alternatively, the host animal may be a transgenic
mammal having transgenes encoding human immunoglobulin genes and
having inactivated endogenous immmunoglobulin loci. The transgenic
mammal responds to immunogens by producing human antibodies. (See,
e.g., Lonberg et al., WO 93/12227 (1993), U.S. Pat. No. 5,877,397,
and Nature 148:1547-1553 (1994); and Kucherlapati et al. (1991) WO
91/10741.) Alternatively, lymphocytes may be immunized in vitro and
then fused with myeloma cells using a suitable fusing agent, such
as polyethylene glycol, to form a hybridoma cell. (See, e.g.,
Goding (1986) Monoclonal Antibodies: Principles and Practice,
2.sup.nd Edition, Academic Press, pp. 59-103.) Alternatively, human
somatic cells capable of producing antibody, specifically B
lymphocytes, are suitable for fusion with myeloma cell lines. While
B lymphocytes from biopsied spleens, tonsils or lymph nodes of an
individual may be used, the more easily accessible peripheral blood
B lymphocytes are preferred. In addition, human B cells may be
directly immortalized by the Epstein-Barr virus. (See, e.g., Cole
et al. (1995) Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96.)
[0103] Preferred myeloma cell lines for use in hybridoma-producing
fusion procedures are those that fuse efficiently, support stable
high-level expression of antibody by the selected
antibody-producing cell, have enzyme deficiencies that render them
incapable of growing in certain selective media which support the
growth of the desired hybridomas, and that do not themselves
produce antibody. Examples of myeloma cell lines that may be used
for the production of hybridomas in the present invention include
P3X63Ag8, P3X63Ag8-653, NS1/1.Ag 4.1, Sp210-Ag14, FO, NSO/U,
MPC-11, MPC11-X45-GTG 1.7, S194/5XX0 Bul, all derived from mice;
R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210, all derived from rats;
and U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6, all derived from
humans. (See, e.g., Goding (1986) Monoclonal Antibodies: Principles
and Practice, 2.sup.nd Edition, Academic Press, pp. 65-66; and
Campbell (1984) In: Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 13 (Burden
and Von Knippenberg, eds.) Amsterdam, Elseview, pp. 75-83.)
[0104] The hybridoma cells are seeded and grown in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, parental myeloma
cells. For example, if the parental myeloma cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT),
the culture medium for the hybridomas typically will include a
substance such as hypoxanthine, aminopterin, and thymidine (HAT
medium) that prevents the growth of HGPRT-deficient cells.
[0105] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
CTGF or fragments of CTGF. Preferably, the binding specificity is
determined by affinity chromatography, immunoprecipitation or by an
in vitro binding assay, such as radioimmunoassay (RIA) or
enzyme-linked immunoabsorbent assay (ELISA), or by
fluorescence-activated cell sorting (FACS) analysis. The monoclonal
antibodies of the invention are those that bind to CTGF, and
additionally those that neutralize a CTGF biological activity, as
exemplified infra.
[0106] The antibodies produced, e.g., as described supra, are
optionally screened to detect antibodies that bind to substantially
the N-terminal fragment of CTGF. In one embodiment, the antibodies
are directed toward a fragment of CTGF extending from about residue
24 to about residue 180 of SEQ ID NO:1. In another embodiment, the
antibodies are directed toward a fragment of CTGF extending from
about residue 96 to about residue 180 of SEQ ID NO:1. In a
particular embodiment, the screen detects antibodies that bind to
substantially the same epitope recognized by antibody mAb1 as
determined, e.g., by competition assays of the sort described
infra. In another particular embodiment, the screen detects
antibodies that bind to substantially the same epitope recognized
by antibody CLN1 as determined, e.g., by competition assays of the
sort described infra. It should be kept in mind that "same epitope"
does not mean the exact amino acid or carbohydrate to which the
benchmark antibody binds, as may be determined, for example, by
epitope mapping using alanine scanned variants of CTGF. "Same
epitope" means the CTGF domain that is blocked by the binding to
CTGF of the native benchmark antibody in intact form. Of course,
"same epitope" includes the CTGF domain residues or carbohydrate
that structurally interacts or binds to the benchmark
complementarity determining regions (CDRs) of mAb1 or CLN1.
[0107] In a preferred embodiment of the invention, the monoclonal
antibody will have an affinity that is equal to or greater than
that of mAb1, as determined, for example, by the Scatchard analysis
of Munson and Pollard (1980, Anal Biochem 107:220).
[0108] After hybridoma cells are identified that produce
neutralizing antibodies of the desired specificity and affinity,
the clones typically are subcloned by limiting dilution procedures
and grown by standard methods. (Goding (1986) Monoclonal
Antibodies: Principles and Practice, 2.sup.nd Edition, Academic
Press, pp. 59-104.) Suitable culture media for this purpose
include, for example, Dulbecco's Modified Eagle's Medium or
RPMI-1640 medium. In addition, the hybridoma cells may be grown in
vivo as ascites tumors in an animal.
[0109] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0110] DNA encoding the monoclonal antibodies of the invention is
readily isolated and sequenced using conventional procedures, e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the antibody heavy and light chains.
Once isolated, the DNA can be ligated into expression or cloning
vectors, which are then transfected into host cells such as simian
COS cells, Chinese Hamster ovary (CHO) cells, or myeloma cells that
do not otherwise produce immunoglobulin protein. The cells so
transformed are cultured under conditions suitable for the
synthesis of monoclonal antibodies in the recombinant host cell
culture. An exemplary cell line is defined by ATCC Accession No.
PTA-6006, deposited with the ATCC on 20 May 2004.
[0111] The DNA optionally is modified in order to change the
character of the encoded immunoglobulin. Variants of
immunoglobulins are well known. For example, chimeric antibodies
are made by substituting the coding sequence for heavy and light
chain constant domains from one species, e.g., mouse, with the
homologous sequences from another species, e.g., human. (See, e.g.,
Boss et al., International Publication No. WO 84/03712; Cabilly et
al., U.S. Pat. No. 4,816,567; or Morrison et al. (1984) Proc Nat
Acad Sci 81:6851.) In a particular embodiment, humanized forms of
murine antibodies can be made by substituting the complementarity
determining regions (CDRs), i.e., variable domains, of a mouse
antibody into a framework domain, i.e., constant region, of a human
antibody. (See, e.g., International Publication No. WO 92/22653.)
In some embodiments, selected murine framework residues also are
substituted into the human recipient immunoglobulin. In addition,
the Fc domain chosen can be any of IgA, IgD, IgE, IgG-1, IgG-2,
IgG-3, IgG-4, or IgM. The Fc domain optionally is capable of
effector functions such as complement binding.
[0112] Anti-CTGF antibodies of the present invention may also be
fused to moieties that provide additional capabilities, such as
detection or cytotoxic effects. Fusions of the immunoglobulins of
this invention and cytotoxic moieties are made, for example, by
ligating to the immunoglobulin coding sequence all or part of the
coding sequence for a cytotoxic non-immunoglobulin polypeptide.
Such non-immunoglobulin polypeptides include polypeptide toxins
such as ricin, diphtheria toxin, or Pseudomonas exotoxin. The
conjugates can also be prepared by in vitro methods. For example,
immunotoxins may be constructed using a disulfide exchange reaction
or by forming a thioether bond between the immunoglobulin and the
toxin polypeptide. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate. Typically
such non-immunoglobulin fusion polypeptides are substituted for the
constant domains of an antibody of the invention. Alternatively,
they are substituted for the variable domains of one
antigen-combining site of an antibody of the invention.
[0113] Substitution of the Fv or CDRs of an antibody having
specificity for a non-CTGF antigen will create a chimeric antibody
comprising one antigen-combining site having specificity for CTGF
and another antigen-combining site having specificity for a
different antigen. In such embodiments, the light chain is deleted
and the Fv of the heavy chain is substituted with the desired
polypeptide. These antibodies are termed bivalent or polyvalent,
depending upon the number of immunoglobulin "arms" possessed by the
Fc domain employed; for example, IgGs will be bivalent and IgMs
will be polyvalent. Aside from the non immunoglobulins mentioned
above, the antibody also is rendered multivalent by recombination
of antibodies that have more than one specificity. For instance,
the antibody in some embodiments is capable of binding CTGF as
described elsewhere herein, but is also capable of binding a second
growth factor, e.g., TGF.beta., VEGF, FGF, other CCN family
members, e.g., CYR61, and the like, or a cytokine. Exemplary
antibodies directed against these factors are well-known. The
multispecific, multivalent antibodies are made by cotransforming a
cell with DNA encoding the heavy and light chains of both
antibodies and the proportion of expressed antibodies having the
desired structure recovered by immunoaffinity chromatography or the
like. Alternatively, such antibodies are made from monovalent
antibodies that are recombined in vitro in conventional
fashion.
[0114] Monovalent antibodies also are made by techniques that are
conventional per se. Recombinant expression of light chain and a
modified heavy chain is suitable. The heavy chain is truncated
generally at any point in the Fc region so as to prevent heavy
chain crosslinking. Alternatively, the relevant cysteines are
substituted with another residue or deleted so as to prevent
crosslinking. In vitro methods also are used to produce monovalent
antibodies, e.g., Fab fragments are prepared by enzymatic cleavage
of intact antibody.
Diagnostics
[0115] The antibodies of the present invention can be used to
quantitatively and qualitatively detect CTGF in a sample. Samples
can be from any source, including conditioned media from cells
grown in culture; tissue samples, e.g., tissue biopsies and organ
transplants; body fluids including blood, urine, blister fluid,
cerebrospinal fluid, vitreous, and synovial fluid; etc. In one
embodiment, detection of CTGF is used to diagnose the state of
cells grown in culture, e.g., with regard to differentiation,
matrix production, etc. CTGF has various autocrine and paracrine
effects on cultured cells, and the level of CTGF associated with
the cell layer or present in conditioned media may be indicative of
the current state of the cell or predictive of the future state of
the cell. (See, e.g., International Publication No. WO 96/38168.)
In other embodiments, detection of CTGF is used to determine the
state of a tissue or organ. For example, an organ destined for
transplant can be evaluated by measuring CTGF levels, wherein the
level of CTGF expressed by cells in the organ indicate the relative
health of the organ and suitability for transplant. CTGF levels can
also be determined in biopsied tissue to determine the status of an
organ, or the stage and potential metastatic potential of a
cancer.
[0116] In preferred embodiments, the antibodies are used to
diagnose a disease or disorder associated with CTGF. (See, e.g.,
International Publication No. WO 03/024308.) In one aspect, the
invention provides antibodies for diagnosing a CTGF-associated
disorder by obtaining a sample, detecting and quantitating the
level of CTGF in the sample, and comparing the level of CTGF in the
sample to that of a standard amount of CTGF, wherein an increased
or decreased amount of CTGF in the sample is indicative of the
presence of a CTGF-associated disorder. Disorders associated with
aberrant (e.g., increased or decreased) levels of CTGF include, but
are not limited to, disorders associated with altered expression
and deposition of extracellular matrix-associated proteins. Such
disorders include, for example, cancers such as breast, pancreatic,
and gastrointestinal cancer; atherosclerosis, arthritis,
retinopathies such as diabetic retinopathy; nephropathies such as
diabetic nephropathy; cardiac, pulmonary, liver, and kidney
fibrosis, and diseases associated with chronic inflammation and/or
infection. CTGF-associated disorders are also associated with
conditions such as myocardial infarction, diabetes, peritoneal
dialysis, chronic and acute transplant rejection, chemotherapy,
radiation therapy, and surgery.
[0117] In another aspect, the invention provides antibodies for
identifying whether or not an individual has a predisposition to
develop a CTGF-associated disorder. A predisposition may be
initially indicated by hyperglycemia, hypertension, or obesity in a
subject. Additionally, a predisposition may be suspected due to an
event, e.g., a myocardial infarction, surgery, orthopedic or
paralytic immobilization, congestive heart failure, pregnancy, or
varicosities in the subject.
[0118] In another aspect, the invention provides antibodies for
monitoring the progression of a CTGF-associated disorder or
monitoring the therapeutic efficacy of treatment of a
CTGF-associated disorder. For example, a method of using the
antibodies may comprise obtaining samples from a subject over time;
detecting and quantitating the level of CTGF in each sample; and
comparing the level of CTGF in subsequent samples with CTGF levels
in earlier or previous samples. A change in CTGF level between
samples over time is indicative of the progression of the
CTGF-associated disorder or the therapeutic efficacy of treatment
of the CTGF-associated disorder.
[0119] For diagnostic applications, the antibodies of the invention
typically will be labeled with a detectable moiety. The detectable
moiety can be any moiety capable of producing, either directly or
indirectly, a detectable signal. For example, the detectable moiety
may be a radioisotope, such as .sup.3H, .sup.14C, .sup.32P,
.sup.35S, or .sup.125I, a fluorescent or chemiluminescent compound,
such as fluorescein isothiocyanate, rhodamine, or luciferin; or an
enzyme, such as alkaline phosphatase, beta-galactosidase or
horseradish peroxidase.
[0120] Any method known in the art for separately conjugating the
antibody to the detectable moiety may be employed. (See, e.g.,
Hunter et al. (1962) Nature 144:945; David et al. (1974)
Biochemistry 13:1014; Pain et al. (1981) J Immunol Meth 40:219; and
Nygren (1982) J Histochem Cytochem 30:407.) The antibodies of the
present invention may be employed in any known assay method, such
as competitive binding assays, direct and indirect sandwich assays,
and immunoprecipitation assays. (Zola (1987) In: Monoclonal
Antibodies: A Manual of Techniques, CRC Press, Inc., pp.
147-158.)
[0121] Competitive binding assays rely on the ability of a labeled
standard (which may be CTGF or an immunologically reactive portion
thereof) to compete with the test sample analyte (CTGF) for binding
with a limited amount of antibody. The amount of CTGF in the test
sample is inversely proportional to the amount of standard that
becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies may conveniently be
separated from the standard and analyte that remain unbound.
[0122] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, or epitope,
of the protein to be detected. In a sandwich assay, the test sample
analyte is bound by a first antibody which is immobilized on a
solid support, and thereafter a second antibody binds to the
analyte, thus forming an insoluble three part complex. (David and
Greene, U.S. Pat. No. 4,376,110.) The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be
measured using an anti-immunoglobulin antibody that is labeled with
a detectable moiety (indirect sandwich assay). For example, one
type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme. An exemplary assay in which the
antibodies of the invention may be used, e.g., is described in
International Publication No. WO 03/024308.
[0123] The antibodies of the invention also are useful for in vivo
imaging, wherein an antibody labeled with a detectable moiety such
as a radio-opaque agent, radioisotope, or fluorescent moiety such
as green fluorescent protein (GFP) is administered to a host,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host is assayed. This imaging technique
is useful in the staging and treatment of CTGF-associated disorders
such as fibrotic disorders. The antibody may be labeled with any
moiety that is detectable in a host, whether by nuclear magnetic
resonance, radiology, or other detection means known in the
art.
Therapeutics
[0124] The present invention provides antibodies for treatment of
various diseases and disorders associated with CTGF. The antibodies
of the invention have been found to reduce the deleterious effects
of CTGF production or activity in several disorders, as exemplified
below. Further, the antibodies show favorable pharmacokinetics
making them superior therapeutic agents for the treatment of
disorders associated with CTGF.
