U.S. patent application number 13/863896 was filed with the patent office on 2013-08-22 for use of il-20 antagonists for treating rheumatoid arthritis and osteoporosis.
This patent application is currently assigned to National Cheng Kung University. The applicant listed for this patent is National Cheng Kung University. Invention is credited to Ming-Shi CHANG.
Application Number | 20130216534 13/863896 |
Document ID | / |
Family ID | 43730798 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130216534 |
Kind Code |
A1 |
CHANG; Ming-Shi |
August 22, 2013 |
USE OF IL-20 ANTAGONISTS FOR TREATING RHEUMATOID ARTHRITIS AND
OSTEOPOROSIS
Abstract
The invention features methods and compositions for preventing
or treating rheumatoid arthritis and osteoporosis by administering
an antagonist of IL-20. The IL-20 antagonist may be an anti-IL-20
antibody, such as mAB 7E, that is capable of binding human IL-20
and blocking IL-20 interaction with its receptors.
Inventors: |
CHANG; Ming-Shi; (Tainan
City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Cheng Kung University; |
|
|
US |
|
|
Assignee: |
National Cheng Kung
University
Tainan City
TW
|
Family ID: |
43730798 |
Appl. No.: |
13/863896 |
Filed: |
April 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12869224 |
Aug 26, 2010 |
8454956 |
|
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13863896 |
|
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61238661 |
Aug 31, 2009 |
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Current U.S.
Class: |
424/133.1 ;
424/142.1; 424/158.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2887 20130101; C07K 16/244 20130101; A61P 3/14 20180101;
C07K 2317/76 20130101; A61P 37/02 20180101; C07K 2317/70 20130101;
A61K 39/3955 20130101 |
Class at
Publication: |
424/133.1 ;
424/158.1; 424/142.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24 |
Claims
1-27. (canceled)
28. A method for treating bone loss in a subject, comprising
administering to a subject in need thereof an anti-IL-20 antibody
in an amount effective in treating bone loss in the subject.
29. The method of claim 28, wherein the subject is a human subject
having rheumatoid arthritis.
30. The method of claim 29, wherein the human subject has bone
erosion.
31. The method of claim 28, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in treating bone
damage.
32. The method of claim 28, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in inhibiting
bone mineral density reduction.
33. The method of claim 28, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in inhibiting
osteoclast differentiation.
34. The method of claim 29, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in treating bone
damage.
35. The method of claim 29, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective inhibiting bone
mineral density reduction.
36. The method of claim 29, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in inhibiting
osteoclast differentiation.
37. The method of claim 28, wherein the anti-IL-20 antibody binds
human IL-20.
38. The method of claim 28, wherein the anti-IL-20 antibody is
selected from the group consisting of an intact antibody and an
antigen-binding fragment thereof.
39. The method of claim 28, wherein the anti-IL-20 antibody is
selected from the group consisting of a human antibody, a humanized
antibody, and a chimeric antibody that comprises a heavy chain
constant region and a light chain constant region from a human
antibody.
40. The method of claim 28, wherein the anti-IL-20 antibody is
co-administered with a TNF.alpha. antagonist.
41. The method of claim 40, wherein the TNF.alpha. antagonist is
selected from the group consisting of an etanercept polypeptide,
infliximab, and adalimumab.
42. The method of claim 1, wherein the anti-IL-20 antibody is
administered parenterally.
43. A method for treating bone erosion in rheumatoid arthritis,
comprising administering to a subject in need thereof an effective
amount of an anti-IL-20 antibody in an amount effective in
inhibiting bone erosion in the subject.
44. The method of claim 43, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective to inhibit
structural joint damage.
45. A method of inhibiting osteoclast differentiation, comprising
administering to a subject in need thereof an anti-IL-20 antibody
in an amount effective in inhibiting osteoclast differentiation in
the subject, wherein the subject is a rheumatoid arthritis
patient.
46. The method of claim 45, wherein the anti-IL-20 antibody is
administered to the subject in an amount effective in inhibiting
bone erosion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Application Ser.
No. 61/238,661, filed Aug. 31, 2009, entitled "Use of Anti-IL-20
Antibody for Treating Rheumatoid Arthritis and Osteoporosis", the
contents of which are incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to the use of an IL-20
antagonist for the prevention, delay of onset or treatment of
rheumatoid arthritis and osteoporosis.
BACKGROUND OF THE INVENTION
[0003] Osteoporosis is a disease characterized by low bone mass and
loss of bone tissue, resulting in weak and fragile bones. Net bone
loss can be induced by various factors, e.g., low levels of
estrogen, inadequate up take of calcium and vitamin D, and
inflammation. Bone resorption is a major pathological factor in
postmenopausal osteoporosis. Osteoporosis is a disorder of impaired
bone strength that causes skeletal fragility and increases fracture
risk (Theill, L E, et al. (2002) Annu Rev Immunol 20:795-823;
Boyle, W J, et al. (2003) Nature 423;337-342). Estrogen deficiency
at menopause and androgen deficiency in men both cause an
unbalanced increase in bone turnover, in which bone resorption
exceeds bone formation. Relatively rapid bone loss occurs and is
accompanied by the destruction of bone micro-architecture (Simonet,
W S, et al. (1997) Cell 89:309-319; McClung, M, (2007) Arthritis
Res Ther 9 Suppl 1:S3). In most instances, low bone mass is caused
by an increase in the number of osteoclasts or by excessive
osteoclast activity (Walsh, N C, et al. (2005) Immunol Rev
208:228-251). Osteoclasts are multinucleated giant cells that
express tartrate-resistant acid phosphatase (TRAP) and calcitonin
receptors. Osteoclast formation requires two factors: macrophage
colony-stimulation factor (M-CSF) and the receptor activator of
NF-.kappa.B ligand (RANKL) (Takayanagi, H, et al. (2005) Immunol
Rev 208:181-193; Ross, F P & Teitelbaum, S L, (2005) Immunol
Rev 208:88-105). M-CSF, which mediates the survival and
proliferation of monocyte/macrophage precursors, is produced
primarily by stromal fibroblasts, osteoblasts, and activated T
cells. RANK, is the sole signaling receptor for RANKL, which
induces the development and activation of osteoclasts (Suda, T, et
al., (1999) Endocr Rev 20:345-357). The in vivo significance of the
RANKL-RANK signaling pathway has been verified by observations that
the deficiency of either gene in mice causes severe osteoporosis
(increased bone mass) and the disappearance of osteoclasts (Kong, Y
Y, et al., (1999) Nature 397:315-323; Li, J, et al., (2000) Proc
Natl Acad Sci USA 97:1566-1571). Several proinflammatory cytokines,
such as TNF-.alpha., IL-1.beta., IL-15, IL-17, and IL-23, induce
the multinucleation of osteoclast precursors, or their commitment
to the osteoclast phenotype, and may act synergistically with RANKL
(Feldmann, M, et al. (2001) Curr Dir Autoimmun 3:188-199; O'
Gradaigh, D, et al. (2004) Ann Rheum Dis 63:354-359; Sato, K, et
al., (2006) J Exp Med 203:2673-2682; Ju, J H, et al., (2008) J
Immunol 181:1507-1518).
[0004] The pleiotropic inflammatory cytokine IL-20, a member of the
IL-10 family--IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26
(Blumberg, H, et al., (2001) Cell 104:9-19; Pestka, S, et al.,
(2004) Annu Rev Immunol 22:929-979)--is expressed in monocytes,
epithelial cells, and endothelial cells. IL-20 acts on multiple
cell types by activating a heterodimer receptor complex of either
IL-20R1/IL-20R2 or IL-22R1/IL-20R2 (Dumoutier, L., et al., (2001) J
Immunol 167:3545-3549). It is involved in various inflammatory
diseases (Wei, C C, et al., (2006) J Biomed Sci 13:601-612), such
as psoriasis (Blumberg, H, et al., (2001) Cell 104:9-19; Sa, S M,
et al., (2007) J Immunol 178:2229-2240; Wei, C C, et al., (2005)
Clin Immunol 117:65-72), rheumatoid arthritis (Hsu, Y H, et al.,
(2006) Arthritis Rheum 54:2722-2733), atherosclerosis (Caligiuri,
G, et al. (2006) Arterioscler Thromb Vasc Biol 26:1929-1930; Chen,
W Y, et al. (2006) Arterioscler Thromb Vasc Biol 26:2090-2095),
ischemic stroke (Chen, W Y & Chang, M S, (2009) J Immunol
182:5003-5012), and renal failure (Li, H H, et al., (2008) Genes
Immun 9:395-404). IL-20 is regulated by hypoxia and inflammatory
stimuli such as IL-1.beta. and LPS (Chen, W Y & Chang, M S,
(2009) J Immunol 182:5003-5012; Otkjaer, K, et al., (2007) J Invest
Dermatol). IL-20 has recently been reported (Heuze-Vourc'h, N, et
al., (2005) Biochem Biophys Res Commun 333:470-475; Hsieh, M Y, et
al., (2006) Genes Immun 7:234-242; Tritsaris, K, et al., (2007)
Proc Natl Acad Sci USA 104:15364-15369) to have regulated
angiogenesis. In experimental rheumatoid arthritis, IL-20 induces
synovial fibroblasts to secrete MCP-1, IL-6, and IL-8, and it acts
as a proinflammatory cytokine (Hsu, Y H, et al., (2006) Arthritis
Rheum 54:2722-2733).
[0005] IL-20 has been shown to be involved in rheumatoid arthritis
and IL-20 soluble receptors have been shown to block IL-20, which
reduces the severity of collagen-induced arthritis (Hsu, Y H, et
al., (2006) Arthritis Rheum 54:2722-2733). Therefore, IL-20 is a
promoting factor during the progression of rheumatoid arthritis.
Little is known, however, about the function of IL-20 in bone
resorption, or about the function of IL-20 in RANKL-RANK
signaling-mediated osteoclastogenesis.
SUMMARY OF THE INVENTION
[0006] The invention provides a method for treating, delaying the
onset of, or preventing osteoporosis in an individual comprising
administering to the individual an effective amount of an IL-20
antagonist.
[0007] The invention also provides a method for treating, delaying
the onset of, or preventing rheumatoid arthritis in an individual
comprising administering to the individual an effective amount of
an IL-20 antagonist in conjunction with a TNF.alpha. antagonist
(such as an etanercept polypeptide).
[0008] Any IL-20 antagonist described herein may be used to treat,
delay the onset of, or prevent osteoporosis or rheumatoid
arthritis. In some embodiments, the IL-20 antagonist is an
anti-IL-20 antibody, such as mAb 7E or a functional equivalent
thereof.
[0009] The details of one or more embodiments of the invention are
set forth in the description below. Other features or advantages of
the present invention will be apparent from the following drawings
and detailed description of several embodiments, and also from the
appending claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a chart showing the incidence of severe hind paw
swelling in healthy rats and collagen-induced-arthritic rats
treated with PBS, mIgG, mAb 7E, Etanercept, or both mAb 7E and
Etanercept.
[0011] FIG. 2 is a number of charts showing the levels of
TNF-.alpha. (panel A), (panel B) and IL-20 (panel C) in healthy
rats and in collagen-induced-arthritic rats treated with mIgG, mAb
7E, Etanercept, or both mAb 7E and Etanercept.
[0012] FIG. 3a is a chart showing the serum level of IL-20 was
upregulated in the OVX-group mice but downregulated in OVX-mice
after treatment with mAb 7E. *P<0.05 compared to sham control.