[0125] The anti-CTGF antibodies of the present invention inhibit
development of fibrosis in animal models of, e.g., lung and kidney
fibrosis. Specifically, the antibodies attenuate bleomycin-induced
lung fibrosis in mice by 60-70%, as determined by inhibition of
pulmonary hydroxyproline (collagen) accumulation and histological
examination of tissue preparations. Further, the antibodies reduce
the accumulation of collagen in a rat remnant kidney (i.e.,
nephrectomy) model, and in mice following unilateral ureter
obstruction (UUO). The antibodies also reduce fibrosis induced by
combined subcutaneous or intraperitoneal infusion of CTGF and
TGF.beta. in newborn mice. Additionally, the antibodies reduce
complications associated with organ failure, e.g., improved kidney
function in various models of chronic and acute kidney failure. No
toxicity has been observed with these antibodies in animals. As
CTGF is overexpressed in a wide variety of fibrotic diseases
including diffuse and limited scleroderma, osteoarthritis, diabetic
nephropathy and retinopathy, etc., the invention contemplates
treating patients with a CTGF-associated disorder with a CTGF
antibody to improve or stabilize the pathology, restore organ
function, improve the quality of life, and extend survival.
[0126] Therefore, the antibodies of the invention are especially
useful in therapeutic applications, to prevent or treat
CTGF-associated disorders in a subject. Such disorders include, but
are not limited to, angiogenesis and other processes which play a
central role in conditions such as atherosclerosis, glaucoma, etc.;
and in cancer, including acute lymphoblastic leukemia,
dermatofibromas, breast cancer, breast carcinoma, glioma and
glioblastoma, rhabdomyosarcoma and fibrosarcoma, desmoplasia,
angiolipoma, angioleiomyoma, desmoplastic cancers, and prostate,
ovarian, colorectal, pancreatic, gastrointestinal, and liver cancer
and other tumor growth and metastases.
[0127] Additionally, the antibodies of the invention are useful in
therapeutic applications to prevent or treat CTGF-associated
disorders involving fibrosis. In one aspect, the antibodies of the
invention are administered to a subject to prevent or treat a
CTGF-associated disorder including, but are not limited to,
disorders exhibiting altered expression and deposition of
extracellular matrix-associated proteins, e.g., fibrotic disorders.
In various aspects, the fibrosis may be localized to a particular
tissue, such as epithelial, endothelial, or connective tissue; or
to an organ, such as kidney, lung, or liver. Fibrosis can also
occur in the eye and joints. In other aspects, the fibrosis may be
systemic and involve multiple organ and tissue systems.
CTGF-associated disorders include, for example, atherosclerosis,
arthritis, retinopathies such as diabetic retinopathy;
nephropathies such as diabetic nephropathy; cardiac, pulmonary,
liver, and kidney fibrosis, and diseases associated with chronic
inflammation and/or infection.
[0128] In another aspect, the invention provides antibodies for
preventing a CTGF-associated disorder in a subject having a
predisposition to develop such a disorder. A predisposition may
include, e.g., hyperglycemia, hypertension, or obesity in the
subject. Such disorders may occur, e.g., due to diabetes, obesity,
etc., and include diabetic nephropathy, retinopathy, and
cardiovascular disease. Additionally, a predisposition may be
suspected due to an event, e.g., a myocardial infarction, surgery,
peritoneal dialysis, chronic and acute transplant rejection,
chemotherapy, radiation therapy, trauma, orthopedic or paralytic
immobilization, congestive heart failure, pregnancy, or
varicosities in the subject.
[0129] In particular embodiments, as exemplified herein, the
antibodies of the present invention are administered to a subject
to treat fibrosis of an organ, e.g., lung or kidney. The antibodies
are shown herein to provide benefit in various models of lung and
kidney fibrosis. (See, e.g., Examples 7 to 9.) In another
particular embodiment, the antibodies of the present invention are
administered to a subject to reduce local or systemic sclerosis.
(See, e.g., Examples 11 and 12.) In additional embodiments, the
antibodies are administered to a subject to treat or prevent ocular
disorders such as proliferative vitreoretinopathy, diabetic
retinopathy, macular degeneration, etc. As CTGF is implicated in a
wide variety of disorders, the invention further contemplates
treating patients having a CTGF-associated disorder using an
antibody of the invention to improve or stabilize pathology and
organ function, improve the quality of life, and extend
survival.
[0130] For therapeutic applications, the antibodies of the
invention are administered to a mammal, preferably a human, in a
pharmaceutically acceptable dosage form. The antibodies may be
administered intravenously as a bolus or by continuous infusion
over a period of time, and/or by intramuscular, subcutaneous,
intra-articular, intrasynovial, intrathecal, intravitreal,
intracranial, oral, topical, or inhalation routes. When the
antibody possesses the suitable activity, intratumoral,
peritumoral, intralesional, or perilesional routes of
administration can also be utilized to exert local as well as
systemic therapeutic effects.
[0131] Such dosage forms encompass pharmaceutically acceptable
carriers that are inherently nontoxic and nontherapeutic. Examples
of such carriers include ion exchangers, alumina, aluminum
stearate, lecithin; serum proteins such as human serum albumin;
buffers such as phosphate or glycine; sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water, salts; or electrolytes such as protamine sulfate,
sodium chloride, metal salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulosic polymers, and
polyethylene glycol. Carriers for topical or gel-based forms of
antibody include polysaccharides such as sodium
carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,
polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,
polyethylene glycol, and wood wax alcohols. Conventional depot
forms include, for example, microcapsules, nano-capsules,
liposomes, plasters, sublingual tablets, and polymer matrices such
as polylactide:polyglycolide copolymers. When present in an aqueous
dosage form, rather than being lyophilized, the antibody typically
will be formulated at a concentration of about 0.1 mg/ml to 100
mg/ml, although wide variation outside of these ranges is
permitted.
[0132] For the prevention or treatment of disease, the appropriate
dosage of antibody will depend on the type of disease to be
treated, as defined above, the severity and course of the disease,
whether the antibodies are administered for preventive or
therapeutic purposes, the course of previous therapy, the patient's
clinical history and response to the antibody, and the discretion
of the attending physician. The antibody is suitably administered
to the patient at one time or over a series of treatments.
[0133] Depending on the type and severity of the disease, about
0.015 to 15 mg of antibody/kg of patient weight is an initial
candidate dosage for administration to the patient, whether, for
example, by one or more separate administrations, or by continuous
infusion. For repeated administrations over several days or longer,
depending on the condition, the treatment is repeated until a
desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful and are not excluded from the present
invention.
[0134] According to another embodiment of the invention, the
effectiveness of the antibody in preventing or treating disease may
be improved by administering the antibody serially or in
combination with another agent that is effective for the same
clinical objective, such as another antibody directed against a
different epitope than the principal antibody, or one or more
conventional therapeutic agents known for the intended therapeutic
indication, e.g. prevention or treatment of conditions associated
with excessive extracellular matrix production such as fibrosis or
sclerosis, inhibition of tumor cell growth or metastasis,
inhibition of neovascularization, or reduction of inflammation.
Such agents may ameliorate symptoms or improve outcome via a
similar mechanism of action, e.g., anti-TGF.beta. antibodies, or by
a different mechanism, e.g., interferon-.gamma.. Such agents may
additionally ameliorate symptoms directly or indirectly associated
with a CTGF-associated disorder or a predisposition to develop a
CTGF-associated disorder, e.g., angiotensin-converting enzyme (ACE)
inhibitors and angiotensin receptor blockers (Arbs).
[0135] For example, scleroderma patients receiving infusions of the
stable prostacyclin agonist Iloprost frequently report an
improvement in skin tightness consistent with an inhibitory effect
on scar tissue formation by skin fibroblasts. Prostanoids have been
shown to exert an inhibitory effect on collagen synthesis, and
several lines of evidence demonstrate that Iloprost blocks CTGF
induction in scleroderma. (Korn et al. (1980) J Clin Invest
65:543-554; Goldstein and Polger (1982) J Biol Chem 257:8630-8633;
and Stratton et al. (2001) J Clin Invest 108:241-250.) CTGF is
elevated seven-fold in blister fluid in patients with scleroderma
compared with healthy controls, however patients receiving
intravenous administration of Iloprost show a marked decrease in
CTGF in blister fluid. (Stratton et al. (2001) J Clin Invest
108:241-250.) Taken together, these results suggest that some of
the benefits of Iloprost therapy in scleroderma might derive from
antifibrotic effects mediated via reduction in CTGF levels. As
there are concerns regarding the use of a potent vasodilatory and
anti-platelet prostacyclin analog in chronic systemic
administration in scleroderma patients, a therapy utilizing an
anti-CTGF antibody alone or in conjunction with reduced levels of
Iloprost could provide a safe and effective treatment for
scleroderma.
Additional Uses
[0136] The antibodies of the invention also are useful as affinity
purification agents. In this process, the antibodies against CTGF
are immobilized on a suitable support, such as Sephadex resin or
filter paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the CTGF to be
purified, and thereafter the support is washed with a suitable
solvent that will remove substantially all the material in the
sample except the CTGF that is bound to the immobilized antibody.
Finally, the support is washed with another suitable solvent, such
as glycine buffer (pH 5.0), that will release the CTGF from the
antibody.
EXAMPLES
[0137] The invention will be further understood by reference to the
following examples, which are intended to be purely exemplary of
the invention. These examples are provided solely to illustrate the
claimed invention. The present invention is not limited in scope by
the exemplified embodiments, which are intended as illustrations of
single aspects of the invention only. Any methods which are
functionally equivalent are within the scope of the invention.
Various modifications of the invention in addition to those
described herein will become apparent to those skilled in the art
from the foregoing description and accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
Example 1
Production of Recombinant Human CTGF
[0138] A recombinant human CTGF baculovirus construct was produced
as described in Segarini et al. ((2001) J Biol Chem
276:40659-40667). Briefly, a CTGF cDNA comprising only the open
reading frame was generated by PCR using DB60R32 (Bradham et al.
(1991) J Cell Biol 114:1285-94) as template and the primers
5'-gctccgcccgcagtgggatccATGaccgccgcc-3' and
5'-ggatccggatccTCAtgccatgtctccgta-3', which add BamHI restriction
enzyme sites to the ends of the amplified product. The native start
and stop codons are indicated in capital letters.
[0139] The resulting amplified DNA fragment was digested with
BamHI, purified by electrophoresis on an agarose gel, and subcloned
directly into the BamHI site of the baculovirus PFASTBAC1
expression plasmid (Invitrogen Corp., Carlsbad Calif.). The
sequence and orientation of the expression cassette was verified by
DNA sequencing. The resulting CTGF expression cassette was then
transferred to bacmid DNA by site-specific recombination in
bacteria. This bacmid was then used to generate a fully recombinant
CTGF baculovirus in Spodoptera frugiperda SF9 insect cells
according to protocols supplied by the manufacturer (BAC-TO-BAC
Expression System manual; Invitrogen). Expansion of recombinant
baculovirus titers in Sf9 insect cells was performed using standard
procedures known in the art.
[0140] Hi5 insect cells were adapted for suspension growth by
serial passage of cells in shake flask culture accompanied by
enrichment at each passage for separated cells. Suspension Hi5
cells were cultured in IL SF900II SFM media (Invitrogen)
supplemented with 20 .mu.g/ml gentamicin (Mediatech, Inc., Herndon
Va.) and 1.times. lipid (Invitrogen) in disposable 2.8 L Fernbach
culture flasks (Corning Inc., Acton Mass.) on a shaker platform at
110 rpm at 27.degree. C. Once cells reached a density of
1.0-1.5.times.10.sup.6 cells/ml with a viability of >95%, they
were infected with recombinant baculovirus at a multiplicity of
infection (MOI) of 10. The cultures were then incubated at
27.degree. C. for an additional 40 to 44 hours. The conditioned
media, which contains rhCTGF, was collected, chilled on ice, and
centrifuged at 5000.times.g. The supernatant was then passed
through a 0.45 mm filter.
[0141] Alternatively, recombinant rat CTGF was produced by
inserting clone 2-4-7, which encodes rat CTGF (Schmidt et al., U.S.
Pat. No. 6,348,329), into pMK33 expression vector (constructed by
Michael Koelle, Stanford University Ph.D. dissertation, 1992). The
rat CTGF expression construct was transfected into Schneider 2
cells (American Type Culture Collection, Manassas Va.; Schneider
(1972) J Embryol Exp Morphol 27:353-365) using CELLFECTIN reagent
(Invitrogen Corp., Carlsbad Calif.). Cells were grown in media
containing 300 .mu.g/ml hygromycin B for 6 weeks, and were then
grown without selection for three days. Expression of CTGF was
induced by the addition of 500 .mu.M CuSO.sub.4 and 100 .mu.M
ZnSO.sub.4, and after four days the medium was harvested and
clarified by centrifugation and filtration as above.
[0142] CTGF produced by either method described above was purified
as follows. Four liters of conditioned media was loaded over a 5 ml
HI-TRAP heparin column (Amersham Biosciences Corp., Piscataway
N.J.) pre-equilibrated with 50 mM Tris (pH7.5), 150 mM NaCl. The
column was washed with 10 column volumes of 350 mM NaCl, 50 mM Tris
(pH 7.5). CTGF was eluted from the column with an increasing NaCl
salt gradient. Eluted fractions were screened by SDS-PAGE, and
those containing CTGF were pooled.
[0143] Heparin purified CTGF was diluted to a final conductivity of
5.7 mS with non-pyrogenic double-distilled water and the pH was
adjusted to 8.0. A Q-SEPHAROSE strong anion exchange column
(Amersham Biosciences) containing approximately 23 ml resin
connected in tandem with a carboxymethyl (CM) POROS polystyrene
column (Applied Biosystems) containing approximately 7 ml resin was
utilized for endotoxin removal, and capture and elution of purified
rhCTGF. Prior to the sample load, the tandem column was washed with
0.5 M NaOH, followed by 0.1 M NaOH, and finally equilibration
buffer. The load sample was passed over the tandem column, the
Q-Sepharose column was removed, and CTGF was eluted from the CM
POROS column (Applied Biosystems) with an increasing 350 mM to 1200
mM NaCl gradient. The purity of the eluted fractions containing
CTGF was evaluated by SDS-PAGE analysis before forming a final
sample pool.
Example 2
CTGF N-Terminal and C-Terminal Fragment Production
[0144] N-terminal fragments and C-terminal fragments of CTGF were
prepared as follows. Recombinant human CTGF, prepared and purified
as described above, was digested at room temperature for 6 hours by
treatment with chymotrypsin beads (Sigma Chemical Co., St. Louis,
Mo.) at 1.5 mg of CTGF per unit of chymotrypsin. The mixture was
centrifuged, the chymotrypsin beads were discarded, and the
supernatant, containing enyzmatically-cleaved rhCTGF, was diluted
1:5 with 50 mM Tris, pH 7.5. The diluted supernatant was applied to
a Hi-Trap heparin column. The flow-through, containing N-terminal
fragments of CTGF, was collected. The heparin column was washed
with 350 mM NaCl, and bound C-terminal fragments of CTGF were
eluted with a linear gradient of 350 mM to 1200 mM NaCl, as
described above. The fractions were analyzed by SDS-PAGE, and
fractions containing C-terminal fragments of CTGF were pooled.
[0145] The heparin column flow-through, which contained N-terminal
fragments of CTGF, was adjusted to 0.5 M ammonium sulfate/50 mM
Tris, pH 7.5 and then loaded onto a 15 ml phenyl sepharose HP
column (Amersham-Pharmacia), which had been pre-equilibrated with
0.5 M ammonium sulfate/50 mM Tris, pH 7.5. The column was washed
with 15 column volumes of 0.5 M ammonium sulfate/50 mM Tris, pH
7.5, and bound N-terminal fragments of CTGF were eluted with a
linear gradient of 0.5 M to 0 M ammonium sulfate/50 mM Tris, pH
7.5, over approximately 15 column volumes. Fractions were analyzed
by SDS-PAGE, and fractions containing N-terminal fragments of CTGF
were pooled. The pooled solution was concentrated and the buffer
exchanged with 50 mM Tris, 400 mM NaCl (pH 7.2), using an ULTRACEL
AMICON YM10 ultrafiltration membrane (Millipore Corp., Bedford
Mass.).
Example 3
Production of Human Anti-CTGF Monoclonal Antibodies
[0146] Fully human monoclonal antibodies to human CTGF were
prepared using HUMAB mouse strains HCo7, HCo12 and HCo7+HCo12
(Medarex, Inc., Princeton N.J.). Mice were immunized by up to 10
intraperitoneal (IP) or subcutaneous (Sc) injections of 25-50 mg
recombinant human CTGF in complete Freund's ajuvant over a 2-4
weeks period. The immune response was monitored by retroorbital
bleeds. Plasma was screened by ELISA (as described below), and mice
with sufficient titers of anti-CTGF immunogolobulin were used for
fusions. Mice were boosted intravenously with antigen 3 and 2 days
before sacrifice and removal of the spleen.
[0147] Single cell suspensions of splenic lymphocytes from
immunized mice were fused to one-fourth the number of P3X63-Ag8.653
nonsecreting mouse myeloma cells (American Type Culture Collection
(ATCC), Manassas Va.) with 50% PEG (Sigma, St. Louis Mo.). Cells
were plated at approximately 1.times.105 cells/well in flat bottom
microtiter plate and incubated for about two weeks in high-glucose
DMEM (Mediatech, Herndon Va.) containing L-glutamine and sodium
pyruvate, 10% fetal bovine serum, 10% P388D1 (ATCC) conditioned
medium, 3-5% origen (Igen International, Gaithersburg Md.), 5 mM
HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/ml gentamycin, and
1.times.HAT (Sigma). After 1-2 weeks, cells were cultured in medium
in which the HAT was replaced with HT. Individual wells were then
screened by ELISA (described below). Antibody secreting hybridomas
were replated, screened again, and, if still positive for anti-CTGF
antibodies, were subcloned at least twice by limiting dilution. The
stable subclones were then cultured in vitro to generate small
amounts of antibody in tissue culture medium for characterization.
One clone from each hybridoma that retained the reactivity of the
parent cells was used to generate 5-10 vial cell banks stored in
liquid nitrogen.
[0148] ELISA assays were performed as described by Fishwild et al.
(1996, Nature Biotech 14:845-851). Briefly, microtiter plates were
coated with 1-2 .mu.g/ml purified recombinant CTGF in PBS at 50
.mu.l/well, incubated at 4.degree. C. overnight, then blocked with
200 .mu.l/well 5% chicken serum in PBS/Tween (0.05%). Dilutions of
plasma from CTGF-immunized mice or hybridoma culture supernatants
were added to each well and incubated for 1-2 hours at ambient
temperature. The plates were washed with PBS/Tween and then
incubated with a goat-anti-human IgG Fc polyclonal antibody
conjugated with horseradish peroxidase (HRP) for 1 hour at room
temperature. After washing, the plates were developed with 0.22
mg/nil ABTS substrate (Sigma) and analyzed by spectrophotometer at
415-495 nm.
Example 4
Antibody Characterization
[0149] Hybridomas that produced antibodies to human CTGF were
prepared as described in Example 3. Cloned hybridoma cells were
grown in Dulbecco's Modified Eagle Medium-High Glucose/RPMI 1640
(50:50) with 8 mM L-Glutamine, 1/2.times. Nonessential Amino Acids,
and 10% Fetal Bovine Serum. Cells expanded for antibody preparation
were grown in the same media with 1.5% Low IgG Fetal Bovine Serum
for 4-9 days at 37.degree. C. and 6% CO.sub.2. The resulting
conditioned media was cleared of cells and concentrated using a
tangential flow filtering/concentrating system. The concentrate was
passed over a protein-A column and bound monoclonal antibodies
eluted with 100 mM glycine, pH 3. The eluate was neutralized with 1
M Tris, pH 8.0, and dialyzed against PBS.
4.1 Epitope Mapping
[0150] Epitope mapping of antibodies by competitive binding
experiments is well known by those skilled in the field of
immunology. (See, e.g., Van Der Geld et al. (1999) Clinical and
Experimental Immunology 118:487-96.) Each antibody population
isolated from cells propagated from a unique cloned hybridoma cell
was mapped and assigned to a specific binding domain on human CTGF
using standard binding and blocking experiments. (See, e.g.,
Antibodies: A Laboratory Manual (1988) Harlow and Lane (eds), Cold
Spring Harbor Laboratory Press; Tietz Textbook of Clinical
Chemistry, 2.sup.nd ed., (1994) Chapter 10 (Immunochemical
Techniques), Saunders; and Clinical Chemistry: Theory, Analysis,
Correlation (1984) Chapter 10 (Immunochemical Techniques) and
Chapter 11 (Competitive Binding Assays), C.V. Mosby, St. Louis.)
Independent binding domains were initially defined by antibody
competition experiments in which two different antibodies were
incubated in sequential order on CTGF coated plates. If steric
hindrance from the first antibody prevented the second antibody
from binding to CTGF, then the two antibodies were assigned to the
same binding domain. It should be understood, however, that two
antibodies might have distinct epitopes yet be near enough to each
other to be designated as members of the same binding domain.
[0151] Binding domains spanning all four exons of human CTGF were
identified. All of the binding domains are conformationally
defined, such that the antibodies bind to CTGF under non-reducing
conditions in western blot assays. Some of the antibodies also
bound to CTGF under reducing conditions in western blot assays,
suggesting that each of these antibodies bound to a linear epitope
on the CTGF protein. Also, antibodies representing a subset of the
binding domains show cross-reactivity to mouse CTGF in western blot
analysis. The antibody from each group having the highest affinity
for whole CTGF was used for further characterization and
analysis.
[0152] More refined epitope mapping was performed by ELISA analysis
using specific recombinantly expressed fragments of CTGF. For
example, antibodies that recognized epitopes on the N-terminal
domain of CTGF were identified by ELISA analysis against
immobilized fragments obtained from recombinant expression of exon
2 and/or exon 3 of the CTGF gene. In this manner, antibodies that
specifically recognize N-terminal domains or N-terminal fragments
of CTGF were selected and further characterized. Antibodies that
specifically recognize C-terminal domains or C-terminal fragments
of CTGF were also selected and further characterized.
[0153] The epitope group defined by mAb1 binds to a linear epitope
on the N-terminal fragment of CTGF encoded by exon 3. A series of
truncated synthetic peptides covering regions encoded by the
polynucleotide of exon 3 were generated, and ELISA tests using
these peptides were conducted to further define the epitope of
mAb1. The results are summarized in Table 1; a "+" indicates
binding between the peptide and mAb1, whereas a "-" indicates mAb1
does not bind to the peptide. A boldfaced italic "C" indicates a
cysteine residue in the peptide that was essential for mAb1
binding. An underlined "C" indicates a cysteine residue added to
the end and not a part of the native CTGF sequence.
TABLE-US-00001 TABLE 1 mAb1 binding to truncated peptide series
encoded by exon 3. mAb1 SEQ Peptide Sequence binding ID NO: N-CTGF
+ 27 Exon 3 + 28 Pep135 CPLCSMDVRLPSPDCPFPRRVKLP + 22 PC5444
PLSSMDVRLPSPDS - 33 PC5445 RLPSPDSPFPRRVKLPGK + 23 PEP5
RLPSPDCPFPRRVKL + 24 P40340 RLPSPD PFPRRV + 25 P40341 RLPSPDSPFPRRV
- 34 P40342 LPSPD PFPRRVKL + 26 10MER SPDSPFPRRV - 35 10MER2
SPDCPFPRRV - 36 9MER PDSPFPRRV - 37 9MER2 CPFPRRVKL - 38 8MER
DSPFPRRV - 39 8MER2 CFPRRVKL - 40 7MER CPRRVKL - 41 6MER CRRVKL -
42 5MER CRVKL - 43
[0154] Therefore, mAb1 is a member of an antibody class that binds
to the N-terminal region of CTGF. The linear epitope on CTGF
necessary and sufficient for binding of mAb1 is defined by amino
acid residue L143 through V154 of human CTGF (SEQ ID NO:2). Further
confirmation of mAb1 binding specificity for this peptide was
obtained by RIA and affinity chromatography. Antibodies that share
this epitope, in part or in whole, are specifically included in the
present invention. Additionally, antibodies that compete with mAb1
for binding to CTGF or a fragment thereof are also specifically
included in the present invention.
4.2 Antibody Affinity for CTGF
[0155] Antibody affinity is defined as the strength of the total
noncovalent interactions between a single antigen-binding site on
an antibody and a single epitope on an antigen. Affinity is
calculated by measuring the association constant (K.sub.a), such
that
Affinity = K a = [ Ab Ag ] [ Ab ] [ Ag ] = 1 K d ##EQU00002##
where [Ab] is the concentration of tree antigen binding site on the
antibody, [Ag] is the concentration of free antigen, [AbAg] is the
concentration of antigen binding site on the antibody occupied by
antigen, and K.sub.d is the dissociation constant of the
antibody-antigen complex.
[0156] The affinity of each antibody population identified by
epitope mapping was measured using RIA, wherein whole rhCTGF was
radio-iodinated and added to wells containing immobilized
monoclonal antibody, as follows. Recombinant human CTGF was
radiolabeled with .sup.125I using the chloramine-T method. (See,
Greenwood et al. (1963) Biochem J 89:114-123.) Typically, at least
60% of the .sup.125I was incorporated and the specific activity of
the labeled CTGF was at least 1.times.10.sup.5 cpm/ng, although
labeled CTGF of lower specific activity can be used in the
radioimmunoassay. Goat anti-human IgG, .gamma.Fc-specific capture
antibody (Jackson ImmunoResearch) in Ca.sup.2+- and Mg.sup.2+-free
DPBS (Mediatech, Herndon Va.) was added to the wells of a MAXISORP
BREAKAPART microtiter plate (Nalge Nunc International, Rochester
N.Y.) and allowed to bind overnight at 4.degree. C. The wells were
then blocked with 1% BSA in Ca.sup.2+- and Mg.sup.2+-free DPBS for
at least 4 hours at 4.degree. C. The blocking solution was removed
and 100 .mu.l of test antibody at 2-50 ng/ml in Ca.sup.2+- and
Mg.sup.2+-free DPBS was added and allowed to bind overnight at
4.degree. C. Mixtures of serial dilutions of unlabelled CTGF in a
constant amount of [.sup.125I]rhCTGF were added to wells and
incubated at room temperature for 4 to 8 hours. Wells were then
washed four times with 0.1% Tween 20 in Ca.sup.2+- and
Mg.sup.2+-free PBS (Mediatech), and the wells of the microtiter
plate were separated and counted in a gamma counter.
[0157] Affinity was estimated graphically by the method of
Scatchard (1948, Ann NY Acad Sci 51:660-72). The total
concentration of labeled CTGF applied to the plate was calculated
as
[ CTGF ] total = cpm_applied cpm / fmol 1 0.1 _ml + [ CTGF ] cold _
stock dilution ##EQU00003##
where cpm_applied are counts obtained from control vials, which are
loaded with CTGF mixtures in parallel with the wells of the
microtiter plate; cpm/fmol is the specific activity of the
[.sup.125I]CTGF, [CTGF].sub.cold.sub._.sub.stock is the
concentration of unlabelled CTGF added to each well, and dilution
is the dilution factor for the unlabelled CTGF.
[0158] The concentration of CTGF bound to antibody is calculated
from the proportion of counts bound to the wells and the total
concentration of CTGF applied to the wells.
[ CTGF ] bound = ( cpm_bound - blank ) cpm_total [ CTGF ] total
##EQU00004##
[0159] The concentration of free (unbound) CTGF is the difference
between the total concentration of CTGF applied and the
concentration of bound CTGF.
[CTGF].sub.free[CTGF].sub.total-[CTGF].sub.bound
[0160] Scatchard plots of affinity determinations for antibodies of
the invention are shown in FIG. 2. FIG. 2A plots the binding of an
antibody of the invention, mAb2, to [.sup.125I]rhCTGF in the
presence of increasing concentrations of unlabeled rhCTGF. FIG. 2B
plots the binding of an exemplary antibody of the invention, mAb1,
to [.sup.125I]rhCTGF in the presence of increasing concentrations
of unlabeled rhCTGF. Greater weight is given points with similar
proportions of bound and unbound CTGF, because these points will
have bound counts in substantial excess of the blanks (hence,
well-determined bound counts), but still substantially less than
the total counts applied (hence, well-determined free counts).
Maximum binding (B.sub.max) and K.sub.d are represented as the
x-intercept and y-intercept, respectively.
[0161] The affinity (K.sub.d) of mAb1 for CTGF is less than
10.sup.-9M, the affinity typically found in commercially successful
antibody therapeutics. (See, e.g., Maini et al. (1998) Arthritis
Rheum 41:1552-1563; Targan et al. (1997) N Engl J Med
337:1029-1035; Bumgardner et al. (2001) Transplantation 72:839-45;
and Kovarik et al. (1999) Transplantation 68:1288-94.) Thus, mAb1
is a suitable candidate for therapeutic use, and antibodies that
share epitope binding with mAb1, as described above, and have an
affinity for CTGF that is similar to or greater than mAb1 (that is,
a K.sub.d.ltoreq.10.sup.-9) are likewise suitable candidates for
therapeutic use. Antibodies sharing epitope binding with mAb1, but
have lower affinity (i.e., higher K.sub.d) than mAb1, are also
embodied within the present invention and are potentially useful in
various assays and diagnostic applications as described herein.
Such antibodies may additionally be useful in therapeutic
applications, especially if they have a high avidity for antigen,
as described below.
4.3 Antibody Avidity
[0162] For antibodies with more than one antigen-binding site
(multivalency), the affinity at one binding site does not always
reflect the true strength of the antibody-antigen interaction. When
a multivalent antibody binds to an antigen having multiple
repeating epitopes, the interaction of one antigen interaction at
one binding site on the antibody increases the chance of antigen
interaction with the additional binding sites. Avidity measures the
functional combining strength of an antibody with its antigen,
which is related to both the affinity of the reaction between the
epitopes and paratopes, and the valencies of the antibody and
antigen. Thus, avidity provides a more accurate measure of an
antibody's tendency to dissociate.