#P<0.05 compared with the OVX-mIgG group. FIG. 3b shows
representative figures of micro-CT analysis of the right tibia of
mice 2 months after OVX with treatments: sham controls (Healthy),
ovariectomized without treatment (OVX), and ovariectomized mice
treated with 17.beta.-estradiol, OVX+mIgG, OVX+7E (3 mg/kg), or
OVX+7E (6 mg/kg). FIG. 3c is a chart showing bone mineral density
in the knee joints of each experimental group. Values are
means.+-.standard deviation.
[0013] FIG. 4a is a schematic of the culture system for osteoclast
differentiation. FIG. 4b shows representative tartrate-resistant
acid phosphatase (TRAP) staining of osteoclasts for the treatments
of macrophage colony-stimulating factor (M-CSF) and soluble
NF-.kappa.B ligand receptor activator (sRANKL) combined with tumor
necrosis factor (TNF)-.alpha., mIgG, or mAb 7E. FIG. 4c is a chart
showing the number of TRAP+ osteoclasts per well. FIG. 4d is a
schematic of the osteoclast differentiation culture system for
early mAb 7E treatment. FIG. 4e shows TRAP staining of osteoclasts.
FIG. 4f is a chart showing the number of TRAP+ osteoclasts per
well. Representative results from 3 independent experiments are
shown.
[0014] FIG. 5a is a chart showing IL-20 expression in bone
marrow-derived hematopoietic stem cells (HSCs) with or without
MCSF. FIG. 5b shows flow cytometric analysis of the surface
expression of RANK protein IL-20-treated HSCs. Isotype indicates
cells stained with a negative control of isotype antibody. FIG. 5c
is a chart showing that RANK mRNA expression was upregulated in
HSCs after they had been treated with IL-20. FIG. 5d is a chart
showing that mAb 7E inhibited IL-20-induced RANK mRNA expression in
OC precursor cells as measured by real time-PCR.
[0015] FIG. 6a shows the expression of IL-20 and its receptors in
MC3T3-E1 osteoblasts by RT-PCR. FIG. 6b shows cell staining of
IL-20 and its receptors in MC3T3-E1 cells: red (IL-20 and
receptors, AEC), blue (nuclei). FIG. 6c shows western blot analysis
of cells incubated with IL-20 for the indicated time periods using
the following specific antibodies: phospho-JNK (JNK), phospho-ERK
(ERK), phospho-AKT (AKT), phospho-p38 (p38), phospho-STAT3 (STAT3),
and .beta.-actin (.beta.-actin). FIG. 6d shows RT-PCR analysis of
IL-17 mRNA expression in MC3T3-E1 cells treated with IL-20. FIG. 6e
is a chart showing RANKL mRNA expression in MC3T3-E1 cells treated
with IL-20 and measured by Real time-PCR. FIG. 6f is a chart
showing RANKL protein expression in MC3T3-E1 cells treated with
IL-20.
[0016] FIG. 7 is a chart showing mAb 7E inhibited IL-20-induced
RANKL expression in MC3T3-E1 osteoblasts. Representative results
from 3 independent experiments are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is based on the discovery that IL-20
is a novel osteoclastogenic cytokine that caused RANK expression on
osteoclast precursors and RANKL expression on osteoblasts.
Antagonists of IL-20; for example, the IL-20 specific monoclonal
antibody mAb 7E, abolished IL-20-induced RANK and RANKL expression.
These results showed that IL-20 antagonists may be used to inhibit
osteoclast differentiation and protect individuals from
osteoporotic bone loss in vivo. The invention is also based on the
discovery that the IL-20 specific monoclonal antibody alone or
combined with etanercept significantly reduced the severity of
arthritis by decreasing hind-paw thickness and swelling, prevented
cartilage damage and bone loss in an animal model for rheumatoid
arthritis.
[0018] The invention provides methods of treating, delaying the
onset of, or preventing osteoporosis in an individual by
administering an effective amount of an IL-20 antagonist (such as
an anti-IL-20 antibody or an antigen-binding fragment thereof). In
some embodiments, the IL-20 antagonist is administered in
combination with another therapeutic agent for osteoporosis. In
some embodiments, the osteoporosis is post-menopausal osteoporosis.
In some embodiments, the osteoporosis is associated with a hormone
deficiency. For example, in some cases, the osteoporosis is
associated with hormone ablation treatment. Examples of hormone
ablation treatment include treatments of breast cancer and
treatments of prostate cancer. In some embodiments, the
osteoporosis is steroid-induced or steroid-associated osteoporosis.
In some embodiments, the osteoporosis is associated with rheumatoid
arthritis.
[0019] The invention also provides methods of treating, delaying
the onset of, or preventing rheumatoid arthritis in an individual
in need thereof by administering an effective amount of an IL-20
antagonist (such as an anti-IL-20 antibody or an antigen-binding
fragment thereof) and an effective amount of a TNF.alpha.
antagonist (such as an etanercept polypeptide).
General Techniques
[0020] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook,
et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana
Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed.,
1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed.,
1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.
E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,
Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular
Biology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase
Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: a practical approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
Definitions
[0021] An "antibody" (interchangeably used in plural form) is an
immunoglobulin molecule capable of specific binding to a target,
such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.,
through at least one antigen recognition site, located in the
variable region of the immunoglobulin molecule. As used herein, the
term encompasses not only intact polyclonal or monoclonal
antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2,
Fv), single chain (ScFv), mutants thereof, fusion proteins
comprising an antibody portion, humanized antibodies, chimeric
antibodies, diabodies, linear antibodies, single chain antibodies,
multispecific antibodies (e.g., bispecific antibodies) and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity.
An antibody includes an antibody of any class, such as IgG, IgA, or
IgM (or sub-class thereof), and the antibody need not be of any
particular class. Depending on the antibody amino acid sequence of
the constant domain of its heavy chains, immunoglobulins can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG1,
IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains
that correspond to the different classes of immunoglobulins are
called alpha, delta, epsilon, gamma, and mu, respectively. The
subunit structures and three-dimensional configurations of
different classes of immunoglobulins are well known.
[0022] A "monoclonal antibody" refers to a homogeneous antibody
population wherein the monoclonal antibody is comprised of amino
acids (naturally occurring and non-naturally occurring) that are
involved in the selective binding of an antigen. A population of
monoclonal antibodies is highly specific, being directed against a
single antigenic site. The term "monoclonal antibody" encompasses
not only intact monoclonal antibodies and full-length monoclonal
antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2,
Fv), single chain (ScFv), mutants thereof, fusion proteins
comprising an antibody portion, humanized monoclonal antibodies,
chimeric monoclonal antibodies, and any other modified
configuration of the immunoglobulin molecule that comprises an
antigen recognition site of the required specificity and the
ability to bind to an antigen. It is not intended to be limited as
regards to the source of the antibody or the manner in which it is
made (e.g., by hybridoma, phage selection, recombinant expression,
transgenic animals, etc.). The term includes whole immunoglobulins
as well as the fragments etc. described above under the definition
of "antibody."
[0023] Humanized antibodies refer to forms of non-human (e.g.
murine) antibodies that are specific chimeric immunoglobulins,
immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Antibodies may have Fc regions modified as described in WO
99/58572. Other forms of humanized antibodies have one or more CDRs
(one, two, three, four, five, six) which are altered with respect
to the original antibody, which are also termed one or more CDRs
"derived from" one or more CDRs from the original antibody.
[0024] "Chimeric" antibodies refer to antibodies having a variable
region or part of variable region from a first species and a
constant region from a second species. Typically, in these chimeric
antibodies, the variable region of both light and heavy chains
mimics the variable regions of antibodies derived from one species
of mammals, while the constant portions are homologous to the
sequences in antibodies derived from another. In some embodiments,
amino acid modifications can be made in the variable region and/or
the constant region.
[0025] An antibody or a polypeptide that "specifically binds" or
"binds" (used interchangeably herein) to a target or an epitope is
a term well understood in the art, and methods to determine such
specific binding are also well known in the art. A molecule is said
to exhibit "specific binding" if it reacts or associates more
frequently, more rapidly, with greater duration and/or with greater
affinity with a particular target than it does with alternative
targets. An antibody or a polypeptide "specifically binds" to a
target if it binds with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other substances. For
example, an antibody that specifically or preferentially binds to
an IL-20 epitope is an antibody that binds this IL-20 epitope with
greater affinity, avidity, more readily, and/or with greater
duration than it binds to other IL-20 epitopes or non-IL-20
epitopes. It is also understood by reading this definition that,
for example, an antibody (or moiety) that specifically binds to a
first target may or may not specifically or preferentially bind to
a second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0026] As used herein, the term "IL-20" and refers to
interleukin-20 and variants thereof that retain at least part of
the activity of IL-20. As used herein, IL-20 includes all mammalian
species of native sequence IL-20, including human, canine, feline,
equine, or bovine.
[0027] An "IL-20 receptor" refers to one or more polypeptides that
is bound by or activated by IL-20. In some cases, IL-20 binds to a
complex formed by IL-20R1 and IL-20R2. In other cases, IL-20 binds
to a complex formed by IL-20R2 and IL-22R1. As such, IL-20
receptors include IL-20R1, IL-20R2 and IL-22R1 of any mammalian
species, including, but are not limited to, human, canine, feline,
equine, primate, or bovine. Examples of human IL-20 receptors
include hIL-20R1 (GenBank Accession No. NM.sub.--014432.2),
hIL-20R2 (GenBank Accession No. NM.sub.--144717.2) and hIL-22R1
(NM.sub.--181309.1). Sequences of human IL receptors have been
described; for example, in U.S. Pat. Nos. 6,610,286; 7,122,632;
7,393,684; and 7,537,761; and U.S. Pat. App. Pub. Nos. 2006/0263850
A1; 2006/0263851 A1; 2008/0247945 A1, and 2009/0074661 A1.
[0028] An "IL-20 antagonist" refers to any molecule that blocks,
suppresses or reduces (including significantly) IL-20 biological
activity, including downstream pathways mediated by IL-20
signaling, such as receptor binding and/or elicitation of a
cellular response to IL-20. The term "antagonist" implies no
specific mechanism of biological action whatsoever, and is deemed
to expressly include and encompass all possible pharmacological,
physiological, and biochemical interactions with IL-20 whether
direct or indirect. Exemplary IL-20 antagonists include, but are
not limited to, an anti-IL-20 antibody or fragment thereof, an
anti-sense molecule directed to an IL-20 (including an anti-sense
molecule directed to a nucleic acid encoding IL-20), a small
interfering RNA (siRNA) directed toward an IL-20 nucleic acid, a
microRNA directed toward an IL-20 nucleic acid, an IL-20 inhibitory
compound. For purpose of the present invention, it will be
explicitly understood that the term "antagonist" encompass all the
previously identified terms, titles, and functional states and
characteristics whereby the IL-20 itself, an IL-20 biological
activity (including but not limited to its ability to mediate any
aspect osteoporosis), or the consequences of the biological
activity, are substantially nullified, decreased, or neutralized in
any meaningful degree. In some embodiments, an IL-20 antagonist
binds IL-20 and prevents IL-20 receptor complex formation. In other
embodiments, an IL-20 antagonist inhibits or reduces IL-20
synthesis and/or production (release). Examples of types of IL-20
antagonists are provided herein.