[0163] High avidity can compensate for low affinity. For example,
IgM antigen-binding sites are generally lower affinity than IgG,
but the multivalency of IgM gives it a high avidity, thus enabling
it to bind antigen effectively.
[0164] To determine the avidity of antibodies of the invention, Fab
fragments were first prepared by conventional papain digestion of
the corresponding immunoglobulin. Immobilized Protein A was then
used to separate the Fab fragments from the Fc and undigested
antibody.
[0165] Approximately 1 ml immobilized papain slurry containing 0.5
ml settled gel, 250 .mu.g papain, and 3.5 BAEE units was washed
3.times.1 ml and 1.times.10 ml with Digestion Buffer (DB; 20 mM
sodium phosphate, 10 mM EDTA, 20 mM cysteine, pH 7.0). The slurry
was then resuspended with 0.3 ml DB, mixed with 1.1 ml antibody
(approximately 5 mg, pH 7), and agitated overnight at 37.degree. C.
The antibody digest was then separated from the resin, and Fab
fragments were separated from Fc fragments and undigested antibody
by affinity chromatography using Protein A. Purity of Fab fragment
was monitored by SDS-PAGE (FIG. 3A).
[0166] Monovalent binding was distinguished from bivalent binding
by eluting antigen-bound antibodies with varying concentrations of
thiocyanate. By increasing chaotropic ion (thiocyanate)
concentration in the solution, lower affinity associations (e.g.,
monovalent binding of Fab to antigen) are disrupted first, while
higher affinity associations (e.g., bivalent binding of IgG to
ligand) remain undisturbed. Thus, by increasing thiocyanate
concentration, two different bindings can be distinguished.
[0167] Plates were coated with 10 .mu.g/ml CTGF or CTGF peptides in
50 mM bicarbonate buffer (pH 8.5) at 4.degree. C. overnight,
blocked with blocker casein/TBS at 4.degree. C. overnight, and then
incubated with 100 .mu.g/ml antibody or corresponding Fab in
blocker casein/TBS at room temperature overnight with agitation.
Plates were then incubated with dilutions (1:1) of thiocyanate
(0-7.6 M) in 100 mM phosphate buffer (pH 6.0) for 15 minutes at
room temperature with agitation, followed by an alkaline
phosphatase-mouse anti-human (Fab').sub.2 conjugate (1:1000
dilution) at room temperature for 45 minutes. Alkaline phosphatase
substrate (1 mg/ml; Sigma) in 1 M diethanolamine, 0.5 mM MgCl.sub.2
(pH9.8) was added, plates were incubated at room temperature, and
the absorbance at 405 nm was determined after 2, 10, 20, and 60
minutes.
[0168] The affinity index is the concentration of chaotropic agent
(thiocyanate) that produces a 50% reduction in initial absorbance.
For an exemplary antibody of the invention, mAb1, the affinity
index for dissociation of Fab from CTGF was 0.46 M, whereas the
affinity index for dissociation of intact IgG from CTGF was 1.8 M
(FIG. 3B). Thus, mAb1 binds to antigen predominantly bivalently
(avidity), and dissociates from antigen much more slowly than an
antibody that binds monovalently. Additional antibodies of the
invention, which share epitope-binding parameters with mAb1, may be
similary bivalent or they may be mono- or multi-valent. Any of the
antibodies of the invention may be manipulated to improve avidity,
e.g., by combining epitope-binding sites into a single antibody
construct, e.g., a tribody, etc. (See, e.g., Schoonjans et al.
(2000) J Immunol 165:7050-7057.)
4.4 Cross-Reactivity
[0169] The radioimmunoassay described above (Example 4.2) was used
to determine cross-reactivity of the antibodies, except unlabelled
rhCTGF was replaced with another unlabelled competitor, rat CTGF
derived from normal rat kidney (NRK) cells. NRK cells were grown
until confluent and then the culture media was changed to
serum-free media containing 2 ng/ml TGF-.beta.2, 50 .mu.g/ml
heparin, and 250 .mu.g/ml BSA. Conditioned medium was collected
after two days of culture, centrifuged to remove debris, and
incubated with heprin-sepharose beads ( 1/100 v/v bead
suspension:medium) for 2 hrs at 4.degree. C. with agitation. The
mixture was then centrifuged; the beads were collected and washed
with PBS, and then lysed in SDS buffer.
[0170] A Scatchard plot of the binding of mAb2 to [.sup.125I]rhCTGF
in the presence of increasing concentrations of unlabeled rat CTGF
is shown in FIG. 4A; and a Scatchard plot of the binding of mAb1 to
[.sup.125I]rhCTGF in the presence of increasing concentrations of
unlabeled rat CTGF is shown in FIG. 4B. As can be seen in the
figure, mAb1 binds to both human and rat CTGF, while mAb2 binds to
human but does not bind to rat CTGF.
[0171] For mAb1, the Scatchard plots for competition with rat CTGF
(FIG. 4B) have shallower slopes, lower apparent affinity, and
higher apparent B.sub.max than the plots for competition with
rhCTGF (FIG. 2B). Thus, although rat CTGF is able to compete with
human CTGF for binding to mAb1, the antibody has higher affinity
for recombinant human CTGF than for recombinant rat CTGF. mAb1 also
cross-reacts with mouse and monkey CTGF (data not shown).
Antibodies showing suitable affinity for CTGF from other species
may be used in treatment and prevention of disorders in those
species. For example, an antibody of the invention that shows a
suitable K.sub.d for canine CTGF could be used to treat a
CTGF-associated disorder in dogs. Antibodies of the invention that
show cross-species affinity, such as mAb1, are also useful as
research tools, to study CTGF-associated disorders in various
animal models.
4.5 Glycosylation
[0172] The radioimmunoassay described above (Example 4.2) was used
to determine the effect of antibody glycosylation on antigen
binding affinity. Antibody mAb1 was treated for 8 days at
37.degree. C. in PBS, 0.5 M EDTA, pH 8.0, with peptide
N-glycosidase F (PNGase F), which cleaves oligosaccharides from
N-linked glycoproteins. After incubation, the reaction solution was
either used directly or fractionated on a protein A-SEPHAROSE
FASTFLOW column (Amersham Bioscience, Piscataway N.J.) and eluted
with 0.1 M glycine-HCl, pH 2.5. Antibody recovery after
fractionation was approximately 87%, and the endotoxin level was
0.30 EU/mg. Deglycosylation was confirmed by SDS-PAGE. Binding
activity of deglycosylated antibody to human recombinant CTGF was
identical within experimental error to the binding activity of the
glycosylated form of the antibody.
[0173] As various cells produce different glycosylation patterns,
production of recombinant proteins, e.g., antibodies, in cultured
cells or non-homologous species may generate non-native
glycosylation. Some proteins require specific glycosylation for
activity, and altered glycosylation reduces activity; e.g., in the
case of antibodies, affinity for antigen is reduced. Protein
production in certain systems, e.g., plants and chicken eggs, may
also produce glycosylation patterns that are immunogenic, thus
reducing the ability to use the proteins in certain applications.
The ability of the present antibodies to show the same activity in
a glycosylated and non-glycosylated form demonstrate that the
invention is not limited by the presence of glycosylation,
particularly a species-specific glycosylation.
Example 5
Cell Migration Assay
[0174] Cell migration is a normal and important cellular event,
e.g., during development and wound healing. Cell migration is also
a factor in the pathology of disorders such as formation of
fibrotic lesions, and cells isolated from fibrotic lesions are more
responsive to chemotactic stimulants than cells from corresponding
normal tissue.
[0175] Antibodies of the present invention were analyzed for their
ability to inhibit CTGF-stimulated chemotactic migration of smooth
muscle cells using a Boyden chamber assay as follows. Rat arterial
smooth muscle cells (ASMCs) in media containing 0.1% fetal calf
serum (FCS) were added to the upper compartment of a Boyden
chamber, and media containing either 300 ng/ml rhCTGF, 10% FCS, or
0.1% FCS alone was added to the lower compartment. A
collagen-coated filter having pores with a diameter of 8 .mu.m
separated the upper chamber from the lower chamber. Cells were
allowed to adhere to and migrate through the filter for 2-3 hours.
The filter was then removed, the cells on the filter were fixed and
stained, and the cells that migrated through the filter were
counted. Incubation with 300 ng/ml rhCTGF increased the number of
cells migrating through the filter approximately 5-fold relative to
0.1% FCS controls. The increase in migration stimulated by CTGF was
approximately 27% of the chemotactic effect seen with 10% FCS,
which contains multiple chemotactic factors.
[0176] The antibodies of the invention were tested for their
ability to inhibit CTGF-mediated cell migration using the assay
described above, except either anti-CTGF antibody (at 30 and 300
mg/ml) or pooled human IgG was also added to the lower chamber.
Four fields of cells from each of 3 separate filters were counted
for each sample in each assay. Results are shown in Table 2.
TABLE-US-00002 TABLE 2 Inhibition of CTGF-mediated cell migration.
Cell Migration (%) Antibody Average SD hIgG 100 10 7 (30 .mu.g/ml)
77 13 19 (30 .mu.g/ml) 71 14 19 (300 .mu.g/ml) 43 12
[0177] As can be seen in Table 2, antibodies that bind to CTGF
within the epitope defined by mAb1 inhibit CTGF-mediated cell
migration in a dose-dependent manner. The antibodies of this
epitope group were the only anti-CTGF antibodies tested that
repeatedly and reproducibly inhibited CTGF-induced migration.
[0178] Various processes, such as angiogenesis, chondrogenesis, and
oncogenesis require alterations in cell adhesion and migration.
CTGF has been associated with both cell adhesion and migration, and
the ability of antibodies directed against CTGF to differentially
affect one activity versus another provides a diverse repertoire of
therapeutic agents for the treatment of CTGF-associated conditions.
The antibodies provided by the present invention clearly
demonstrate differential activity relating to neutralization of
CTGF activities. As exemplified below, these abilities provide
unique therapeutic potential in this class of anti-CTGF
antibody.
Example 6
Pulmonary Disorders
[0179] The intratracheal (IT) instillation of bleomycin in mice is
a model system widely used for studying lung fibrosis and for
screening potentially desirable antifibrotic agents. Antibodies of
the invention were tested for their ability to reduce
bleomycin-induced lung fibrosis in vivo using the procedure
described by Wang et al. (2000) Biochem Pharmacol 60:1949-1958, as
follows.
[0180] Male C57BL/6 mice were randomly divided into two groups.
Mice were anesthetized with isofluorane, and then injected
intratracheally with either a single dose of bleomycin in 0.9%
saline at 0.1 unit/50 .mu.l/mouse or 0.9% saline alone. Each group
was divided and treated immediately and thereafter once every other
day for a total of seven doses with either saline or antibody
administered intraperitoneally (IP). Fourteen days after the IT
instillation, mice were euthanized by exsanguination of the
descending abdominal aorta under anesthesia and lung tissue was
harvested.
[0181] Lung collagen content was analyzed by measuring the level of
hydroxyproline and proline using the method of Palmerini et al.
(1985; J Chromatogr 339:285-292), except that L-azetidine
2-carboxylic acid (Aldrich) was substituted for 3,4-dehydroproline
as the internal standard. Briefly, tissue samples were hydrolysed
in 6 N HCl for 22 hours at 105.degree. C. Samples underwent
pre-column derivitization with o-phthaladehyde and then
4-chloro-7-nitrobenzofuran (Aldrich) to form fluorescent adducts of
proline and hydroxyproline. The fluorescent adducts were separated
by reverse phase HPLC followed by fluorometric detection.
[0182] FIG. 5 shows the result of therapeutic administration of
saline (SA), an exemplary antibody of the invention, mAb1, and a
pool of CTGF-specific antibodies (AbsJ) were compared for their
ability to suppress lung fibrosis following bleomycin treatment. As
can be seen in FIG. 5A, bleomycin treatment (BL+SA) significantly
increased lung hydroxyproline content 168% over the control group
(SA+SA; 220.+-.15 .mu.g/lung). However, subsequent treatment with
pooled antibodies of the invention (BL+AbsJ) showed a 60% decrease
in lung hydroxyproline compared to the bleomycin-treatment alone.
Similarly, subsequent treatment with mAb1 (BL+mAb1) showed a 70%
decrease in lung hydroxyproline compared to bleomycin alone.
[0183] Histological examination of the mouse lungs revealed normal
pulmonary parenchymal tissue in the control group (not shown). In
bleomycin treated lungs, however, an increase in regions of
fibrosis was clearly seen (FIG. 5B; arrows). Therapeutic
administration of an antibody of the invention subsequent to
bleomycin treatment showed a clear reduction in fibrosis (FIG. 5C),
although some lobes still showed a mild degree of interstitial
fibrosis. Thus, antibodies of the invention provide therapeutic
benefit when administered to patients at risk for or suffering from
a pulmonary disorder such as idiopathic pulmonary fibrosis
(IPF).
Example 7
Renal Disorders
7.1. Renal Failure
[0184] Tubulointerstitial fibrosis is a major component of several
kidney diseases associated with the progression to end-stage renal
failure. (Sharma et al. (1993) Kidney Int 44:774-788.) Unilateral
ureteral obstruction (UUO), characterized by decreased renal
function and increased interstitial fibrosis, has been used as an
experimental model to induce tubulointerstitial damage and
fibrosis. (Fern et al. (1999) J Clin Invest 103:39-46.)
[0185] Mice were anesthetized with isofluorane, and then ligation
of the left ureter was performed according to the method described
by Moriyama et al. (1998; Kidney Int 54:110-119). Mice were treated
immediately following surgery and thereafter once every other day
for a total of seven doses with either saline or antibody
administered intraperitoneally (IP). Fourteen days after UUO,
animals were anesthetized and sacrificed by exsanguination of the
descending abdominal aorta. Both the right and left kidneys were
separately decapsulated and weighed. Half of each kidney was fixed
in 10% formalin for histology (trichrome stain) and the other half
was weighed and stored at -70.degree. C. for hydroxyproline
determination. Hydroxyproline and proline were determined as
described above.
[0186] As can be seen in FIG. 6A, UUO increased the kidney collagen
content approximately 4-fold, as measured by the hydroxyproline to
proline ratio of the obstructed left kidney relative to the
unobstructed right kidney in each mouse. Treatment with an antibody
of the invention, mAb1, resulted in a statistically significant
dose-dependent reduction in fibrosis of the obstructed kidney (FIG.
6A). However, an antibody that binds to a C-terminal epitope on
CTGF, mAb3, did not show significant effect. Trichrome staining of
UUO kidney identifies regions of increased collagen accumulation
(FIG. 6B, arrows), whereas treatment with an antibody of the
invention shows a considerable reduction in collagen staining in
the obstructed kidney (FIG. 6C).
[0187] Alternatively, kidney fibrosis can be studied in the rat
remnant kidney model of progressive renal failure. The model, which
involves 2/3 unilateral nephrectomy combined with complete renal
ablation contralaterally ( total nephrectomy), induces degenerative
parenchymal changes associated with chronic renal failure in the
renal remnant, and animals become uremic and exhibit marked
albuminuria, glomerulosclerosis, interstitial fibrosis and tubular
atrophy. (See, e.g., Frazier et al. (2000) Vet Pathol 37:328-335;
and Gandhi et al. (1998) Kidney Int 54:1157-1165.)