[0029] As used herein, an "anti-IL-20 antibody" refers to an
antibody which is able to bind to IL-20 and inhibit IL-20
biological activity and/or downstream pathway(s) mediated by IL-20
signaling.
[0030] The term "anti-IL-20 antibody 7E" refers to monoclonal
antibody mAb 7E and its functional variants. MAb 7E is produced by
the hybridoma cell line deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
U.S.A. and assigned a deposit number PTA-8687. This hybridoma cell
line will be released to the public irrevocably and without
restriction/condition upon granting a US Patent on this
application, and will be maintained in the ATCC for a period of at
least 30 years from the date of the deposit for the enforceable
life of the patent or for a period of 5 years after the date of the
most recent.
[0031] A "functional equivalent" of mAb 7E is an antibody that (1)
specifically binds to human IL-20, and (2) contains a heavy chain
variable region (VH) at least 70% (e.g., 80%, 90%, or 95%)
identical to that of mAb 7E (shown below as SEQ ID NO: 2, encoded
by the nucleotide sequence of SEQ ID NO:1) and a light chain
variable region (VL) at least 70% (e.g., 80%, 90%, or 95%)
identical to that of mAb 7E (shown below as SEQ ID NO: 4, encoded
by the nucleotide sequence of SEQ ID NO:3). See U.S. patent
application Ser. No. 11/763,812.
[0032] As used herein, "percent homology" of two amino acid
sequences is determined using the algorism described in Karlin and
Altschul, Proc, Natl. Acad. Sci. USA 87:2264-2268, 1990, modified
as described in Karlin and Altschul, Proc, Natl. Acad. Sci. USA
5873-5877, 1993. Such an algorism is incorporated into the NBLAST
and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403-410,
1990. BLAST protein searches are performed with the XBLAST program,
score=50, wordlength=3, to obtain amino acid sequences homologous
to a reference polypeptide. To obtain gapped alighments for
comparison purposes, Gapped BLAST is utilized as described in
Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. When
utilizing the BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) are
used. See www.ncbi.nlm.nih.gov.
[0033] The term "treating" as used herein refers to the application
or administration of a composition including one or more active
agents to a subject, who has rheumatoid arthritis or osteoporosis,
a symptom of either diseases, or a predisposition toward the
disease, with the purpose to cure, heal, alleviate, relieve, alter,
remedy, ameliorate, improve, or affect the disease, the symptoms of
the disease, or the predisposition toward the disease.
[0034] "An effective amount" as used herein refers to the amount of
each active agent required to confer therapeutic effect on the
subject, either alone or in combination with one or more other
active agents. Effective amounts vary, as recognized by those
skilled in the art, depending on route of administration, excipient
usage, and co-usage with other active agents
[0035] As used therein, "delaying" the development of a disease
(such as osteoporosis or rheumatoid arthritis) means to defer,
hinder, slow, retard, stabilize, and/or postpone progression of the
disease. This delay can be of varying lengths of time, depending on
the history of the disease and/or individuals being treated. As is
evident to one skilled in the art, a sufficient or significant
delay can, in effect, encompass prevention, in that the individual
does not develop the disease. A method that "delays" development of
the symptom is a method that reduces probability of developing the
symptom in a given time frame and/or reduces extent of the symptoms
in a given time frame, when compared to not using the method. Such
comparisons are typically based on clinical studies, using a number
of subjects sufficient to give a statistically significant
result.
[0036] "Development" or "progression" of a disease (such as
osteoporosis, rheumatoid arthritis) means initial manifestations
and/or ensuing progression of the disorder. Development of the
disease can be detectable and assessed using standard clinical
techniques as well known in the art. However, development also
refers to progression that may be undetectable. For purpose of this
invention, development or progression refers to the biological
course of the symptoms. "Development" includes occurrence,
recurrence, and onset. As used herein "onset" or "occurrence" of
osteoporosis includes initial onset and/or recurrence.
[0037] As used herein, "agent" refers to a biological,
pharmaceutical, or chemical compound. Non-limiting examples include
simple or complex organic or inorganic molecule, a peptide, a
protein, an oligonucleotide, an antibody, an antibody derivative,
antibody fragment, a vitamin derivative, a carbohydrate, a toxin,
or a chemotherapeutic compound. Various compounds can be
synthesized, for example, small molecules and oligomers (e.g.,
oligopeptides and oligonucleotides), and synthetic organic
compounds based on various core structures. In addition, various
natural sources can provide compounds for screening, such as plant
or animal extracts, and the like. A skilled artisan can readily
recognize that there is no limit as to the structural nature of the
agents of the present invention.
[0038] As used herein, "co-administration" or "administration in
conjunction with" includes simultaneous administration and/or
administration at different times. Co-administration also
encompasses administration as a co-formulation (i.e., the IL-20
antagonist and an agent are present in the same composition) or
administration as separate compositions. As used herein,
co-administration is meant to encompass any circumstance wherein an
agent and IL-20 antagonist are administered to an individual, which
can occur simultaneously and/or separately. As further discussed
herein, it is understood that the IL-20 antagonist and an agent can
be administered at different dosing frequencies or intervals. For
example, an anti-IL-20 antibody can be administered weekly, while
the agent can be administered more frequently. It is understood
that the IL-20 antagonist and the agent can be administered using
the same route of administration or different routes of
administration.
[0039] An "individual" or a "subject" is a mammal, more preferably
a human. Mammals include, but are not limited to, farm animals,
sport animals, pets, primates, horses, dogs, cats, mice and
rats.
[0040] With respect to all methods described herein, reference to
an IL-20 antagonist also includes compositions comprising one or
more of these agents. These compositions may further comprise
suitable excipients, such as pharmaceutically acceptable excipients
(carriers) including buffers, which are well known in the art. The
present invention can be used alone or in combination with other
conventional methods of treatment.
[0041] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural reference unless the
context clearly indicates otherwise. For example, reference to an
"antibody" is a reference to from one to many antibodies, such as
molar amounts, and includes equivalents thereof known to those
skilled in the art, and so forth.
[0042] It is understood that aspect and variations of the invention
described herein include "consisting" and/or "consisting
essentially of aspects and variations.
IL-20 Antagonists
[0043] The present invention is useful for treating, delaying
development of and/or preventing osteoporosis and rheumatoid
arthritis in an individual in need thereof, both human and
non-human mammalian.
[0044] The methods of the invention use an IL-20 antagonist, which
refers to any molecule that blocks, suppresses or reduces
(including significantly) IL-20 biological activity, including
downstream pathways mediated by IL-20 signaling, such as receptor
binding and/or elicitation of a cellular response to IL-20. An
example of an IL-20 is human IL-20. The amino acid sequence of a
human IL-20 (SEQ ID NO:6) is as follows:
TABLE-US-00001 MKASSLAFSLLSAAFYLLWTPSTGLKTLNLGSCVIATNLQEIRNGFSEI
RGSVQAKDGNI DIRILRRTESLQDTKPANRCCLLRHLLRLYLDRVFKNYQTPDHYTLRKI
SSLANSFLTIK KDLRLCHAHMTCHCGEEAMKKYSQILSHFEKLEPQAAVVKALGELDILL
QWMEETE (the signal peptide is undelined).
[0045] Exemplary IL-20 antagonists include, but are not limited to,
an anti-IL-20 antibody or fragment thereof, an anti-sense molecule
directed to an IL-20 (including an anti-sense molecule directed to
a nucleic acid encoding IL-20), a small interfering RNA (siRNA)
directed toward an IL-20 nucleic acid, a microRNA directed toward
an IL-20 nucleic acid, an IL-20 inhibitory compound, and a
polypeptide comprising a extracellular portion of an IL-20
receptor. For purpose of the present invention, it will be
explicitly understood that the term "antagonist" encompass all the
previously identified terms, titles, and functional states and
characteristics whereby the IL-20 itself, an IL-20 biological
activity (including but not limited to its ability to mediate any
aspect osteoporosis, inflammatory diseases), or the consequences of
the biological activity, are substantially nullified, decreased, or
neutralized in any meaningful degree. In some embodiments, an IL-20
antagonist binds IL-20 and prevents IL-20 from forming a complex
with one or more of its receptors. In other embodiments, an IL-20
antagonist inhibits or reduces IL-20 synthesis and/or production
(release). Accordingly, in some embodiments, an IL-20 antagonist
binds (physically interacts with) IL-20. In some embodiments, the
IL-20 antagonist is a polypeptide which binds to IL-20. In some
embodiments, the IL-20 antagonist is a peptide or a modified
peptide (such as IL-20 binding peptide including soluble receptors
of IL-20 fused to a Fc domain). See for example; U.S. Pat. Nos.
6,610,286; 7,189,394; 7,364,732; 7,393,684; and 7,537,761; and U.S.
Patent Application Pub. Nos. 2006/0263850 A1; 2006/0263851 A1;
2008/0171041 A1; and US 2008/0233115 A1. In other embodiments, the
IL-20 antagonist is an anti-IL-20 antibody. In still other
embodiments, the anti-IL-20 antibody is humanized. In some
embodiments, the anti-IL-20 antibody is antibody mAb 7E (as
described herein) or a functional equivalent of mAb 7E. In other
embodiments, the anti-IL-20 antibody comprises one or more CDR(s)
of antibody mAb 7E (such as one, two, three, four, five, or, in
some embodiments, all six CDRs from mAb 7E). In other embodiments,
the antibody is a human antibody. In still other embodiments, the
anti-IL-20 antibody comprises the amino acid sequence of the heavy
chain variable region (SEQ ID NO:2) and/or the amino acid sequence
of the light chain variable region (SEQ ID NO:4). In still other
embodiments, the antibody comprises a modified constant region,
such as a constant region that is immunologically inert, e.g., does
not trigger complement mediated lysis, or does not stimulate
antibody-dependent cell mediated cytotoxicity (ADCC). In other
embodiments, the constant region is modified as described in Eur.
J. Immunol. (1999) 29:2613-2624; PCT Application No.
PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In
other embodiments, an IL-20 antagonist inhibits (reduces) IL-20
synthesis and/or release.
[0046] Nucleotide sequence (SEQ ID NO:1) and amino acid sequence
(SEQ ID NO:2) of mAb 7E heavy chain variable region
TABLE-US-00002 gaa ttg aag ctt gag gag tct gga gga ggc ttg gtg cag
cct gga 45 E L K L E E S G G G L V Q P G 15 gga tcc atg aaa ctc tct
tgt gct gcc tct gga ttc act ttt agt 90 G S M K L S C A A S G F T F
S 30 gac gcc tgg atg gac tgg gtc cgc cag tct cca gag aag ggg ctt
135 D A W M D W V R Q S P E K G L 45 gag tgg att gct gaa att aga
agc aaa gct aat aat tat gca aca 180 E W I A E I R S K A N N Y A T
60 tac ttt gct gag tct gtg aaa ggg agg ttc acc atc tca aga gat 215
Y F A E S V K G R F T I S R D 75 gat tcc aaa agt ggt gtc tac ctg
caa atg aac aac tta aga gct 270 D S K S G V Y L Q M N N L R A 90
gag gac act ggc att tat ttc tgt acc aag tta tca cta cgt tac 315 E D
T G I Y F C T K L S L R Y 105 tgg ttc ttc gat gtc tgg ggc gca ggg
acc acg gtc acc gtc tcc 360 W F F D V W G A G T T V T V S 120 tca
363 S 121
[0047] Nucleotide sequence (SEQ ID NO:3) and amino acid sequence
(SEQ ID NO:4) of mAb 7E light chain variable region
TABLE-US-00003 gat ttt gtg atg acc cag act cca ctc act ttg tcg gtt
acc att 45 D F V M T Q T P L T L S V T I 15 gga caa cca gcc tcc atc
tct tgc aag tca agt cag agc ctc ttg 90 G Q P A S I S C K S S Q S L
L 30 gat agt gat gga aag aca tat ttg aat tgg ttg tta cag agg cca
135 D S D G K T Y L N W L L Q R P 45 ggc cag tct cca aag cac ctc
atc tat ctg gtg tct aaa ctg gac 180 G Q S P K H L I Y L V S K L D
60 tct gga gtc cct gac agg ttc act ggc agt gga tca ggg acc gat 215
S G V P D R F T G S G S G T D 75 ttc aca ctg aga atc agc aga gtg
gag gct gag gat ttg gga gtt 270 F T L R I S R V E A E D L G V 90
tat tat tgc tgg caa agt aca cat ttt ccg tgg acg ttc ggt gga 315 Y Y
C W Q S T H F P W T F G G 105 ggc acc aag ctg gaa atc aaa cgg 339 G
T K L E I K R 113
[0048] Anti-IL-20 Antibodies
[0049] In some embodiments of the invention, the IL-20 antagonist
comprises an anti-IL-20 antibody. Anti-IL-20 antibodies are known
in the art, see, e.g., U.S. Pat. Nos. 7,435,800; 7,115,714;
7,119,175; 7,151,166; and 7,393,684; and PCT publications WO
2007/081465; WO 99/27103; WO 2004/085475; and WO 2005052000.
[0050] In another embodiment, the anti-IL-20 antibody comprises one
or more CDR(s) of antibody mAb 7E (such as one, two, three, four,
five, or, in some embodiments, all six CDRs from mAb 7E). In some
embodiments, the anti-IL-20 antibody comprises the three CDRs from
the heavy chain and the three CDRs from the light chain of the
antibody produced by the cell line having ATCC No. PTA-8587 or
progeny thereof. In some embodiments, the anti-IL-20 antibody
comprises the three heavy chain CDRs from the amino acid sequence
shown in SEQ ID NO:2 and the three light chain CDRs from the amino
acid sequence shown in SEQ ID NO:4.
[0051] Determination of CDR regions is well within the skill of the
art. CDR(s) may be Kabat, Chothia, or a combination of Kabat and
Chothia. There are at least two techniques for determining CDRs:
(1) an approach based on cross-species sequence variability (i.e.,
Kabat et al. Sequences of Proteins of Immunological Interest, (5th
ed., 1991, National Institutes of Health, Bethesda Md.)); and (2)
an approach based on crystallographic studies of antigen-antibody
complexes (Chothia et al. (1989) Nature 342:877; Al-lazikani et al
(1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR may
refer to CDRs defined by either approach or by a combination of
both approaches.
[0052] The antibodies useful in the present invention can encompass
monoclonal antibodies, polyclonal antibodies, antibody fragments
(e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies,
bispecific antibodies, heteroconjugate antibodies, single chain
(ScFv), mutants thereof, fusion proteins comprising an antibody
portion, humanized antibodies, and any other modified configuration
of the immunoglobulin molecule that comprises an antigen
recognition site of the required specificity, including
glycosylation variants of antibodies, amino acid sequence variants
of antibodies, and covalently modified antibodies. The antibodies
may be murine, rat, human, or any other origin (including chimeric
or humanized antibodies). For purposes of this invention, the
antibody reacts with IL-20 in a manner that inhibits IL-20 and/or
downstream pathways mediated by the IL-20 signaling function. In
one embodiment, the antibody is a human antibody, a humanized
antibody or a chimeric antibody which recognizes one or more
epitopes on human IL-20. In some embodiments, the anti-IL-20
antibody binds to the same epitope on human IL-20 as antibody mAb
7E. In other embodiments, the antibody comprises a modified
constant region, such as a constant region that is immunologically
inert, e.g., does not trigger complement mediated lysis, or does
not stimulate antibody-dependent cell mediated cytotoxicity (ADCC).
ADCC activity can be assessed using methods disclosed in U.S. Pat.
No. 5,500,362. In other embodiments, the constant region is
modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT
Application No. PCT/GB99/01441; and/or UK Patent Application No.
9809951.8.
[0053] The binding affinity of an anti-IL-20 antibody to IL-20
(such as human IL-20) can be less than any of about 100 nM, about
50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or
about 50 pM to any of about 2 pM. Binding affinity can be expressed
K.sub.D or dissociation constant, and an increased binding affinity
corresponds to a decreased K.sub.D. One way of determining binding
affinity of antibodies to IL-20 is by measuring binding affinity of
monofunctional Fab fragments of the antibody. To obtain
monofunctional Fab fragments, an antibody (for example, IgG) can be
cleaved with papain or expressed recombinantly. The affinity of an
anti-IL-20 Fab fragment of an antibody can be determined by surface
plasmon resonance (BIAcore3000.TM. surface plasmon resonance (SPR)
system, BIAcore, INC, Piscaway N.J.). Kinetic association rates
(k.sub.on) and dissociation rates (k.sub.off) (generally measured
at 25.degree. C.) are obtained; and equilibrium dissociation
constant (K.sub.D) values are calculated as k.sub.off/k.sub.on.
[0054] In some embodiments, the antibody binds human IL-20, and
does not significantly bind an IL-20 from another mammalian
species. In some embodiments, the antibody binds human IL-20 as
well as one or more IL-20 from another mammalian species. In still
other embodiments, the antibody binds IL-20 and does not
significantly cross-react with other cytokines (such as the related
cytokines IL-10, IL-17A, IL-19, IL-22, IL-24 and IL-26). The
epitope(s) bound by the antibody can be continuous or
discontinuous. In one embodiment, the antibody binds essentially
the same human IL-20 epitopes as antibody mAb 7E.
[0055] The anti-IL-20 antibodies may be made by any method known in
the art. For example, antibodies that can inhibit IL-20 may be made
by immunization using full length or partial sequence of IL-20 as
immunogens The route and schedule of immunization of the host
animal are generally in keeping with established and conventional
techniques for antibody stimulation and production, as further
described herein. General techniques for production of mouse,
humanized and human antibodies are known in the art and are
described herein.
[0056] It is contemplated that any mammalian subject including
humans or antibody producing cells therefrom can be manipulated to
serve as the basis for production of mammalian, including human,
hybridoma cell lines. Typically, the host animal is inoculated
intraperitoneally, intramuscularly, orally, subcutaneously,
intraplantar, and/or intradermally with an amount of immunogen,
including as described herein.
[0057] Hybridomas can be prepared from the lymphocytes and
immortalized myeloma cells using the general somatic cell
hybridization technique of Kohler, B. and Milstein, C. (1975)
Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro,
18:377-381 (1982). Available myeloma lines, including but not
limited to X63-Ag8.653 and those from the Salk Institute, Cell
Distribution Center, San Diego, Calif., USA, may be used in the
hybridization. Generally, the technique involves fusing myeloma
cells and lymphoid cells using a fusogen such as polyethylene
glycol, or by electrical means well known to those skilled in the
art. After the fusion, the cells are separated from the fusion
medium and grown in a selective growth medium, such as
hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate
unhybridized parent cells. Any of the media described herein,
supplemented with or without serum, can be used for culturing
hybridomas that secrete monoclonal antibodies. As another
alternative to the cell fusion technique, EBV immortalized B cells
may be used to produce the anti-IL-20 monoclonal antibodies of the
subject invention. The hybridomas are expanded and subcloned, if
desired, and supernatants are assayed for anti-immunogen activity
by conventional immunoassay procedures (e.g., radioimmunoassay,
enzyme immunoassay, or fluorescence immunoassay).
[0058] Hybridomas that may be used as source of antibodies
encompass all derivatives, progeny cells of the parent hybridomas
that produce monoclonal antibodies specific for IL-20, or a portion
thereof.
[0059] Hybridomas that produce such antibodies may be grown in
vitro or in vivo using known procedures. The monoclonal antibodies
may be isolated from the culture media or body fluids, by
conventional immunoglobulin purification procedures such as
ammonium sulfate precipitation, gel electrophoresis, dialysis,
chromatography, and ultrafiltration, if desired. Undesired activity
if present, can be removed, for example, by running the preparation
over adsorbents made of the immunogen attached to a solid phase and
eluting or releasing the desired antibodies off the immunogen.
Immunization of a host animal with a human IL-20, or a fragment
containing the target amino acid sequence conjugated to a protein
that is immunogenic in the species to be immunized, e.g., keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor using a bifunctional or derivatizing agent, for
example maleimidobenzoyl sulfosuccinimide ester (conjugation
through cysteine residues), N-hydroxysuccinimide (through lysine
residues), glutaraldehyde, succinic anhydride, SOCl2, or
R1N.dbd.C.dbd.NR, where R and R1 are different alkyl groups, can
yield a population of antibodies (e.g., monoclonal antibodies).
[0060] If desired, the anti-IL-20 antibody (monoclonal or
polyclonal) of interest (e.g., produced by a hybridoma) may be
sequenced and the polynucleotide sequence may then be cloned into a
vector for expression or propagation. The sequence encoding the
antibody of interest may be maintained in vector in a host cell and
the host cell can then be expanded and frozen for future use. In an
alternative, the polynucleotide sequence may be used for genetic
manipulation to "humanize" the antibody or to improve the affinity,
or other characteristics of the antibody. For example, the constant
region may be engineered to more resemble human constant regions to
avoid immune response if the antibody is used in clinical trials
and treatments in humans. It may be desirable to genetically
manipulate the antibody sequence to obtain greater affinity to
IL-20 and greater efficacy in inhibiting IL-20. It will be apparent
to one of skill in the art that one or more polynucleotide changes
can be made to the anti-IL-20 antibody and still maintain its
binding ability to IL-20.
[0061] "Humanized" antibodies generally refer to a molecule having
an antigen binding site that is substantially derived from an
immunoglobulin from a non-human species and the remaining
immunoglobulin structure of the molecule based upon the structure
and/or sequence of a human immunoglobulin. The antigen binding site
may comprise either complete variable domains fused onto constant
domains or only the complementarity determining regions (CDRs)
grafted onto appropriate framework regions in the variable domains.
Antigen binding sites may be wild type or modified by one or more
amino acid substitutions, e.g., modified to resemble human
immunoglobulin more closely. Some forms of humanized antibodies
preserve all CDR sequences (for example, a humanized mouse antibody
which contains all six CDRs from the mouse antibodies). Other forms
of humanized antibodies have one or more CDRs (one, two, three,
four, five, six) which are altered with respect to the original
antibody. In some instances, framework region (FR) residues or
other residues of the human immunoglobulin replaced by
corresponding non-human residues. Furthermore, humanized antibodies
may comprise residues which are not found in the recipient antibody
or in the donor antibody. Humanization can also include affinity
maturation.
[0062] In yet another alternative, fully human antibodies may be
obtained by using commercially available mice that have been
engineered to express specific human immunoglobulin proteins.
Transgenic animals that are designed to produce a more desirable
(e.g., fully human antibodies) or more robust immune response may
also be used for generation of humanized or human antibodies.
Examples of such technology are Xenomouse.RTM. from Amgen, Inc.
(Fremont, Calif.) and HuMAb-Mouse.RTM. and TC Mouse.TM. from
Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies
may be made recombinantly by phage display technology. See, for
example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and
6,265,150; and Winter et al., (1994) Annu. Rev. Immunol.