[0188] The nephrectomy was performed according to Frazier et al.
(2000, Vet Pathol 37:328-335). Five-week-old male Sprague-Dawley
rats (Harlan, Indianapolis Ind.) averaging 120 g were anesthetized
with ketamine and xylazine, and the cranial 1/3 and caudal 1/3 of
the left kidney was incised. A gauze sponge was briefly applied to
provide hemostasis, the abdomen was rinsed with saline, 0.2 ml
butorphenol, and the animal was sutured. One week after the initial
surgery, the contralateral kidney was removed completely.
[0189] Rats were divided into saline and antibody treated groups
with treatment initiated 2 weeks following nephrectomy. Saline or
antibody at a dosage of 5 mg/kg was administered by IP injection
(0.5 mL each) every 3 days for 15 days (a total of 5 injections).
Blood and urine samples were taken weekly from random
nephrectomized rats to follow the development of renal disease and
to correlate renal functional disturbance with histologic changes.
Results from renal fibrosis analysis, urinalysis and serum
chemistry assays were compared between the groups at 18 and 28 days
after treatment initiation.
[0190] Renal fibrosis was evaluated independently by two
pathologists in a blinded fashion; three histologic sections from
each kidney were examined using three distinct morphologic stains:
hematoxylin/eosin, Masson's trichrome and picric acid-sirius red.
In addition, immunohistochemistry was performed on frozen sections
to assess the type of collagen deposition at each location in the
kidney. Quantitative collagen evaluation (hydroxyproline/proline
ratio) was performed and renal function was assessed using both
urinalysis and serum chemistry of samples collected at the time of
euthanasia.
[0191] Histologically, moderate differences in fibrosis were noted
between untreated and antibody treated remnant kidneys (FIG. 7). At
3 days post-treatment, blinded subjective evaluation resulted in a
mean fibrosis score of 12.6 in saline treated group versus 10.7 in
antibody treated group (p<0.05). Statistically significant
differences in histologic fibrosis grade between antibody and
saline treated rats were maintained at 14 days post-treatment, with
a mean fibrosis score of 16.9 in the saline treated group versus
14.4 in the antibody treated group (p<0.05). The quantitative
hydroxyproline content analysis of collagen also demonstrated a
trend towards decreased fibrosis in the antibody-treated group
relative to the saline-treated group, but the difference was not
statistically significant.
[0192] Qualitative differences were also noted between treatment
groups. While most of the collagen deposition in the antibody
treated groups was limited to the corticomedullary and medullary
interstitium, the fibrosis in the saline treated rats was
multifocal to diffusely distributed throughout the cortex and
medulla. The most marked histopathologic differences were in the
amount of glomerular fibrosis. Many of the saline treated group had
moderate to severe glomerulosclerosis with pericapsular fibrosis,
thickened Bowman's membrane, synechia, and glomerular obsolescence.
These changes were minimal to mild in the other groups, including
the antibody treated rat kidneys. Collagen accumulation was
visualized with Masson's trichrome and picric acid-sirius red
stains.
[0193] In both models of progressive renal failure, antibody of the
invention reduced tissue degradation and improved kidney function.
Thus, antibodies of the invention provide therapeutic benefit when
administered to patients at risk for or suffering from a renal
disorder such as glomerulonephritis, IgA nephropathy,
glomerulosclerosis; and kidney failure and tubule destruction due
to toxins, etc.
7.2. Diabetic Nephropathy
[0194] Diabetes leads to failure of multiple organs including, but
not limited to, kidney, heart, and eye. A major component of the
pathological progression of diabetic organ failure is fibrosis. An
established model of diabetic nephropathy is a mouse carrying a
loss-of-function mutation in the leptin receptor (Ob-R; encoded by
the db gene). Key features in common between the db/db mouse and
human diabetic nephropathy include renal hypertrophy, glomerular
enlargement, albuminuria, and mesangial matrix expansion.
[0195] Antibodies of the invention were tested using the db/db
mouse model of diabetic nephropathy as follows. Eight-week-old
db/db mice (Harlan, Indianapolis Ind.) and their heterozygous db/+
littermates were treated by intraperitoneal injection with either
antibody of the invention (CLN1; see below) or control human IgG
(cIgG). In all animals, an initial injection of 300 .mu.g of
antibody was followed by 100 .mu.g doses administered 3 times per
week for 60 days. Blood samples were collected and body weights
were measured at the beginning of and periodically throughout the
treatment period. Food consumption was also recorded.
[0196] By 11 weeks, clear distinction existed between the diabetic
(db/db) animals and the non-diabetic (db/+) animals with respect to
body weight, blood glucose levels, and food consumption. Treatment
with either antibody of the invention or control antibody did not
significantly affect any of these parameters. However, various
measurements of kidney function demonstrated a clear difference
between diabetic and non-diabetic mice. As can be seen in Table 3,
diabetic mice showed increased kidney weight, creatinine clearance,
and albumin excretion rate (AER) relative to non-diabetic mice.
However, diabetic animals treated with antibody of the invention
showed normalized values for all the parameters. All data are
expressed as Mean.+-.SEM. The number of mice per group (n) ranged
from 9 to 15.
TABLE-US-00003 TABLE 3 Kidney function in db/db and db/+ mice.
Animal Treat- Kidney wt Creatinine AER Group ment (mg) (ml/h)
(.mu.g/24 h) db/+ cIgG 133.8 .+-. 5.1 2.17 .+-. 0.29 0.30 .+-. 0.02
db/+ mAb1 141.0 .+-. 4.3 2.37 .+-. 0.19 0.23 .+-. 0.04 db/db cIgG
207.8 .+-. 3.9** 5.39 .+-. 0.36** 2.52 .+-. 0.20** db/db mAb1 177.4
.+-. 4.5* .sup. 2.76 .+-. 0.31.sup..DELTA. .sup. 0.98 .+-.
0.09.sup..quadrature. **P < 0.01 vs. db/+ mice. *P < 0.01 vs.
db/+ mice and P < 0.05 cIgG-treated db/db mice. .sup..DELTA.P
< 0.01 vs. cIgG-treated db/db mice. .sup..quadrature.P < 0.01
vs. db/+ mice and cIgG-treated db/db mice.
[0197] As CTGF is induced by high glucose and mediates various
activities including ECM production in tissues as a result of
damage, e.g., due to advanced glycation endproduct (AGE) formation
and accumulation, etc., pathologies associated with diabetes, such
as diabetic nephropathy, may be prevented using the antibodies of
the invention.
Example 8
Ocular Disorders
[0198] Increased expression of CTGF has been associated with
various ocular disorders including proliferative vitreoretinopathy
(PVR), macular degeneration, and diabetic retinopathy. (See, e.g.,
Hinton et al. (2002) Eye 16:422-428; He et al. (2003) Arch
Ophthalmol 121:1283-1288; and Tikellis et al. (2004) Endocrinology
145:860-866.) The role of CTGF and the use of anti-CTGF
therapeutics has been proposed. (See International Publication No.
WO 03/049773.) The antibodies of the present invention represent a
unique, therapeutically efficacious class of anti-CTGF therapeutic
for use in such ocular disorders. The ability of antibodies of the
present invention to ameliorate complications in ocular disorders
is tested in models of ocular disease as follows.
8.1. Diabetic Retinopathy
[0199] Animal models of diabetes, e.g., db/db mice, are described
in Example 7.2, above. Any of these models can be used to
demonstrate the efficacy of treatment of diabetic retinopathy using
the antibodies of the invention. A particular model for diabetic
retinopathy is provided below, wherein animals are injected with
streptozotocin (STZ), a known toxin of the insulin-secreting
pancreatic .beta.-islet cells.
[0200] Diabetes is induced in rats (e.g., Long-Evans,
Sprague-Dawley, etc.) by injection, e.g., intraperitoneally, of
streptozotocin (STZ), e.g., at about 60 to 85 mg/kg body weight. To
improve survival, rats may be given 10% sugar water for 24 hours
and/or 2 to 4 units insulin per day following STZ injection.
Various factors including, e.g., body weight, urinary albumin
excretion rate, blood glucose, glycated hemoglobin, blood pressure,
etc., are measured after, e.g., 4, 8, and 12 weeks. Control animals
injected with buffer alone are followed concurrently. One half of
the STZ-treated and control rats are additionally treated with
antibody of the invention injected, e.g., intravenously,
intraperitoneally, or intraocularly. Throughout the study animals
are given access to food and water ad libitum. Animals are
sacrificed at 12 weeks, and eyes are harvested and examined for
histological changes.
[0201] A reduction in pathological changes in antibody-treated
animals relative to non-treated controls is indicative of
therapeutic efficacy in diabetic retinopathy. As CTGF is induced by
high glucose and mediates various activities including ECM
production in tissues as a result of damage, e.g., due to advanced
glycation endproduct (AGE) formation and accumulation, etc.,
pathologies associated with diabetes, such as diabetic retinopathy,
may be prevented using anti-CTGF therapeutics. (See, e.g.,
International Publication No. WO 03/049773.) The antibodies of the
present invention represent a unique, therapeutically efficacious
class of anti-CTGF therapeutic for use in ocular disorders, e.g.,
diabetic retinopathy.
8.2. PVR
[0202] Rabbit retinal pigmented epithelial (RPE) cells are isolated
from adult rabbit eyes and cultured in DMEM supplemented with 10%
fetal bovine serum. Subconfluent cultures (typically at passage 2
to 3) are used for all subsequent injections. At the time of
injection, cultured RPE cells are collected and suspended in PBS to
approximately 2.5.times.10.sup.6 cells/ml. Approximately 0.2 mls of
aqueous humor is removed from each recipient rabbit eye using a
25-gauge needle, and then RPE cells are injected through the sclera
to a point three millimeters posterior to the limbus just over and
above the optic disc using a 27-gauge needle. Following injection
of RPE cells, either 0.1 ml PDGF BB (50-150 ng), CTGF (200-400 ng),
or PDGF and CTGF in PBS is injected through the same entrance site.
The non-injected eye of each animal serves as a control.
Optionally, CTGF can additionally be injected on day 7 and/or day
14 following first injection. One half of the animals are
additionally treated with antibody of the invention injected, e.g.,
intravenously, intraperitoneally, or intraocularly. Depending on
the administration site, antibody may be provided daily or
administered less frequently, e.g., on days 7, 10, 14, etc.
[0203] Animals are examined using indirect ophthalmoscopic
procedures to monitor development and extent of PVR, which is
classified according to parameters described by Fastenberg.
(Fastenberg et al. (1982) Am J Ophthalmol 93:565-572.) Animals are
then sacrificed and eyes are analyzed by histological examination
for both extent of membrane formation and fibrosis. Additionally,
retina and fibrotic membrane may be collected for measurement of
collagen content.
[0204] Alternatively, PVR is induced in rabbit eyes using
subretinal injection of dispase, using the model and a procedure
adapted from Frenzel et al. (1998, Invest Ophthamol Vis Sci
39:2157-2164.) A subretinal bleb is formed using 50 ml (0.05 U) of
Dispase (Sigma Chemical Co.) in PBS. One half of the animals are
additionally treated with antibody of the invention injected, e.g.,
intravenously, intraperitoneally, or intraocularly. Retinal
detachment is induced in approximately 75% of the injected rabbits
not receiving antibody of the invention one week after surgery, and
in approximately 100% of these animals two weeks following surgery.
Epiretinal membranes are examined for extent of fibrosis.
[0205] A reduction in pathological changes in antibody-treated
animals relative to non-treated controls is indicative of
therapeutic efficacy in PVR. As CTGF has been associated with
tissue damage in models of PVR, anti-CTGF agents have been proposed
as therapeutics for use in such disorders. (See, e.g.,
International Publication No. WO 03/049773.) The antibodies of the
present invention represent a unique, therapeutically efficacious
class of anti-CTGF therapeutic for use in ocular disorders, e.g.,
PVR.
Example 9
Sclerosis
[0206] Sclerosis is generally characterized by diffuse fibrosis,
degenerative changes, and vascular abnormalities in the skin
(scleroderma), joints, and internal organs, especially the
esophagus, GI tract, lung, heart, and kidney.
9.1. Localized Granuloma Induction
[0207] Newborn mice develop a persistent localized fibrosis when
administered a combination of human-derived TGF-.beta.2 and CTGF by
subcutaneous injection over 7 consecutive days. (Mori et al. (1999)
J Cell Physiol 181:153-159; Shinozaki et al. (1997) Biochem Biophys
Res Commun 237:292-297.)
[0208] One day after birth, mice were divided into three treatment
groups and administered 40 .mu.l 1% mouse serum albumin (MSA), PBS
containing either 800 ng TGF-.beta.2, 400 ng CTGF, or both
TGF-.beta.2 and CTGF by subcutaneous injection into the subscapular
region for 7 consecutive days. The combination TGF-.beta.2 and CTGF
group was further divided into two groups, with one group
additionally receiving 40 antibody of the invention, mAb1. On Day
11, animals were sacrificed and sections of the injection sites
were processed and stained with Mason's trichrome for histological
assessment. The slides were randomized and evaluated qualitatively
in a blinded fashion by three scientists; scoring ranged from 0 (no
change) to 4 (fibrotic tissue) based on the degree of fibrosis or
connective tissue expansion (see FIG. 8). Cumulative scores from
each slide from all the individual reviewers were then calculated
and the mean value compared among the groups using ANOVA test.
[0209] Group mean scores for the vehicle control, TGF-.beta.2, and
TGF-.beta.2 and CTGF combination were 0.75, 6.83 and 9.00,
respectively (Table 4).
TABLE-US-00004 TABLE 4 Histological scoring of granuloma in
neonatal mice Treatment Mean Score Std Err Group Size Vehicle 0.75
0.48 4 TGF-.beta.2 6.83 0.65 6 TGF-.beta.2 + rhCTGF 9.00 0.72 7
TGF.beta.2 + CTGF + mAb1 6.17 1.40 6 TGF.beta.2 + CTGF + FG-3025
7.50 1.50 4 .sup.1Group scoring of slides from 3 different
readers.
[0210] Group mean score for antibody treatment was 6.17, a
statistically significant reduction when compared with the
corresponding TGF-.beta.2 and CTGF combination (p<0.05), while
treatment with a C-terminally directed, non-neutralizing anti-CTGF
antibody, mAb3, did not reduce fibrosis. Thus, antibodies of the
present invention are particularly effective at reducing local
sclerotic damage to tissues.
9.2. Neonatal Systemic Fibrosis
[0211] Newborn mice were divided into groups and administered daily
intraperitoneal injections for 21 consecutive days with 300
.mu.g/kg/day TGF.beta., 300 .mu.g/kg/day CTGF, a combination of 300
.mu.g/kg/day each TGF.beta. and CTGF, or the combination of
TGF.beta. and CTGF preceded by IP administration of 5 mg/kg
antibody of the invention, mAb1, 30 min prior to growth factor
treatment. Pups remained with their mothers during the course of
treatment. On Day 21, animals were sacrificed, major organs were
removed, and total proline and hydroxyproline were measured as
above.