12:433-455. Alternatively, the phage display technology (McCafferty
et al., (1990) Nature 348:552-553) can be used to produce human
antibodies and antibody fragments in vitro, from immunoglobulin
variable (V) domain gene repertoires from unimmunized donors.
[0063] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the monoclonal
antibodies). The hybridoma cells serve as a preferred source of
such DNA. Once isolated, the DNA may be placed into one or more
expression vectors, which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. See, e.g., PCT
Publication No. WO 87/04462. The DNA also may be modified, for
example, by substituting the coding sequence for human heavy and
light chain constant domains in place of the homologous murine
sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851,
or by covalently joining to the immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin
polypeptide. In that manner, "chimeric" or "hybrid" antibodies are
prepared that have the binding specificity of an anti-IL-20
monoclonal antibody herein.
[0064] Anti-IL-20 antibodies may be characterized using methods
well known in the art. For example, one method is to identify the
epitope to which it binds, or "epitope mapping." There are many
methods known in the art for mapping and characterizing the
location of epitopes on proteins, including solving the crystal
structure of an antibody-antigen complex, competition assays, gene
fragment expression assays, and synthetic peptide-based assays, as
described, for example, in Chapter 11 of Harlow and Lane, Using
Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1999. In an additional example,
epitope mapping can be used to determine the sequence to which an
anti-IL-20 antibody binds. The epitope can be a linear epitope,
i.e., contained in a single stretch of amino acids, or a
conformational epitope formed by a three-dimensional interaction of
amino acids that may not necessarily be contained in a single
stretch (primary structure linear sequence). Peptides of varying
lengths (e.g., at least 4-6 amino acids long) can be isolated or
synthesized (e.g., recombinantly) and used for binding assays with
an anti-IL-20 antibody. In another example, the epitope to which
the anti-IL-20 antibody binds can be determined in a systematic
screening by using overlapping peptides derived from the IL-20
sequence and determining binding by the anti-IL-20 antibody.
According to the gene fragment expression assays, the open reading
frame encoding IL-20 is fragmented either randomly or by specific
genetic constructions and the reactivity of the expressed fragments
of IL-20 with the antibody to be tested is determined. The gene
fragments may, for example, be produced by PCR and then transcribed
and translated into protein in vitro, in the presence of
radioactive amino acids. The binding of the antibody to the
radioactively labeled IL-20 fragments is then determined by
immunoprecipitation and gel electrophoresis. Certain epitopes can
also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage
libraries). Alternatively, a defined library of overlapping peptide
fragments can be tested for binding to the test antibody in simple
binding assays. In an additional example, mutagenesis of an antigen
binding domain, domain swapping experiments and alanine scanning
mutagenesis can be performed to identify residues required,
sufficient, and/or necessary for epitope binding. For example,
domain swapping experiments can be performed using a mutant IL-20
in which various fragments of the IL-20 polypeptide have been
replaced (swapped) with sequences from a closely related, but
antigenically distinct protein (such as another member of the
neurotrophin protein family). By assessing binding of the antibody
to the mutant IL-20, the importance of the particular IL-20
fragment to antibody binding can be assessed.
[0065] Yet another method which can be used to characterize an
anti-IL-20 antibody is to use competition assays with other
antibodies known to bind to the same antigen, i.e., various
fragments on IL-20, to determine if the anti-IL-20 antibody binds
to the same epitope as other antibodies. Competition assays are
well known to those of skill in the art.
Other IL-20 Antagonists
[0066] IL-20 antagonists other than anti-IL-20 antibodies may be
used. In some embodiments of the invention, the IL-20 antagonist
comprises at least one antisense molecule capable of blocking or
decreasing the expression of a functional IL-20. Nucleotide
sequences of the IL-20 are known and are readily available from
publicly available databases. See for example, Genbank accession
numbers NM 018724.3 and NP 061194.2. It is routine to prepare
antisense oligonucleotide molecules that will specifically bind
IL-20 mRNA without cross-reacting with other polynucleotides.
Exemplary sites of targeting include, but are not limited to, the
initiation codon, the 5' regulatory regions, the coding sequence
and the 3' untranslated region. In some embodiments, the
oligonucleotides are about 10 to 100 nucleotides in length, about
15 to 50 nucleotides in length, about 18 to 25 nucleotides in
length, or more. The oligonucleotides can comprise backbone
modifications such as, for example, phosphorothioate linkages, and
2'-O sugar modifications well know in the art.
[0067] Alternatively, IL-20 expression and/or release can be
decreased using gene knockdown, morpholino oligonucleotides, small
interfering RNA (siRNA or RNAi), microRNA or ribozymes, methods
that are well-known in the art.
[0068] In other embodiments, the IL-20 antagonist comprises at
least one IL-20 inhibitory compound. As used herein, "IL-20
inhibitory compound" refers to a compound other than an anti-IL-20
antibody that directly or indirectly reduces, inhibits,
neutralizes, or abolishes IL-20 biological activity. An IL-20
inhibitory compound should exhibit any one or more of the following
characteristics: (a) binds to IL-20 and inhibits IL-20 biological
activity and/or downstream pathways mediated by IL-20 signaling
function; (b) prevents, ameliorates, or treats any aspect of
osteoporosis or rheumatoid arthritis; (c) blocks or decreases IL-20
receptor activation; (d) increases clearance of IL-20; (e) inhibits
(reduces) IL-20 synthesis, production or release. One skilled in
the art can prepare other small molecules IL-20 inhibitory
compounds.
[0069] In some embodiments, an IL-20 inhibitory compound is an
IL-20 mutant which can bind to an IL-20 receptor but can not elicit
signal transduction. In some embodiments, the IL-20 inhibitory
compound is an IL-20 mutant which blocks binding of wild type IL-20
to an IL-20 receptor thus preventing IL-20 signal transduction.
[0070] In some embodiments, IL-20 inhibitory compounds comprise
small molecules, a small molecule can have a molecular weight of
about any of 100 to 20,000 daltons, 500 to 15,000 daltons, or 1000
to 10,000 daltons. Libraries of small molecules are commercially
available. The small molecules can be administered using any means
known in the art, including inhalation, intraperitoneally,
intravenously, intramuscularly, subcutaneously, intrathecally,
intraventricularly, orally, enterally, parenterally, intranasally,
or dermally. In general, when the IL-20-antagonist according to the
invention is a small molecule, it will be administered at the rate
of 0.1 to 300 mg/kg of the weight of the patient divided into one
to three or more doses. For an adult patient of normal weight,
doses ranging from 1 mg to 5 g per dose can be administered.
[0071] In some embodiments, the IL-20 antagonists include a
polypeptide comprising an extracellular portion of an IL-20
receptor (such as IL-20 R1, IL-20R2, or IL-22R1), wherein the
polypeptide specifically binds to 11-20 and blocks its interaction
with one or more IL-20 receptors. In some embodiments, the
extracellular portion of the IL-20 receptor is fused to a Fc domain
of antibody. Examples of the soluble receptors are described in PCT
WO 01/46232.
Identification of IL-20 Antagonists
[0072] Anti-IL-20 antibodies and other IL-20 antagonists can be
identified or characterized using methods known in the art, whereby
reduction, amelioration, or neutralization of an IL-20 biological
activity is detected and/or measured. For example, an ELISA-type
assay may be suitable for qualitative or quantitative measurement
of IL-20 mediated kinase activation by measuring the
phosphorylation of proteins activated through an IL-20 cascade.
Examples include JNK, ERK, AKT, p38, STAT3 and TRAF6.
[0073] The IL-20 antagonists can also be identified by incubating a
candidate agent with IL-20 and monitoring any one or more of the
following characteristics: (a) binding to IL-20 and inhibiting
IL-20 biological activity and/or downstream pathways mediated by
IL-20 signaling function; (b) preventing, ameliorating, or treating
any aspect of osteoporosis or rheumatoid arthritis; (c) blocking or
decreasing IL-20 receptor activation; (d) increasing clearance of
IL-20; (e) inhibiting (reducing) IL-20 synthesis, production or
release. In some embodiments, an IL-20 antagonist is identified by
incubating a candidate agent with IL-20 and monitoring binding and
attendant reduction or neutralization of a biological activity of
IL-20. The binding assay may be performed with purified IL-20
polypeptide(s), or with cells naturally expressing, or transfected
to express, IL-20 polypeptide(s). In one embodiment, the binding
assay is a competitive binding assay, where the ability of a
candidate antibody to compete with a known IL-20 antagonist for
IL-20 binding is evaluated. The assay may be performed in various
formats, including the ELISA format. In other embodiments, an IL-20
antagonist is identified by incubating a candidate agent with IL-20
and monitoring attendant inhibition of IL-20R1/IL-20R2 complex
formation or IL-20R2/IL-22R1 complex formation. Following initial
identification, the activity of a candidate anti-IL-20 antagonist
can be further confirmed and refined by bioassays, known to test
the targeted biological activities. Alternatively, bioassays can be
used to screen candidates directly.
[0074] The examples provided below provide a number of assays that
can be used to screen candidate IL-20 antagonists. Bioassays
include but are not limited to MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
assays for proliferation of HUVEC cells; analysis of candidate
agents on osteoclast differentiation, for example, as measured by
TRAP staining; flow cytometry of determine competitive binding of
IL-20 to cells in the presence of candidate IL-20 antagonists; and
inhibition of IL-20-induced apoptosis in renal epithelial cells. In
addition, RT-PCR or Real-time PCR which can be used to directly
measure IL-20 expression or to measure expression of genes
upregulated by IL-20 such as TNF-.alpha., MCP-1, IL-1.beta., IL-6
and VEGF.
Compositions for Use in the Methods of the Invention
[0075] The compositions used in the methods of the invention
comprise an effective amount of one or more IL-20 antagonists (such
as anti-IL-20 antibody), and, in some embodiments, further comprise
a pharmaceutically acceptable excipient. In some embodiments, the
composition is for use in any of the methods described herein.
Examples of IL-20 antagonists are described herein. It is
understood that the compositions can comprise more than one IL-20
antagonist. For example, a composition can comprise more than one
member of a class of IL-20 antagonist (e.g., a mixture of
anti-IL-20 antibodies that recognize different epitopes of IL-20),
as well as members of different classes of IL-20 antagonists (e.g.,
an anti-IL-20 antibody and an IL-20 inhibitory compound). Other
exemplary compositions comprise more than one anti-IL-20 antibodies
that recognize the same epitope(s), different species of anti-IL-20
antibodies that bind to different epitopes of IL-20, or different
IL-20 inhibitory compounds.
[0076] The composition used in the present invention can further
comprise pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and Practice of Pharmacy 20th
Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations used, and may comprise
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrans; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0077] The IL-20 antagonist and compositions thereof can also be
used in conjunction with other agents that serve to enhance and/or
complement the effectiveness of the agents.
Administration of an IL-20 Antagonist and Assessment of
Treatment
[0078] The invention provides methods to treat, delay the onset of,
or prevent osteoporosis in an individual. As discussed above, IL-20
is an osteoclastogenic cytokine that acts upstream RANKL-RANK
signaling cascade in the development and activation of osteoclasts.
Overexpression of IL-20 may stimulate osteoclast differentiation
thereby reducing the capacity to repair bone damage associated with
osteoporosis.