[0212] Daily injections of TGF.beta. induced minor systemic
fibrosis, whereas CTGF alone produced no response. The combination
of TGF.beta. and CTGF induced systemic fibrosis with extensive
collagen deposition in several organs (FIG. 10) including liver,
lung, heart, GI tract, diaphragm and kidney; extensive intestinal
adhesions; and a 25% increase in mortality. Administration of the
antibody of the invention in conjunction with growth factor
treatment reduced or prevented organ fibrosis (FIG. 10) and
intestinal adhesions, and prevented mortality. Thus, antibodies of
the present invention are additionally effective when administered
systemically at reducing sclerotic damage to various tissues and
organs. The results presented in Examples 10.1 and 10.2 clearly
demonstrate that the antibodies of the invention, when administered
locally or systemically, are therapeutically efficacious for the
treatment of sclerotic conditions.
9.3. Scleroderma
[0213] The antibodies of the invention can be used to ameliorate
fibrosis associated with scleroderma. Methods that measure the
extent and severity of skin disease in scleroderma are known within
the art. (See, e.g., Rodnan et al. (1979) Arthritis Rheum
22:130-40; Aghassi et al. (1995) Arch Dermatol 131:1160-1166;
Brennan et al. (1982) Br J Rheumatol 31:457-460; Kahaleh et al.
(1986) Clin Exp Rheumatol 4:367-369; Falanga and Bucalo (1993) J Am
Acad Derm 29:47-51; Seyger et al. (1997) J Am Acad Derm 37:793-796;
Seyger et al. (1998) J Am Acad Dermatol 39: 220-225; Black (1969)
Br J Dermatol 81:661-666; Ballou et al. (1990) J Rheumatol
17:790-794; and Enomoto et al. (1996) J Am Acad Dermatol
35:381-387.)
[0214] For example, modified Rodnan skin score measures skin
hardness using a Type OO Rex DD-3 digital durometer (Rex Gauge
Company, Buffalo Grove Ill.) in standardized durometer units with
0.1 unit resolution. Durometer measurements are performed at all
the same skin sites as measured by Rodnan skin scoring. Skin scores
and durometer readings are performed at baseline screening, prior
to administration of an antibody of the invention, and every three
months throughout the dosing and follow-up periods. Each
measurement is repeated four times, and a structured analysis of
variance and calculation of intraclass correlation coefficients is
used to determine between repetition variability in relation to
site and patient variability. (Fleiss (1971) Psychol Bull
76:378-382.) Correlation techniques are also used to assess the
concordance between skin scores and durometer scores, both for
overall scores and for sub-group scores, at a given point in time.
Lagged correlation analyses (e.g., relating durometer scores at
entry to skin scores at time t+3 months, or t+6 months of treatment
with antibody) is also performed. Disease activity and functional
status information can also be collected, including collagen
synthesis data (PIIINP measurements). Reduction in symptoms and/or
complications of scleroderma as measured using any of the methods
described above demonstrates therapeutic efficacy of the antibodies
of the present invention.
Example 10
Osteoarthritis
[0215] Antibodies of the present invention are tested in one of the
following models to demonstrate therapeutic efficacy in
osteoarthritis. For the following examples, concentration of
antibody used is in the range of about 0.015 to 15 mg antibody per
kilogram of subject body weight; e.g., a dosage of about 5 mg
antibody per kilogram body weight, is considered appropriate.
[0216] Animals, e.g., 12-week-old male C57BL/6 mice, are housed in
standard cages and fed a standard diet with tap water ad
libitum.
10.1 Intra-Articular Injection of AdCTGF in Murine Knee Joints
[0217] A CTGF-containing adenovirus expression vector construct
(AdCTGF) is prepared using the ADEASY system (Qbiogene, Carlsbad
Calif.) according to procedures supplied by the manufacturer.
Briefly, a polynucleotide encoding full-length human CTGF is
inserted using standard molecular cloning techniques into a
PSHUTTLE-CMV plasmid (Qbiogene). The pShuttle-CMV-CTGF construct is
then linearized and co-transfected with PADEASY-1 plasmid
(Qbiogene) into competent E. coli BJ-5183 cells by electroporation.
AdCTGF is amplified and purified using procedures described by Kim
et al. (2001, J Biol Chem 276:38781-38786); an empty adenoviral
vector is used as a control. Plaque-forming units (range
1.0-2.1.times.10.sup.10/ml) and virus particles (range
0.9-1.5.times.10.sup.12/ml) are similar for AdCTGF and control
virus.
[0218] AdCTGF or control adenovirus (1.times.10.sup.7
plaque-forming units) is injected intra-articularly; and antibodies
of the invention are administered by intra-articular, intravenous,
intraperitoneal, or subcutaneous injection. Antibody may be
injected at the same time as adenoviral administration or,
alternatively, therapy may begin either before or after injection
of AdCTGF. Animals receiving control adenovirus are similarly
injected with either anti-CTGF antibody or control antibody.
Non-injected knee joints serve as controls for antibody
affects.
[0219] Knee joints are isolated on various days, e.g., 1, 3, 7, 14,
and/or 28 days, after AdCTGF injection, decalcified for 14 days in
EDTA/polyvinylpyrrolidone, and stored at -20.degree. C. using the
procedure described in Stoop et al. (2001, Osteoarthritis Cartilage
9:308-315). Histology of joints is analyzed to measure synovial
thickness and proteoglycan depletion; in situ hybridization and
immunohistochemistry is performed to identify CTGF expression, as
well as expression of additional factors including collagen (type I
and/or III), etc. Synovial fluid is collected to determine levels
of CTGF, metalloproteinases, etc. Efficacy of therapy using
anti-CTGF antibodies is confirmed by a reduction in parameters
associated with osteoarthritis relative to animals injected with
AdCTGF and treated with control antibody.
10.2 Intra-Articular Injection of AdTGF.beta. in Murine Knee
Joints
[0220] Alternatively, antibodies may be tested in the animal model
of osteoarthritis described by Bakker et al. (2001, Osteoarthritis
Cartilage 9:128-136). For example, antibody of the invention or
control antibody may be injected at the same time as, subsequent
to, or in advance of intra-articular injection of 1.times.10.sup.7
pfu TGF.beta.-expressing adenovirus construct (AdTGF.beta.).
Non-injected knee joints serve as controls for antibody effects. On
various days, e.g., Day 3, 7, 14, etc., animals from each group are
sacrificed and tissues are isolated and processed. Histology of
joints is analyzed to measure synovial thickness, proteoglycan
depletion, and osteophyte formation, etc.; in situ hybridization
and immunohistochemistry is performed to identify CTGF expression,
as well as expression of additional factors including collagen
(type I and/or III), etc. Synovial fluid is collected to determine
levels of TGF.beta., CTGF, metalloproteinases, etc. Efficacy of
therapy using antibodies of the invention is confirmed by a
reduction in parameters associated with osteoarthritis relative to
animals injected with AdTGF.beta. and treated with control
antibody.
10.3 Intra-Articular Injection of Papain in Marine Knee Joints
[0221] Alternatively, antibodies may be tested using the procedure
described in van der Kraan et al. (1989, Am J Pathol
135:1001-1014). Intra-articular injection of papain induces
osteophyte formation, fibrosis, and proteoglycan depletion from
articular cartilage. The papain model is initiated by injecting 1
unit of papain solution (Sigma, St. Louis, Mo.) into the right knee
joint of the mice. The left knee joint of each animal serves as an
internal control. Antibodies of the invention are administered by
intra-articular, intravenous, intraperitoneal, or subcutaneous
injection at the same time as, subsequent to, or in advance of
intra-articular injection of papain (0.5%/knee). On various days,
e.g., Day 3, 7, 14, etc., animals from each group are sacrificed
and tissues are isolated and processed. Histology of joints is
analyzed to measure synovial thickness, proteoglycan depletion, and
osteophyte formation, etc.; in situ hybridization and
immunohistochemistry is performed to identify CTGF expression, as
well as expression of additional factors including collagen (type I
and/or III), etc. Synovial fluid is collected to determine levels
of TGF.beta., CTGF, metalloproteinases, etc. Efficacy of therapy
using anti-CTGF antibodies is confirmed by a reduction in
parameters associated with osteoarthritis relative to animals
injected with papain and treated with control antibody.
Example 11
Cloning and Expression
[0222] Although the following example illustrates the cloning and
expression of one particular antibody of the invention, the methods
are generally applicable to all of the antibodies described and
claimed herein.
[0223] An exemplary antibody of the invention, mAb1, was first
identified as part of a complex human antibody secreted by a
hybridoma cell line (8C12-F10; prepared as described in Example
3).
11.1 Cloning and Sequencing of mAb1 Heavy Chain
[0224] Messenger RNA was isolated from a culture of 8C12-F10 cells
using a MICRO-FAST TRACK kit (Invitrogen) following protocols
provided by the manufacturer. Two cDNA pools were then produced by
second strand synthesis using a cDNA cell cycle kit (Invitrogen)
following protocols provided by the manufacturer and one of the
following heavy chain antisense primers:
TABLE-US-00005 AB90 (TGCCAGGGGGAAGACCGATGG; SEQ ID NO: 3) m19
H1504R (GCTGGGCGCCCGGGAAGTATGTA; SEQ ID NO: 4)
[0225] Heavy chain variable region sequences were cloned by PCR
amplification of the AB90-primed cDNA pool using AB90 primer and
one of a series of V-region primers, including primers
corresponding to conserved secretory signal sequences, which encode
the 5' end of the respective coding regions, and framework region 1
sequences, which encode the beginning of the mature
immunoglobulins. Pfu DNA polymerase (Stratagene) was used according
to recommended manufacturer's protocols, with the following
variations: Reactions were typically carried out in 50 .mu.l total
volume, containing 1 .mu.l cDNA, 0.75 .mu.M each forward and
reverse primer, 200 .mu.M each dNTP, and 1 .mu.l Pfu polymerase
(2.5 units per .mu.l). A countdown thermal cycler program was used
with an initial incubation at 94.degree. C. for 2 min prior to
addition of enzyme. The following cycle parameters were then used:
Ten cycles of 94.degree. C. for 45 seconds, 65.degree. C. for 45
seconds, and 72.degree. C. for 1 minute; thirty cycles of
94.degree. C. for 45 seconds, 55.degree. C. for 45 seconds, and
72.degree. C. for 1 minute; and then one cycle at 72.degree. C. for
ten 10 minutes.
[0226] Only one heavy chain signal sequence primer, AB87
(ATGGAGTTTGGRCTGAGCTG; SEQ ID NO:5), which binds to VH3 family
heavy chain V regions, produced significant product. The 453
nucleotide PCR product was cloned into PCR BLUNT II-TOPO vector
(Invitrogen), clones were screened for the correct insert size, and
three clones corresponding to the PCR products were sequenced.
Identical sequences were obtained for all three clones.
[0227] Heavy chain constant and UTR region sequences were cloned by
PCR amplification of the m19 H1504R-primed cDNA pool. A 601
nucleotide PCR fragment corresponding to the 5' end of the heavy
chain segment was amplified using sense primer VH3-33 29-51F
(CGGCGGTGTTTCCATTCGGTGAT; SEQ ID NO:6) and heavy chain constant
region antisense primer m19 H553R (GGGCGCCTGAGTTCCACGACAC; SEQ ID
NO:7). Topoisomerase-mediated cloning was used to clone the PCR
products into PCR-BLUNT II vector (Invitrogen), as directed by the
manufacturer, and the insert was then sequenced. Similarly, a 505
nucleotide PCR fragment was amplified using sense primer m19 H439F
(GTCTTCCCCCTGGCACCCTCCTC; SEQ ID NO:8) and antisense primer m19
H943R (CCCGCGGCTTTGTCTTGGCATTAT; SEQ ID NO:9), and a 503 nucleotide
PCR fragment was amplified using sense primer m19 H1002F
(CTGGCTGAATGGCAAGGAGTA; SEQ ID NO:10) and antisense primer m19
H1504R. Both fragments were separately cloned into PCR-BLUNT II
vector (Invitrogen) and sequenced as described above. A fourth
heavy chain PCR fragment of 586 nucleotides was amplified using
sense primer m19 H645F (GGGCACCCAGACCTACATC; SEQ ID NO:11) and
antisense primer m19 H1230R (CTCCGGCTGCCCATTGCTCTCC; SEQ ID NO:12)
and sequenced directly.
[0228] FIG. 11A shows a diagram of the alignment of the cloned PCR
fragments which provided the full length nucleotide sequence (SEQ
ID NO:13) that encodes the mAb1 heavy chain (SEQ ID NO:14). The
amino acid sequence of the heavy chain variable region most closely
resembles VH3 germ line gene DP-44. Although it was not possible to
tell which D segment had been used, the sequence of mAb1 most
closely resembles the DH4 family. The JH region most closely
matches germ line JH4 and JH5. The heavy chain constant region of
mAb1 matches GenBank Accession No. BC016381, indicating an allotype
of G 1 m(3).
11.2 Cloning and Sequencing of mAb1 Light Chain
[0229] Messenger RNA was isolated from a culture of 8C12-F10 cells
using a MICRO-FAST TRACK kit (Invitrogen) following protocols
provided by the manufacturer. Two cDNA pools were then produced by
second strand synthesis using a cDNA cell cycle kit (Invitrogen)
following protocols provided by the manufacturer and one of the
following light chain antisense cDNA primers:
TABLE-US-00006 AB16 (CGGGAAGATGAAGACAGATG; SEQ ID NO: 15) Ck-760R
(AAGGATGGGAGGGGGTCAGG; SEQ ID NO: 16)
[0230] Light chain variable region sequences were cloned by PCR
amplification of the AB16-primed cDNA pool using AB16 primer and
one of a series of V-region primers including primers corresponding
to conserved secretory signal sequences, which encode the 5' end of
the respective coding regions, and framework region 1 sequences,
which encode the beginning of the mature immunoglobulins. Pfu DNA
polymerase (Stratagene) was used according to recommended
manufacturer's protocols with the variations and cycle parameters
described above.
[0231] Only one light chain signal sequence primer, AB123
(CCCGCTCAGCTCCTGGGGCTCCTG; SEQ ID NO:17), which binds to VK1 family
heavy chain V regions, produced significant product. The 408
nucleotide PCR product was cloned into PCR BLUNT II-TOPO vector
(Invitrogen), clones were screened for the correct insert size, and
three clones corresponding to the PCR products were sequenced.
Identical sequences were obtained for all three clones.
[0232] Light chain constant region sequences were cloned by PCR
amplification of the Ck-760R-primed cDNA pool. The entire coding
sequence and 5' UTR region of the light chain was amplified using
light chain sense primer L15 22m (TCAGWCYCAGTCAGGACACAGC; SEQ ID
NO:18) and Ck-760R. The 788 nucleotide fragment was cloned into PCR
BLUNT II vector (Invitrogen) and sequenced. The resulting plasmid
was designated 41m6.
[0233] FIG. 11B shows a diagram of the alignment of the cloned PCR
fragments which provided the full length nucleotide sequence (SEQ
ID NO:19) that encodes the mAb1 light chain (SEQ ID NO:20). The
amino acid sequence of the light chain variable region most closely
matches regions encoded by germ line Vk L15 and Jk2 nucleotide
sequences. The light chain constant region of mAb1 is identical to
the reported human germ-line kappa light chain immunoglobulin gene
sequence. (Whitehurst et al. (1992) Nucleic Acids Res
20:4929-4930.)