[0079] There are a number of factors that increase the risk of
developing osteoporosis. For example, osteoporosis is associated
with low estrogen levels that occur in postmenopause. Low estrogen
levels may also be the result of early surgical removal of both
ovaries. In addition, chemotherapy can result in early menopause as
a result of the toxic effects of the chemotherapy on the ovaries.
As shown in Examples, an IL-20 antagonist ameliorated the
osteoporotic effects in oviarectomized mice. Thus, the IL-20
antagonists described here may be used to treat, delay the onset
of, or prevent osteoporosis in a postmenopausal individual by
administering an effective dose of an IL-20 antagonist.
[0080] Osteoporosis may also result from hormone ablation
treatment. In both prostate cancer and breast cancer, it is common
for patients to receive hormone ablation therapies; for example,
androgen in the case of prostate cancer and estrogen in the case of
breast cancer, which can lead to a decrease in bone mass and an
increased risk of fractures. Thus, the IL-20 antagonists described
here may be used to treat, delay the onset of, or prevent
osteoporosis in an individual undergoing hormone ablation therapy
by administering an effective dose of an IL-20 antagonist.
[0081] Chronic inflammation due to diseases including but not
limited to rheumatoid arthritis and chronic liver disease can lead
to bone damage. As shown in Examples, an IL-20 antagonist
alleviated bone damage in a rat model of rheumatoid arthritis.
Thus, the IL-20 antagonists described here may be used to treat,
delay the onset of, or prevent osteoporosis in an individual with a
chronic inflammatory condition by administering an effective dose
of an IL-20 antagonist.
[0082] The IL-20 antagonist can be administered to an individual
via any suitable route. For example, the IL-20 antagonist can be
administered orally, intravenously, sublingually, subcutaneously,
intraarterially, intrasynovially, intravescicular (such as via the
bladder), intramuscularly, intracardiacly, intrathoracicly,
intraperitoneally, intraventricularly, sublingually, by inhalation,
by suppository, and transdermally. They can be administered orally,
for example, in the form of tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, lollypops, chewing gum or the like
prepared by art recognized procedures. It should be apparent to a
person skilled in the art that the examples described herein are
not intended to be limiting but to be illustrative of the
techniques available.
[0083] Accordingly, in some embodiments, the IL-20 antagonist, such
as an anti-IL-20 antibody, is administered to a individual in
accordance with known methods, such as intravenous administration,
e.g., as a bolus or by continuous infusion over a period of time,
by intramuscular, intraperitoneal, intracerebrospinal,
subcutaneous, intra-articular, intrasynovial, intrathecal, oral,
inhalation or topical routes. Commercially available nebulizers for
liquid formulations, including jet nebulizers and ultrasonic
nebulizers are useful for administration. Liquid formulations can
be directly nebulized and lyophilized powder can be nebulized after
reconstitution. Alternatively, IL-20 antagonists can be aerosolized
using a fluorocarbon formulation and a metered dose inhaler, or
inhaled as a lyophilized and milled powder.
[0084] In one embodiment, an IL-20 antagonist is administered via
site-specific or targeted local delivery techniques. Examples of
site-specific or targeted local delivery techniques include various
implantable depot sources of the IL-20 antagonist or local delivery
catheters, such as infusion catheters, an indwelling catheter, or a
needle catheter, synthetic grafts, adventitial wraps, shunts and
stents or other implantable devices, site specific carriers, direct
injection, or direct application. See, e.g., PCT Publication No. WO
00/53211 and U.S. Pat. No. 5,981,568.
[0085] Various formulations of an IL-20 antagonist (such as
anti-IL-20 antibody) may be used for administration. In some
embodiments, an IL-20 antagonist may be administered neat. In some
embodiments, the IL-20 antagonist comprises an anti-IL-20 antibody,
and may be in various formulations, including formulations
comprising a pharmaceutically acceptable excipient.
Pharmaceutically acceptable excipients are known in the art, and
are relatively inert substances that facilitate administration of a
pharmacologically effective substance. For example, an excipient
can give form or consistency, or act as a diluent. Suitable
excipients include, but are not limited to, stabilizing agents,
wetting and emulsifying agents, salts for varying osmolarity,
encapsulating agents, buffers, and skin penetration enhancers.
Excipients as well as formulations for parenteral and nonparenteral
drug delivery are set forth in Remington, The Science and Practice
of Pharmacy 20th Ed. Mack Publishing (2000).
[0086] In some embodiments, these agents are formulated for
administration by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Accordingly,
these agents can be combined with pharmaceutically acceptable
vehicles such as saline, Ringer's solution, dextrose solution, and
the like. The particular dosage regimen, i.e., dose, timing and
repetition, will depend on the particular individual and that
individual's medical history.
[0087] An anti-IL-20 antibody can be administered using any
suitable method, including by injection (e.g., intraperitoneally,
intravenously, subcutaneously, intramuscularly, etc.). Anti-IL-20
antibodies can also be administered via inhalation, as described
herein. Generally, for administration of anti-IL-20 antibodies, an
initial candidate dosage can be about 2 mg/kg. For the purpose of
the present invention, a typical daily dosage might range from
about any of 0.1 .mu.g/kg to 3 .mu.g/kg to 30 .mu.g/kg to 300
.mu.g/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on
the factors mentioned above. For repeated administrations over
several days or longer, depending on the condition, the treatment
is sustained until a desired suppression of symptoms occurs or
until sufficient therapeutic levels are achieved to reduce
osteoporosis or rheumatoid arthritis. An exemplary dosing regimen
comprises administering an initial dose of about 2 mg/kg, followed
by a weekly maintenance dose of about 1 mg/kg of the anti-IL-20
antibody, or followed by a maintenance dose of about 1 mg/kg every
other week. However, other dosage regimens may be useful, depending
on the pattern of pharmacokinetic decay that the practitioner
wishes to achieve. For example, dosing from one-four time a week is
contemplated. In some embodiments, dosing ranging from about 3
.mu.g/mg to about 2 mg/kg (such as about 3 .mu.g/mg, about 10
.mu.g/mg, about 30 .mu.g/mg, about 100 .mu.g/mg, about 300
.mu.g/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In some
embodiments, dosing frequency is once every week, every 2 weeks,
every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8
weeks, every 9 weeks, or every 10 weeks; or once every month, every
2 months, or every 3 months, or longer. The progress of this
therapy is easily monitored by conventional techniques and assays.
The dosing regimen (including the IL-20 antagonist(s) used) can
vary over time.
[0088] In general, when it is not an antibody, an IL-20 antagonist
may (in some embodiments) be administered at the rate of about 0.1
to 300 mg/kg of the weight of the patient divided into one to three
doses, or as disclosed herein. In some embodiments, for an adult
patient of normal weight, doses ranging from about 0.3 to 5.00
mg/kg may be administered. The particular dosage regimen, i.e.,
dose, timing and repetition, will depend on the particular
individual and that individual's medical history, as well as the
properties of the individual agents (such as the half-life of the
agent, and other considerations well known in the art).
[0089] For the purpose of the present invention, the appropriate
dosage of an IL-20 antagonist will depend on the IL-20
antagonist(s) (or compositions thereof) employed, the type and
severity of the osteoporosis or rheumatoid arthritis to be treated,
whether the agent is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the agent, and the discretion of the attending
physician. Typically the clinician will administer an IL-20
antagonist, such as an anti-IL-20 antibody, until a dosage is
reached that achieves the desired result.
[0090] Empirical considerations, such as the half-life, generally
will contribute to the determination of the dosage. For example,
antibodies that are compatible with the human immune system, such
as humanized antibodies or fully human antibodies, may be used to
prolong half-life of the antibody and to prevent the antibody being
attacked by the host's immune system. Frequency of administration
may be determined and adjusted over the course of therapy, and is
generally, but not necessarily, based on treatment and/or
suppression and/or amelioration and/or delay of osteoporosis.
Alternatively, sustained continuous release formulations of
anti-IL-20 antibodies may be appropriate. Various formulations and
devices for achieving sustained release are known in the art.
[0091] In one embodiment, dosages for an IL-20 antagonist may be
determined empirically in individuals who have been given one or
more administration(s) of IL-20 antagonist (such as an antibody).
Individuals are given incremental dosages of an IL-20 antagonist,
e.g., anti-IL-20 antibody. To assess efficacy of an IL-20
antagonist, an indicator of osteoporosis (such as bone mineral
density) or rheumatoid arthritis (such as swelling, pain,
stiffness, and tissue destruction in the joints) can be
followed.
[0092] Administration of an IL-20 antagonist in accordance with the
method in the present invention can be continuous or intermittent,
depending, for example, upon the recipient's physiological
condition, whether the purpose of the administration is therapeutic
or prophylactic, and other factors known to skilled practitioners.
The administration of an IL-20 antagonist (for example if the IL-20
antagonist is an anti-IL-20 antibody) may be essentially continuous
over a preselected period of time or may be in a series of spaced
dose, e.g., either before, during, or after developing osteoporosis
or rheumatoid arthritis.
[0093] In some embodiments, more than one IL-20 antagonist, such as
an antibody, may be present. The antagonist can be the same or
different from each other. At least one, at least two, at least
three, at least four, at least five different IL-20 antagonists can
be present. Generally, those IL-20 antagonists have complementary
activities that do not adversely affect each other. IL-20
antagonists can also be used in conjunction with other agents that
serve to enhance and/or complement the effectiveness of the
agents.
[0094] In some embodiments, the IL-20 antagonist is administered in
conjunction with another agent. In some embodiments, the other
agent is an agent for the treatment or amelioration of rheumatoid
arthritis. Examples of anti-rheumatoid arthritis agents include a
TNF.alpha. antagonist, for example, a polypeptide that binds TNF
and inhibits TNF activity as reflected in TNF binding to a
TNF-receptor. Examples of TNF.alpha. antagonists include etanercept
(ENBREL.RTM.) and anti-TNF.alpha. antibodies such as infliximab
(REMICADE.RTM.) and adalimumab (HUMIRA.RTM.). In one example, the
etanercept polypeptide is a fusion protein containing human soluble
TNF receptor (SEQ ID NO:5 shown below) and the Fc component of
human IgG1 (i.e., Etanercept). In some embodiments, the other agent
is an agent for the treatment or amelioration of osteoporosis.
Examples of anti-osteoporosis agents include alendronate,
ibandronate, risedronate, zoledronic acid, calcitonin, estrogen,
selective estrogen receptor modulators, raloxifene, parathyroid
hormone, and teriparatide.
Amino Acid Sequence of Human Soluble TNF Receptor (SEQ ID NO:5)
[0095] aqvaft pyapepgstc rlreyydqta qmccskcspg qhakvfctkt
sdtvcdsced stytqlwnwv peclscgsrc ssdqvetqac treqnrictc rpgwycalsk
qegcrlcapl rkcrpgfgva rpgtetsdvv ckpcapgtfs nttsstdicr phqic
[0096] In some embodiments of the invention, the IL-20 antagonist;
for example, mAb 7E or a derivative thereof, can be used in
combination with an etanercept polypeptide, for treating rheumatoid
arthritis or osteoporosis. The term "etanercept polypeptide" refers
to a fusion protein containing a soluble receptor of tumor necrosis
factor (TNF) and the Fc component of an immunoglobulin. In one
example, the soluble TNF receptor is a human soluble TNF receptor
having the amino acid sequence SEQ ID NO:5 shown below and its
functional equivalent, i.e., a polypeptide having an amino acid
sequence at least 85% (e.g., 90%, 95%, or 98%) identical to SEQ ID
NO:5 and capable of binding to human TNF. The etanercept
polypeptide can be made by conventional recombinant technology.
[0097] Therapeutic formulations of the IL-20 antagonist (such as an
antibody) used in accordance with the present invention are
prepared for storage by mixing an antibody having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington, The Science and
Practice of Pharmacy 20th Ed. Mack Publishing (2000)), in the form
of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and may comprise buffers
such as phosphate, citrate, and other organic acids; salts such as
sodium chloride; antioxidants including ascorbic acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium chloride; benzalkonium chloride,
benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl
parabens, such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0098] Liposomes containing the IL-20 antagonist (such as an
antibody) are prepared by methods known in the art, such as
described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688
(1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980);
and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced
circulation time are disclosed in U.S. Pat. No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid composition comprising
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0099] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy 20th Ed. Mack Publishing (2000).
[0100] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or `poly(v nylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0101] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic
anti-IL-20 antibody compositions are generally placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0102] The compositions according to the present invention may be
in unit dosage forms such as tablets, pills, capsules, powders,
granules, solutions or suspensions, or suppositories, for oral,
parenteral or rectal administration, or administration by
inhalation or insufflation.
[0103] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical carrier,
e.g. conventional tableting ingredients such as corn starch,
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate,
dicalcium phosphate or gums, and other pharmaceutical diluents,
e.g. water, to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention, or a
non-toxic pharmaceutically acceptable salt thereof. When referring
to these preformulation compositions as homogeneous, it is meant
that the active ingredient is dispersed evenly throughout the
composition so that the composition may be readily subdivided into
equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation composition is then subdivided
into unit dosage forms of the type described above containing from
0.1 to about 500 mg of the active ingredient of the present
invention. The tablets or pills of the novel composition can be
coated or otherwise compounded to provide a dosage form affording
the advantage of prolonged action. For example, the tablet or pill
can comprise an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two
components can be separated by an enteric layer that serves to
resist disintegration in the stomach and permits the inner
component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers
or coatings, such materials including a number of polymeric acids
and mixtures of polymeric acids with such materials as shellac,
cetyl alcohol and cellulose acetate.
[0104] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. Tween.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently comprise between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0105] Suitable emulsions may be prepared using commercially
available fat emulsions, such as Intralipid.TM., Liposyn.TM.,
Infonutrol.TM., Lipofundin.TM. and Lipiphysan.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can comprise
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0.
[0106] The emulsion compositions can be those prepared by mixing an
IL-20 antagonist with Intralipid.TM. or the components thereof
(soybean oil, egg phospholipids, glycerol and water).
[0107] Compositions for inhalation or insufflation include
solutions and suspensions in pharmaceutically acceptable, aqueous
or organic solvents, or mixtures thereof, and powders. The liquid
or solid compositions may contain suitable pharmaceutically
acceptable excipients as set out above. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in preferably
sterile pharmaceutically acceptable solvents may be nebulised by
use of gases. Nebulised solutions may be breathed directly from the
nebulising device or the nebulising device may be attached to a
face mask, tent or intermittent positive pressure breathing
machine. Solution, suspension or powder compositions may be
administered, preferably orally or nasally, from devices which
deliver the formulation in an appropriate manner.
[0108] Treatment efficacy can be assessed by methods well-known in
the art.
[0109] Targeted delivery of therapeutic compositions containing an
antisense polynucleotide, expression vector, or subgenomic
polynucleotides can also be used. Receptor-mediated DNA delivery
techniques are described in, for example, Findeis et al., Trends
Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods
And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994);
Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem.
(1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)
87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic
compositions containing a polynucleotide are administered in a
range of about 100 ng to about 200 mg of DNA for local
administration in a gene therapy protocol. In some embodiments,
concentration ranges of about 500 ng to about 50 mg, about 1 .mu.g
to about 2 mg, about 5 .mu.g to about 500 .mu.g, and about 20 .mu.g
to about 100 .mu.g of DNA or more can also be used during a gene
therapy protocol. The therapeutic polynucleotides and polypeptides
of the present invention can be delivered using gene delivery
vehicles. The gene delivery vehicle can be of viral or non-viral
origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51;
Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene
Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).
Expression of such coding sequences can be induced using endogenous
mammalian or heterologous promoters and/or enhancers. Expression of
the coding sequence can be either constitutive or regulated.
[0110] Viral-based vectors for delivery of a desired polynucleotide
and expression in a desired cell are well known in the art.
Exemplary viral-based vehicles include, but are not limited to,
recombinant retroviruses (see, e.g., PCT Publication Nos. WO
90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO
93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB
Patent No. 2,200,651; and EP Patent No. 0 345 242),
alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki
forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC
VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus
(ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and
adeno-associated virus (AAV) vectors (see, e.g., PCT Publication
Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO
95/11984 and WO 95/00655). Administration of DNA linked to killed
adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can
also be employed.
[0111] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.
Additional approaches are described in Philip, Mol. Cell Biol.
(1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994)
91:1581.
[0112] It is also apparent that an expression vector can be used to
direct expression of any of the protein-based IL-20 antagonists
described herein (e.g., anti-IL-20 antibody, immunoadhesin, etc.).
For example, other IL-20 receptor fragments that are capable of
blocking (from partial to complete blocking) IL-20 and/or an IL-20
biological activity are known in the art.
Kits
[0113] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
comprising an IL-20 antagonist (such as an antibody, such as
antibody mAb 7E described herein or its derivatives), and in some
embodiments, further comprise instructions for use in accordance
with any of the methods of the invention described herein. In some
embodiments, the IL-20 antagonist is any IL-20 antagonist described
herein. In other embodiments, the kit comprises an IL-20 antagonist
that is other than an anti-IL-20 antibody. In some embodiment, the
kit comprises an anti-IL-20 antibody (such as antibody mAb 7E
described herein). In other embodiments, the kit comprises an
anti-IL-20 antibody comprising one or more CDR(s) of antibody mAb
7E (such as one, two, three, four, five, or, in some embodiments,
all six CDRs from mAb 7E). In some embodiments, the included
instructions comprise a description of administration of the IL-20
antagonist to treat, delay the onset or prevent osteoporosis or
rheumatoid arthritis according to any of the methods described
herein. The kit may further comprise a description of selecting an
individual suitable for treatment based on identifying whether that
individual has osteoporosis or rheumatoid arthritis. In still other
embodiments, the instructions comprise a description of
administering an IL-20 antagonist to an individual at risk of
osteoporosis or rheumatoid arthritis.
[0114] The instructions relating to the use of an IL-20 antagonist
generally include information as to dosage, dosing schedule, and
route of administration for the intended treatment. The containers
may be unit doses, bulk packages (e.g., multi-dose packages) or
sub-unit doses. Instructions supplied in the kits of the invention
are typically written instructions on a label or package insert
(e.g., a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable.
[0115] The label or package insert indicates that the composition
is used for treating, delaying the onset and/or preventing
osteoporosis. Instructions may be provided for practicing any of
the methods described herein.
[0116] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an IL-20 antagonist, such as an
anti-IL-20 antibody. The container may further comprise a second
pharmaceutically active agent, such as a TNF.alpha. antagonist or
another drug for treating osteoporosis.
[0117] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit comprises a
container and a label or package insert(s) on or associated with
the container.
[0118] In some embodiments, the invention provides articles of
manufacture comprising contents of the kits described above. In
some embodiments, the kits comprise an IL-20 antagonist (such as
anti-IL-20 antibody) with information indicating use to treat
osteoporosis or rheumatoid arthritis.
[0119] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific examples
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever. All publications, references, patents and patent
applications cited herein are incorporated by reference in their
entirety.
EXAMPLES
Example 1
Treating Rheumatoid Arthritis with Monoclonal Antibody 7E (mAb
7E)
[0120] Rats having collagen-induced arthritis (CIA) is a
well-developed animal model for studying human rheumatoid
arthritis. This model was employed in this study to examine the
efficacy of mAb 7E for treating this disease.
[0121] CIA was induced in eight-week-old male Sprague-Dawley rats
as follows. The rats were immunized initially by intradermal
injection (in the dorsum) of 200 .mu.l emulsion containing Freund's
complete adjuvant, 4 mg/ml heat-killed Mycobacterium tuberculosis
(Arthrogen-CIA; Chondrex, Redmond, Wash.), and bovine type II
collagen (CII; 2 mg/ml dissolved in 0.05 M acetic acid) at a ratio
of 1:1:1 (v/v/v). Eight days later, the rats were then injected
subcutaneously with 100 .mu.l of the just-described emulsion in the
roots of the tails to boost their immune responses. CIA was
observed in these rats between day 11 and day 13 after the initial
immunization.
[0122] The following four groups of rats (n=5) were subjected to
this study: Group (1): healthy rats; Group (2): CIA rats, as
described above, administered with PBS (s.c.) one week after CIA
onset; Group (3): CIA rats administered with mAb 7E (3 mg/kg, s.c.)
one week after CIA onset, and; Group (4): Etanercept (Enebrel.RTM.;
Wyeth, USA, 3 mg/kg, s.c.) one week after CIA onset. Hind-paw
thickness of each treated rat was measured with a caliper. All raw
results obtained from this study were subjected to statistical
analysis using statistical software Prism 4.0; GraphPad Software,
San Diego, Calif., USA. The Kruskal-Wallis test was used to compare
the thickness of the hind paws. P-values<0.05 were considered
significant. Significant differences were evaluated using Student's
t-test or one-way analysis of variance (ANOVA). Statistical
significance was set at P<0.05.
[0123] As shown in Table 1 below, mAb 7E significantly reduced
hind-paw thickness in CIA rats (p<0.05) and its efficacy was
close to that of Etanercept, a commercially available
anti-rheumatoid arthritis drug (see Mihara et al., Br J Pharmacol.,
2008, 154:153-164). This result indicates that, like Etanercept,
mAb 7E is also effective in treating rheumatoid arthritis.
TABLE-US-00004 TABLE 1 Hind-Paw Thickness of Control and Treated
Rats Median Hind-Paw 25th-75th GROUP Thickness Percentiles 1
(health control) 0.53 cm 0.52-0.54 cm 2 (PBS-treated) 1.05 cm
1.02-1.13 cm 3 (mAb 7E-treated) 0.84 cm 0.72-0.93 cm 4
(Etanercept-treated) 0.86 cm 0.78-0.91 cm
[0124] Next, the effect of mAb 7E in reducing levels of
inflammatory mediators in synovial tissue was examined as follows.
The synovial tissues surrounding the knee joints in the treated CIA
rats were isolated and suspended in a PBS solution. The tissues
were then homogenized, centrifuged at 3000 rpm for 10 min at
4.degree. C., and the supernatants thus obtained were stored at
80.degree. C., ready for analysis. The levels of TNF-.alpha.,
IL-1.beta. (TNF-.alpha. and IL-1.beta. kits; R&D Systems,
Minneapolis, Minn.), and IL-20 (IL-20 kit; PeproTech Asia/CytoLab,
Rehovot, Israel) were evaluated using a sandwich ELISA assay
according to the manufacturer's instructions. It is known in the
art that the levels of all these inflammatory mediators are
elevated in CIA rats.