11.3 Production of mAb1 Heavy and Light Chain Expression
Constructs
[0234] Full-length mAb1 heavy chain cDNA was generated by overlap
extension PCR in two steps from the heavy chain PCR products
described above and shown in FIG. 11A. The two 5' PCR products were
combined with the distal primers VH3-33 29-51F and m19 H943R in a
PCR overlap extension reaction to produce a single fragment of 991
nucleotides. Similarly, the two 3' PCR products were combined with
the distal primers VH3-33 29-51F and m19 H943R in a PCR overlap
extension reaction to produce a fragment of 860 nucleotides. These
two PCR extension reaction products were then gel-purified and
amplified together using the distal primers VH3-33 29-51F and m19
H1504R to generate the 1407 nucleotide cDNA (residues 441 through
1847 of SEQ ID NO:13) coding sequence of the full-length mAb1 heavy
chain.
[0235] The heavy chain cDNA was then cloned into PCR-BLUNT II TOPO
vector (Invitrogen) to produce plasmid 43a4. The mAb1 heavy chain
coding region was then subcloned by digestion of plasmid 43a4 with
BamHI and XbaI restriction endonucleases, followed by ligation of
the excised insert into PCDNA5-FRT expression vector (Invitrogen),
which had been pre-digested with BamHI and Nhe restriction
endonucleases. The insert of the resulting expression plasmid,
44a1, was sequence verified before being similarly subcloned in
reverse orientation into PBK-CMV vector (Clontech) to produce
plasmid 47a4, and into pCEP-Pu vector (E. Kohfeldt,
Max-Planck-Institut fur Biochemie), a vector derived from pCEP4
vector (Invitrogen), to produce plasmid 49a1.
[0236] The 708 nucleotide cDNA (residue 415 through 1122 of SEQ ID
NO:19) encoding full-length mAb1 light chain was excised from
plasmid 41m6, described above, using HindIII and Xho I restriction
endonucleases, and ligated into PCDNA5-FRT vector (Invitrogen),
which had been pre-digested with HindIII and XhoI restriction
endonucleases, to produce the mammalian expression plasmid 42b2.
The insert of plasmid 42b2 was sequence verified before being
similarly subcloned in reverse orientation into PBK-CMV vector
(Clontech) to produce plasmid 47b3, and into pCEP-Pu vector (E.
Kohfeldt, Max-Planck-Institut fur Biochemie) to produce plasmid
49b1.
11.4 Transfection and Expression of Antibody Chain Constructs
[0237] COS7 cells were transfected with plasmids 44a1 (mAb1 heavy
chain) and 42b2 (mAb1 light chain) in both separate and
co-transfections using standard procedures. Conditioned culture
media was assayed for the presence of antibody as described in
Example 4 (supra). Only medium from cells co-transfected with both
44a1 and 42b2 expressed human antibody having CTGF-binding activity
as measured by ELISA using procedures as described above. The
antibody, herein identified as CLN1, produced by the co-transfected
COS7 cells, binds to the N-terminal half of CTGF with an affinity
of 0.8 nM.
[0238] CLN1 has also been expressed in genetically modified Chinese
Hamster Ovary (CHO) cells. A CHO cell line expressing exemplary
antibody CLN1 was deposited with the American Type Culture
Collection (Manassas Va.) on 19 May 2004 and is identified by ATCC
Accession No. PTA-6006. Cell lines can be optimized and antibody
expression can be enhanced using various techniques known in the
art, e.g., by gene amplification as described by Wigler et al.
(1980; Proc Natl Acad Sci USA 77:3567-3570) with modifications as
described by Ringold et al. (1981; J Mol Appl Genet 1:165-175),
Gasser et al. (1982; Proc Natl Acad Sci USA 79:6522-6526), and
Kaufman et al. (1985; Mol Cell Biol 5:1750-1759).
Example 12
Interaction of CTGF with TGF.beta.
[0239] Antibodies of the invention specifically bind to regions of
CTGF defined by residues encoded by exon 3 (FIG. 1B; nucleotide 418
to nucleotide 669 of SEQ ID NO:1). This region encompasses amino
acid 97 to amino acid 180 of SEQ ID NO:2, and includes the von
Willebrand Type C domain (amino acid 103 to amino acid 164 of SEQ
ID NO:2) and the epitope of mAb1 (amino acid 134 to amino acid 158
of SEQ ID NO:2). Abreu et al. (2002, Nat Cell Biol 4:599-604)
report that a domain corresponding to the VWC domain of CTGF is
important for interaction between CTGF and TGF.beta. or BMP-4, and
that said interaction modulates the activity of TGF.beta. and
BMP-4. The following experiments demonstrate that regions encoded
by exon 3 are necessary and sufficient for binding of CTGF to
TGF.beta., and that antibodies of the invention can block the
interaction between CTGF and TGF.beta..
[0240] Interaction between CTGF and TGF.beta. was assayed using the
following procedure. The wells of a 96-well MAXISORP ELISA plate
(Nalge Nunc) were coated overnight at 4.degree. C. with 10 .mu.g/ml
of either CTGF, the CTGF fragment encoded by exon 3, or the CTGF
fragment encoded by exon 5, in PBS; or with PBS alone. All wells
were then blocked with 1% BSA in PBS, followed by incubation for 1
hour at room temperature in 50 .mu.l solution containing TGF.beta.
at 0, 1, 3.3, 10, 33, 100, 333, or 1000 ng/ml, and MAB612 or
MAB1835 mouse anti-TGF.beta. monoclonal antibody (R&D Systems,
Minneapolis Minn.) at 100, 300, or 1000 ng/ml in PBS, 0.05%
Tween-20. MAB1835 recognizes bovine, mouse, and human TGF-.beta.1,
and .beta.2, and blocks binding of TGF.beta. to mouse thymocytes.
MAB612 recognizes TGF-.beta.2, but does not inhibit TGF.beta.
activities. Wells were washed with PBS, 0.05% Tween-20, and then
incubated for 1 hour at room temperature in a solution containing
an alkaline phosphatase-conjugated goat anti-mouse IgG antibody
diluted in PBS, 0.05% Tween-20. Plates were again washed, and
p-nitrophenyl phosphate (PNPP) in 1 M ethanolamine, 1 mM MgSO4, pH
9.8 was added, wells were incubated for a suitable time to develop,
and the reaction was then terminated by addition of NaOH. The
absorbance at 2 of 405 nm was measured using a
spectrophotometer.
[0241] FIG. 12 shows that CTGF and the CTGF fragment encoded by
exon 3 are capable of interacting with TGF.beta. to an equivalent
degree, whereas the CTGF fragment encoded by exon 5 did not show
any binding activity toward TGF.beta.. Interestingly, the
anti-TGF.beta. antibody MAB612 was able to detect CTGF-bound
TGF.beta. in a dose-dependent manner, but the neutralizing
antibody, MAB1835, was not able to detect CTGF-bound TGF.beta. at
any concentration tested (data not shown). This suggests that CTGF
competes with MAB1835 for binding to TGF.beta..
[0242] Anti-CTGF antibodies were tested for their ability to block
binding between CTGF and TGF.beta.. As shown in FIG. 12,
anti-bodies of the invention, exemplified by mAb4 and mAb1, blocked
TGF.beta. binding of both CTGF and the CTGF fragment encoded by
exon 3, whereas an anti-CTGF antibody directed to a C-terminal
fragment of CTGF did not block binding. These results provide
support for a mechanism of action wherein antibodies of the
invention specifically block an interaction between CTGF and
TGF.beta., and potentially between CTGF and other members of the
TGF.beta. superfamily.
[0243] Various modifications of the invention, in addition to those
shown and described herein, will become apparent to those skilled
in the art from the foregoing description. Such modifications are
intended to fall within the scope of the appended claims.
[0244] All references cited herein are hereby incorporated by
reference in their entirety.
Sequence CWU 1
1
4312075DNAHomo sapiens 1cccggccgac agccccgaga cgacagcccg gcgcgtcccg
gtccccacct ccgaccaccg 60ccagcgctcc aggccccgcg ctccccgctc gccgccaccg
cgccctccgc tccgcccgca 120gtgccaacca tgaccgccgc cagtatgggc
cccgtccgcg tcgccttcgt ggtcctcctc 180gccctctgca gccggccggc
cgtcggccag aactgcagcg ggccgtgccg gtgcccggac 240gagccggcgc
cgcgctgccc ggcgggcgtg agcctcgtgc tggacggctg cggctgctgc
300cgcgtctgcg ccaagcagct gggcgagctg tgcaccgagc gcgacccctg
cgacccgcac 360aagggcctct tctgtgactt cggctccccg gccaaccgca
agatcggcgt gtgcaccgcc 420aaagatggtg ctccctgcat cttcggtggt
acggtgtacc gcagcggaga gtccttccag 480agcagctgca agtaccagtg
cacgtgcctg gacggggcgg tgggctgcat gcccctgtgc 540agcatggacg
ttcgtctgcc cagccctgac tgccccttcc cgaggagggt caagctgccc
600gggaaatgct gcgaggagtg ggtgtgtgac gagcccaagg accaaaccgt
ggttgggcct 660gccctcgcgg cttaccgact ggaagacacg tttggcccag
acccaactat gattagagcc 720aactgcctgg tccagaccac agagtggagc
gcctgttcca agacctgtgg gatgggcatc 780tccacccggg ttaccaatga
caacgcctcc tgcaggctag agaagcagag ccgcctgtgc 840atggtcaggc
cttgcgaagc tgacctggaa gagaacatta agaagggcaa aaagtgcatc
900cgtactccca aaatctccaa gcctatcaag tttgagcttt ctggctgcac
cagcatgaag 960acataccgag ctaaattctg tggagtatgt accgacggcc
gatgctgcac cccccacaga 1020accaccaccc tgccggtgga gttcaagtgc
cctgacggcg aggtcatgaa gaagaacatg 1080atgttcatca agacctgtgc
ctgccattac aactgtcccg gagacaatga catctttgaa 1140tcgctgtact
acaggaagat gtacggagac atggcatgaa gccagagagt gagagacatt
1200aactcattag actggaactt gaactgattc acatctcatt tttccgtaaa
aatgatttca 1260gtagcacaag ttatttaaat ctgtttttct aactggggga
aaagattccc acccaattca 1320aaacattgtg ccatgtcaaa caaatagtct
atcttcccca gacactggtt tgaagaatgt 1380taagacttga cagtggaact
acattagtac acagcaccag aatgtatatt aaggtgtggc 1440tttaggagca
gtgggagggt accggcccgg ttagtatcat cagatcgact cttatacgag
1500taatatgcct gctatttgaa gtgtaattga gaaggaaaat tttagcgtgc
tcactgacct 1560gcctgtagcc ccagtgacag ctaggatgtg cattctccag
ccatcaagag actgagtcaa 1620gttgttcctt aagtcagaac agcagactca
gctctgacat tctgattcga atgacactgt 1680tcaggaatcg gaatcctgtc
gattagactg gacagcttgt ggcaagtgaa tttgcctgta 1740acaagccaga
ttttttaaaa tttatattgt aaatattgtg tgtgtgtgtg tgtgtgtata
1800tatatatata tatgtacagt tatctaagtt aatttaaagt tgtttgtgcc
tttttatttt 1860tgtttttaat gctttgatat ttcaatgtta gcctcaattt
ctgaacacca taggtagaat 1920gtaaagcttg tctgatcgtt caaagcatga
aatggatact tatatggaaa ttctgctcag 1980atagaatgac agtccgtcaa
aacagattgt ttgcaaaggg gaggcatcag tgtcttggca 2040ggctgatttc
taggtaggaa atgtggtagc tcacg 20752349PRTHomo sapiens 2Met Thr Ala
Ala Ser Met Gly Pro Val Arg Val Ala Phe Val Val Leu1 5 10 15Leu Ala
Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly Pro 20 25 30Cys
Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly Val Ser 35 40
45Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu
50 55 60Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly
Leu65 70 75 80Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly
Val Cys Thr 85 90 95Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr
Val Tyr Arg Ser 100 105 110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr
Gln Cys Thr Cys Leu Asp 115 120 125Gly Ala Val Gly Cys Met Pro Leu
Cys Ser Met Asp Val Arg Leu Pro 130 135 140Ser Pro Asp Cys Pro Phe
Pro Arg Arg Val Lys Leu Pro Gly Lys Cys145 150 155 160Cys Glu Glu
Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val Val Gly 165 170 175Pro
Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro Asp Pro 180 185
190Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp Ser Ala
195 200 205Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr
Asn Asp 210 215 220Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu
Cys Met Val Arg225 230 235 240Pro Cys Glu Ala Asp Leu Glu Glu Asn
Ile Lys Lys Gly Lys Lys Cys 245 250 255Ile Arg Thr Pro Lys Ile Ser
Lys Pro Ile Lys Phe Glu Leu Ser Gly 260 265 270Cys Thr Ser Met Lys
Thr Tyr Arg Ala Lys Phe Cys Gly Val Cys Thr 275 280 285Asp Gly Arg
Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro Val Glu 290 295 300Phe
Lys Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met Met Phe Ile305 310
315 320Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn Asp Ile
Phe 325 330 335Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala
340 345321DNAartificial sequenceIg Heavy chain primer 3tgccaggggg
aagaccgatg g 21423DNAartificial sequenceIg heavy chain primer
4gctgggcgcc cgggaagtat gta 23520DNAartificial sequenceIg Heavy
chain primer 5atggagtttg grctgagctg 20623DNAartificial sequenceIg
heavy chain primer 6cggcggtgtt tccattcggt gat 23722DNAartificial
sequenceIg heavy chain primer 7gggcgcctga gttccacgac ac
22823DNAartificial sequenceIg heavy chain primer 8gtcttccccc
tggcaccctc ctc 23924DNAartificial sequenceIg heavy chain primer
9cccgcggctt tgtcttggca ttat 241021DNAartificial sequenceIg heavy
chain primer 10ctggctgaat ggcaaggagt a 211119DNAartificial
sequenceIg heavy chain primer 11gggcacccag acctacatc
191222DNAartificial sequenceIg heavy chain primer 12ctccggctgc
ccattgctct cc 22132197DNAHomo sapiens 13ggtaccttct gaggcggaaa
gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag 60tccccaggct ccccagcagg
cagaagtatg caaagcatgc atctcaatta gtcagcaacc 120aggtgtggaa
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
180tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
tccgcccagt 240tccgcccatt ctccgcccca tggctgacta atttttttta
tttatgcaga ggccgaggcc 300gcctcggcct ctgagctatt ccagaagtag
tgaggaggct tttttggagg cctaggcttt 360tgcaaaaagc ggccgcaagc
taattcgccc ttcggcggtg tttccattcg gtgatcagga 420ctgaacacac
aggaatcacc atggagtttg tgctgagctg ggttttcctt gttgctatat
480taaaaggtgt ccagtgtgag ggtcagctgg tgcaatctgg gggaggcttg
gtacatcctg 540gggggtccct gagactctcc tgtgcaggct ctggattcac
cttcagtagc tatggtatgc 600actgggttcg ccaggctcca ggaaaaggtc
tggagtgggt atcaggtatt ggtactggtg 660gtggcacata ctctacagac
tccgtgaagg gccgattcac catctccaga gacaatgcca 720agaactcctt
gtatcttcaa atgaacagcc tgagagccga ggacatggct gtgtattact
780gtgcaagagg agattactat ggttcgggga gtttctttga ctgctggggc
cagggaaccc 840tggtcaccgt ctcctcagcc tccaccaagg gcccatcggt
cttccccctg gcaccctcct 900ccaagagcac ctctgggggc acagcggccc
tgggctgcct ggtcaaggac tacttccccg 960aaccggtgac ggtgtcgtgg
aactcaggcg ccctgaccag cggcgtgcac accttcccgg 1020ctgtcctaca
gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
1080gcttgggcac ccagacctac atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg 1140acaagagagt tgagcccaaa tcttgtgaca aaactcacac
atgcccaccg tgcccagcac 1200ctgaactcct ggggggaccg tcagtcttcc
tcttcccccc aaaacccaag gacaccctca 1260tgatctcccg gacccctgag
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg 1320aggtcaagtt
caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc
1380gggaggagca gtacaacagc acgtaccggg tggtcagcgt cctcaccgtc
ctgcaccagg 1440actggctgaa tggcaaggag tacaagtgca aggtctccaa
caaagccctc ccagccccca 1500tcgagaaaac catctccaaa gccaaagggc
agccccgaga accacaggtg tacaccctgc 1560ccccatcccg ggaggagatg
accaagaacc aggtcagcct gacctgcctg gtcaaaggct 1620tctatcccag
cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca
1680agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc
aagctcaccg 1740tggacaagag caggtggcag caggggaacg tcttctcatg
ctccgtgatg catgaggctc 1800tgcacaacca ctacacgcag aagagcctct
ccctgtctcc gggtaaatga gtgcgacggc 1860cggcaagccc cgctccccgg
gctctcgcgg tcgcacgagg atgcttggca cgtaccccct 1920gtacatactt
cccgggcgcc cagcaagggc gaattgatcc agacatgata agatacattg
1980atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
tgtgaaattt 2040gtgatgctat tgctttattt gtaaccatta taagctgcaa
taaacaagtt aacaacaaca 2100attgcattca ttttatgttt caggttcagg
gggaggtgtg ggaggttttt taaagcaagt 2160aaaacctcta caaatgtggt
atggctgatt atgatca 219714469PRTHomo sapiens 14Met Glu Phe Val Leu
Ser Trp Val Phe Leu Val Ala Ile Leu Lys Gly1 5 10 15Val Gln Cys Glu
Gly Gln Leu Val Gln Ser Gly Gly Gly Leu Val His 20 25 30Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe 35 40 45Ser Ser
Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60Glu
Trp Val Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp65 70 75
80Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
85 90 95Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val
Tyr 100 105 110Tyr Cys Ala Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe
Phe Asp Cys 115 120 125Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly 130 135 140Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly145 150 155 160Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200
205Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
210 215 220Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys225 230 235 240Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu 245 250 255Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 260 265 270Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 275 280 285Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315
320Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 340 345 350Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 355 360 365Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln 370 375 380Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala385 390 395 400Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440
445Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460Leu Ser Pro Gly Lys4651520DNAartificial sequenceIg Light
chain primer 15cgggaagatg aagacagatg 201620DNAartificial sequenceIg
light chain primer 16aaggatggga gggggtcagg 201724DNAartificial
sequenceIg Light chain primer 17cccgctcagc tcctggggct cctg
241822DNAartificial sequenceIg light chain primer 18tcagwcycag
tcaggacaca gc 22191433DNAHomo sapiens 19ggtaccttct gaggcggaaa
gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag 60tccccaggct ccccagcagg
cagaagtatg caaagcatgc atctcaatta gtcagcaacc 120aggtgtggaa
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
180tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
tccgcccagt 240tccgcccatt ctccgcccca tggctgacta atttttttta
tttatgcaga ggccgaggcc 300gcctcggcct ctgagctatt ccagaagtag
tgaggaggct tttttggagg cctaggcttt 360tgcaaaaagc ggccgcaagc
taattcgccc tttcagtctc agtcaggaca cagcatggac 420atgagggtcc
tcgctcagct cctggggctc ctgctgctct gtttcccagg tgccagatgt
480gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
cagagtcacc 540atcacttgtc gggcgagtca gggtattagc agctggttag
cctggtatca gcagaaacca 600gagaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 660aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 720gaagattttg
caacttatta ctgccaacag tataatagtt accctcccac ttttggccag
780gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat
cttcccgcca 840tctgatgagc agttgaaatc tggaactgcc tctgttgtgt
gcctgctgaa taacttctat 900cccagagagg ccaaagtaca gtggaaggtg
gataacgccc tccaatcggg taactcccag 960gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacg 1020ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
1080ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttagaggga
gaagtgcccc 1140cacctgctcc tcagttccag cctgaccccc tcccatcctt
aagggcgaat tgatccagac 1200atgataagat acattgatga gtttggacaa
accacaacta gaatgcagtg aaaaaaatgc 1260tttatttgtg aaatttgtga
tgctattgct ttatttgtaa ccattataag ctgcaataaa 1320caagttaaca
acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
1380gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tca
143320236PRTHomo sapiens 20Met Asp Met Arg Val Leu Ala Gln Leu Leu
Gly Leu Leu Leu Leu Cys1 5 10 15Phe Pro Gly Ala Arg Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Trp Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys 50 55 60Ala Pro Lys Ser Leu Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Tyr
Asn Ser Tyr Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 115 120
125Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
130 135 140Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn145 150 155 160Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu 165 170 175Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp 180 185 190Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys225 230
2352162PRTartificialsynthetic peptide 21Cys Ile Phe Gly Gly Thr Val
Tyr Arg Ser Gly Glu Ser Phe Gln Ser1 5 10 15Ser Cys Lys Tyr Gln Cys
Thr Cys Leu Asp Gly Ala Val Gly Cys Met 20 25 30Pro Leu Cys Ser Met
Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe 35 40 45Pro Arg Arg Val
Lys Leu Pro Gly Lys Cys Cys Glu Glu Trp 50 55
602224PRTartificialsynthetic peptide 22Cys Pro Leu Cys Ser Met Asp
Val Arg Leu Pro Ser Pro Asp Cys Pro1 5 10 15Phe Pro Arg Arg Val Lys
Leu Pro 202318PRTartificialsynthetic peptide 23Arg Leu Pro Ser Pro
Asp Ser Pro Phe Pro Arg Arg Val Lys Leu Pro1 5 10 15Gly
Lys2415PRTartificialsynthetic peptide 24Arg Leu Pro Ser Pro Asp Cys
Pro Phe Pro Arg Arg Val Lys Leu1 5 10 152513PRTartificialsynthetic
peptide 25Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val1 5
102614PRTartificialsynthetic peptide 26Leu Pro Ser Pro Asp Cys Pro
Phe Pro Arg Arg Val Lys Leu1 5 1027198PRTHomo sapiens 27Met Thr Ala
Ala Ser Met Gly Pro Val Arg Val Ala Phe Val Val Leu1 5 10 15Leu Ala
Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser Gly Pro 20 25 30Cys
Arg Cys Pro Asp Glu Pro
Ala Pro Arg Cys Pro Ala Gly Val Ser 35 40 45Leu Val Leu Asp Gly Cys
Gly Cys Cys Arg Val Cys Ala Lys Gln Leu 50 55 60Gly Glu Leu Cys Thr
Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu65 70 75 80Phe Cys Asp
Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90 95Ala Lys
Asp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser 100 105
110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp
115 120 125Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg
Leu Pro 130 135 140Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu
Pro Gly Lys Cys145 150 155 160Cys Glu Glu Trp Val Cys Asp Glu Pro
Lys Asp Gln Thr Val Val Gly 165 170 175Pro Ala Leu Ala Ala Tyr Arg
Leu Glu Asp Thr Phe Gly Pro Asp Pro 180 185 190Thr Met Ile Arg Ala
Asn 1952884PRTartificialsynthetic peptide 28Ala Lys Asp Gly Ala Pro
Cys Ile Phe Gly Gly Thr Val Tyr Arg Ser1 5 10 15Gly Glu Ser Phe Gln
Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp 20 25 30Gly Ala Val Gly
Cys Met Pro Leu Cys Ser Met Asp Val Arg Leu Pro 35 40 45Ser Pro Asp
Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys Cys 50 55 60Cys Glu
Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val Val Gly65 70 75
80Pro Ala Leu Ala29198PRTBos taurus 29Met Ser Ala Thr Gly Leu Gly
Pro Val Arg Cys Ala Phe Val Leu Leu1 5 10 15Leu Ala Leu Cys Ser Arg
Pro Ala Ser Ser Gln Asp Cys Ser Ala Pro 20 25 30Cys Gln Cys Pro Ala
Gly Pro Ala Pro Arg Cys Pro Ala Gly Val Ser 35 40 45Leu Val Leu Asp
Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gln Leu 50 55 60Ser Glu Leu
Cys Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu65 70 75 80Phe
Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90
95Ala Lys Asp Gly Ala Pro Cys Val Phe Gly Gly Thr Val Tyr Gln Ser
100 105 110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys
Leu Asp 115 120 125Gly Ser Val Gly Cys Val Pro Leu Cys Ser Val Asp
Val Arg Leu Pro 130 135 140Ser Pro Asp Cys Pro Phe Pro Arg Arg Val
Lys Leu Pro Gly Lys Cys145 150 155 160Cys Glu Glu Trp Val Cys Asp
Glu Pro Lys Glu His Thr Val Val Gly 165 170 175Pro Ala Leu Ala Ala
Tyr Arg Pro Glu Asp Thr Phe Gly Pro Asp Pro 180 185 190Thr Met Ile
Arg Ala Asn 19530198PRTSus scrofa 30Met Ser Ala Thr Gly Leu Ser Pro
Val Arg Cys Ala Phe Val Leu Leu1 5 10 15Leu Ala Leu Cys Ser Arg Pro
Ala Ser Gly Gln Asp Cys Ser Gly Gln 20 25 30Cys Gln Cys Ala Ala Gly
Lys Arg Arg Ala Cys Pro Ala Gly Val Ser 35 40 45Leu Val Leu Asp Gly
Cys Gly Cys Cys Arg Leu Cys Ala Lys Gln Leu 50 55 60Gly Glu Leu Cys
Thr Glu Arg Asp Pro Cys Asp Pro His Lys Gly Leu65 70 75 80Phe Cys
Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 85 90 95Ala
Lys Asp Gly Ala Pro Cys Val Phe Gly Gly Thr Val Tyr Arg Ser 100 105
110Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp
115 120 125Gly Ala Val Gly Cys Val Pro Leu Cys Ser Met Asp Val Arg
Leu Pro 130 135 140Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu
Pro Gly Lys Cys145 150 155 160Cys Glu Glu Trp Val Cys Asp Glu Pro
Lys Asp His Thr Val Val Gly 165 170 175Pro Ala Leu Ala Ala Tyr Arg
Leu Glu Asp Thr Phe Gly Pro Asp Pro 180 185 190Thr Met Met Arg Ala
Asn 19531196PRTRattus norvegicus 31Met Leu Ala Ser Val Ala Gly Pro
Val Ser Leu Ala Leu Val Leu Leu1 5 10 15Leu Cys Thr Arg Pro Ala Thr
Gly Gln Asp Cys Ser Ala Gln Cys Gln 20 25 30Cys Ala Ala Glu Ala Ala
Pro Arg Cys Pro Ala Gly Val Ser Leu Val 35 40 45Leu Asp Gly Cys Gly
Cys Cys Arg Val Cys Ala Lys Gln Leu Gly Glu 50 55 60Leu Cys Thr Glu
Arg Asp Pro Cys Asp Pro His Lys Gly Leu Phe Cys65 70 75 80Asp Phe
Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr Ala Lys 85 90 95Asp
Gly Ala Pro Cys Val Phe Gly Gly Ser Val Tyr Arg Ser Gly Glu 100 105
110Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly Ala
115 120 125Val Gly Cys Val Pro Leu Cys Ser Met Asp Val Arg Leu Pro
Ser Pro 130 135 140Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly
Lys Cys Cys Glu145 150 155 160Glu Trp Val Cys Asp Glu Pro Lys Asp
Arg Thr Val Val Gly Pro Ala 165 170 175Leu Ala Ala Tyr Arg Leu Glu
Asp Thr Phe Gly Pro Asp Pro Thr Met 180 185 190Met Arg Ala Asn
19532197PRTMus musculus 32Met Leu Ala Ser Val Ala Gly Pro Ile Ser
Leu Ala Leu Val Leu Leu1 5 10 15Ala Leu Cys Thr Arg Pro Ala Thr Gly
Gln Asp Cys Ser Ala Gln Cys 20 25 30Gln Cys Ala Ala Glu Ala Ala Pro
His Cys Pro Ala Gly Val Ser Leu 35 40 45Val Leu Asp Gly Cys Gly Cys
Cys Arg Val Cys Ala Lys Gln Leu Gly 50 55 60Glu Leu Cys Thr Glu Arg
Asp Pro Cys Asp Pro His Lys Gly Leu Phe65 70 75 80Cys Asp Phe Gly
Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr Ala 85 90 95Lys Asp Gly
Ala Pro Cys Val Phe Gly Gly Ser Val Tyr Arg Ser Gly 100 105 110Glu
Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys Leu Asp Gly 115 120
125Ala Val Gly Cys Val Pro Leu Cys Ser Met Asp Val Arg Leu Pro Ser
130 135 140Pro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly Lys
Cys Cys145 150 155 160Lys Glu Trp Val Cys Asp Glu Pro Lys Asp Arg
Thr Ala Val Gly Pro 165 170 175Ala Leu Ala Ala Tyr Arg Leu Glu Asp
Thr Phe Gly Pro Asp Pro Thr 180 185 190Met Met Arg Ala Asn
1953314PRTartificialsynthetic peptide 33Pro Leu Ser Ser Met Asp Val
Arg Leu Pro Ser Pro Asp Ser1 5 103413PRTartificialsynthetic peptide
34Arg Leu Pro Ser Pro Asp Ser Pro Phe Pro Arg Arg Val1 5
103510PRTartificialsynthetic peptide 35Ser Pro Asp Ser Pro Phe Pro
Arg Arg Val1 5 103610PRTartificialsynthetic peptide 36Ser Pro Asp
Cys Pro Phe Pro Arg Arg Val1 5 10379PRTartificialsynthetic peptide
37Pro Asp Ser Pro Phe Pro Arg Arg Val1 5389PRTartificialsynthetic
peptide 38Cys Pro Phe Pro Arg Arg Val Lys Leu1
5398PRTartificialsynthetic peptide 39Asp Ser Pro Phe Pro Arg Arg
Val1 5408PRTartificialsynthetic peptide 40Cys Phe Pro Arg Arg Val
Lys Leu1 5417PRTartificialsynthetic peptide 41Cys Pro Arg Arg Val
Lys Leu1 5426PRTartificialsynthetic peptide 42Cys Arg Arg Val Lys
Leu1 5435PRTartificialsynthetic peptide 43Cys Arg Val Lys Leu1
5
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