[0125] Results thus obtained indicate that mAb 7E and Etanercept
significantly reduced the levels of TNF-.alpha., IL-1.beta., and
IL-20 as compared with mIgG. More specifically, while in
mIgG-treated CIA rats, the levels of TNF-.alpha., IL-1.beta., and
IL-20 in synovial tissues were much higher than those in the
synovial tissues of healthy control rats, they were significantly
reduced in CIA rats treated with mAb 7E or Etanercept.
Example 2
Treating Rheumatoid Arthritis with Both mAb 7E and Etanercept
[0126] CIA was induced in rats following the method described in
Example 1. The CIA rats were randomly assigned to five groups (n=9
in each group) and treated as follows three times per week after
CIA onset: Group 1: PBS; Group 2: mouse IgG, obtained from Chemicon
International, Inc., Temecula, Calif., USA; Group 3: Etanercept (6
mg/kg, s.c.); Group 4: mAb 7E (6 mg/kg, s.c.); and Group 5: mAb 7E
(3 mg/kg, s,c,) and Etanercept (3 mg/kg, s.c.). First, the
thickness of hind-paw of each treated rats was examined following
the methods described in Example 1 above. The combined treatment of
mAb 7E and Etanercept showed significantly higher effect in
reducing hind-paw thickness as compared to the individual treatment
of mAb 7E and Etanercept.
[0127] Next, the severity of CIA in each hind paw of the rats was
monitored and scored, following the method described in Hsu et al.,
(Arthritis Rheum. 2006, 54:2722-2733). Generally, if a rat has a
severity score higher than 3, that rat is considered as having
severe swelling in its hind paw. The Kruskal-Wallis test was
applied to compare the severity scores obtained from different
groups to assess whether the results were statistical significant.
As shown in Table 2 below, the median severity score of rats
treated with both mAb 7E and Etanercept was much lower than that of
rats treated with mAb 7E alone or with Etanercept alone. These
results were statistically significant (P<0.05).
TABLE-US-00005 TABLE 2 Severity Score of Healthy and CIA Rats
Treated with Various Agents GROUP Median Severity Score 25.sup.th
75th Percentiles Healthy controls 0.2 0.0-0.4 Group 1 (PBS) 4.2
3.9-4.5 Group 2 (mIgG) 4.0 3.5-4.2 Group 3 (mAb 7E) 2.0 0.5-3.1
Group 4 (Etanercept) 2.1 0.7-3.6 Group 5 (mAb 7E + 0.9 0.0-2.2
Etanercept)
[0128] The presence of severe hind-paw swelling was then examined
in each treated CIA rat and the results were shown in FIG. 1.
Unexpectedly, while the incidences of severe swelling in the CIA
rats treated with mAb 7E and Etanercept, individually, were reduced
from 100% to 22% and from 100% to 33%, respectively, the incidence
of severe swelling in the CIA rats treated with both mAb 7E and
Etanercept reduced from 100% to only 6%. These results, which were
statistically significant as analyzed using Fisher's exact test,
indicate that the combined treatment of mAb 7E and Etanercept is
much more efficient than the individual treatment of mAb 7E or
Etanercept.
[0129] In addition, the severity of bone damage in the treated CIA
rats was examined twenty-five days after the initial immunization
with bovine collagen via radio imaging. Severe bone damage was
observed in hind-paw joints in the CIA rats treated with PBS and
mIgG (i.e., the rats of group 1 and group 2). Surprisingly, the
severity of local ankle bone damage was relatively mild in the CIA
rats treated with mAb 7E, Etanercept, or the combination thereof
(rats of groups 3-5). The differences between groups 1 and 2 rats
and groups 3-5 rats were statistically significant
(P<0.01-0.05). These results further confirm that mAb 7E
alleviated bone damage in CIA rats as efficiently as Etanercept and
the combined treatment of mAb 7E and Etanercept was much more
efficient than the corresponding individual treatment.
[0130] Further, a microcomputed tomographic analysis, using a 1076
microCT-40 system (Skyscan, Aartselaar, Belgium) equipped with a
high resolution, low-dose X-ray scanner, was performed to assess
the efficacy of mAb 7E alone and its combination with Etanercept in
protecting bone destruction in CIA rats. The X-ray tube in the
scanner was operated with photon energy of 48 kV, current of 200
uA, and exposure time of 1180 ms through a 0.5-mm-thick filter. The
image pixel size was 17.20 um, and the scanning time was
approximately 15 min. After standardized reconstruction of the
scanned images, the data sets for each tibia sample were resampled
with software (CTAn; Skyscan) to orient each sample in the same
manner. Consistent conditions such as thresholds were applied
throughout all analyses. Bone mineral density, a three-dimensional
bone characteristic parameter, was analyzed in 50 consecutive
slices. The results were calculated as a percentage versus values
relative to an mIgG control.
[0131] The tibias obtained from the CIA rats treated with PBS and
mIgG showed prominent bone damage compared to the intact joints
found in healthy controls. The CIA rats treated with mAb 7E
displayed alleviated bone loss compared to the rats treated with
mIgG. In the rats treated with both mAb 7E and Etanercept, the bone
loss was even less severe relative to the rats treated with either
mAb or Etanercept alone.
[0132] The bone mineral density, a quantitative parameter for
assessing disease severity, was measured in each treated CIA rat as
described above. mAb 7E treatment in CIA rats significantly
inhibited bone loss as compared to the mIgG-treated CIA rats
(P<0.05). The protective effects were drastically increased in
the CIA rats treated with both mAb 7E and Etanercept (P<0.01).
The microCT result supported the radiological data from their ankle
joints. These results provided evidence that mAb 7E not only
reduced the severity of arthritis but also inhibited bone loss.
[0133] Finally, the expression levels of TNF-.alpha., IL-1.beta.,
and IL-20 were examined in the CIA rats treated with both mAb 7E
and Etanercept and the results thus obtained showed that these
cytokines were significantly decreased. See FIG. 2. Expression of
IL-6 was also decreased following treatment with mAb 7E,
Etanercept, and mAb 7E and Etanercept together.
[0134] In sum, the results described above demonstrate that mAb 7E
is effective in treating CIA by both reducing severity of arthritis
and inhibiting bone loss. These results also indicate that the
combined effect of mAb 7E and Etanercept is significantly higher
than the individual effect of either mAb 7E or Etanercept.
Example 3
Treating Osteoporosis with mAb 7E
[0135] Fourteen-week-old female BALB/C mice (Laboratory Animal
Center, National Cheng Kung University, Tainan, Taiwan) were housed
in an environmentally controlled laboratory upon arrival and
acclimatized for 4 days. Animals were allocated in polycarbonate
cages (3 per cage) in a temperature/humidity controlled room (20-25
.degree. C. and 40-45%). The Light:dark cycle was 12-h light:12-h
dark, and feed and water were supplied free to access. The animals
were either dorsal ovariectomized (OVX) or falsely operated (Sham
controls) under general anesthesia by using pentobarbital (50 mg/kg
body weight; Sigma-Aldrich, St. Louis, Mo.). In Sham controls,
bilateral ovaries were exposed and then closed with skin suture not
removed. The mice were recovered for a week after OVX or control
surgery and then randomly assigned to six groups: Group 1: Sham
controls (n=5); Group 2: OVX mice with no further treatment (n=5);
Group 3: OVX mice treated with 17.beta.-estradiol (Sigma-Aldrich,
St. Louis, Mo., 10 .mu.g/kg/day, n=6); Group 4: OVX mice treated
with mIgG (Chemicon International, Inc., Temecula, Calif., USA, 3
mg/kg/three days, n=7)); Group 5: OVX mice treated with mAb 7E (3
mg/kg/three days, n=5); and Group 6: OVX mice treated with mAb 7E
(6 mg/kg/three days, n=5). The dosage of 17.beta.-estradiol
treatment used as a positive control are based on previous
protocols known to be effective in treating OVX mice. See Cano et
al., Osteoporos Int. 2008 June; 19(6):793-800.
[0136] The mice of all groups were sacrificed 2 months later. The
tibia of each mouse was aseptically collected, cleaned to remove
adherent soft tissues, and deposited in a tube filled with 3.7%
formalin. It was then subjected to Microcomputed tomography and
bone mineral density analysis following the methods described in
Example 2 above.
[0137] The serum level of IL-20 was upregulated in the OVX-group
mice but downregulated in OVX-mice treated with mAb 7E (FIG. 3a).
MicroCT scanning of the bone mineral density of the mice tibia
showed levels of bone damage in Groups 2 and 3 (untreated or
treated with mIgG) were much greater than those in Groups 4-6
(treated with 3mg/kg mAb 7E, 6 mg/kg mAb 7E, and
17.beta.-estradiol), indicating that, like 17.beta.-estradiol, mAb
7E also reduced bone loss in OVX mice (FIG. 3b). Further, the bone
mineral densities in mAb 7E-treated and 17.beta.-estradiol-treated
OVX mice were much higher than those in Sham controls and in
mIgG-treated mice (FIG. 3b). A statistically significant (P<0.05
compared with the mIgG controls) dose-response increase in bone
density was observed in those mice (FIG. 3c). Taken together, these
results demonstrate that mAb 7E is effective in treating
osteoporosis by reducing bone loss.
Example 4
IL-20 Antibody mAb 7E Inhibits Osteoclast Differentiation
[0138] Bone formation is tightly regulated by crosstalk between
osteoblasts and osteoclasts. Unbalanced osteoclastogenesis causes
bone loss in osteoporosis and rheumatoid arthritis (Takayanagi, H,
et al. (2005) Immunol Rev 208:181-193; Ross, F P and Teitelbaum, S
L (2005) Immunol Rev 208:88-105). Thus, we wanted to determine
whether mAb 7E protects against bone loss in OVX mice by inhibiting
the differentiation of osteoclasts.
[0139] Bone marrow cells (BMCs) were prepared from the tibias of
mice and incubated for 12 h (37.degree. C./5% CO2). Later,
non-adherent cells were collected and seeded in 24-well plates
(2.times.10.sup.6 cells per well) and cultured in the same medium
supplemented with 30 ng/ml recombinant murine macrophage colony
stimulating factor (M-CSF) (PreproTech). After 48 h, M-CSF-derived
BMCs were cultured with murine M-CSF (40 ng/ml) and sRANKL (100
ng/ml) (PreproTech) until the end of experiment. To test the effect
of mAb 7E, MCSF-derived BMCs were treated with IL-20 (200 ng/ml),
mAb 7E (2 .mu.g/ml), mIgG (2 .mu.g/ml) in .alpha.-MEM with M-CSF
and sRANKL until the end of the experiment.
[0140] For earlier treatment with mAb 7E, the BMCs were cultured
for 12 h. Non-adherent cells were seeded in 24-well plates
(2.times.10.sup.6 cells per well) and cultured in .alpha.-MEM
containing mAb 7E (2 .mu.g/ml) or control mIgG (2 .mu.g/ml), after
which M-CSF (40 ng/ml) was added. After 40 h, the mAb 7E treatment
was ended, the cells were washed with serum-free culture medium and
then incubated until the end of the experiments in .alpha.-MEM (40
ng/ml) and sRANKL (100 ng/ml). To calculate the number of
osteoclasts, the cells were fixed in acetone and stained for TRAP
using an acid phosphatase kit (Sigma-Aldrich).
[0141] Osteoclast precursor cells were prepared from bone
marrow-derived hematopoietic stem cells (HSCs), and both M-CSF and
soluble (s) RANKL were added to the culture to drive OC
differentiation. Two culture protocols were used to analyze the
effect of IL-20 antibody mAb 7E on OC differentiation in the early
and later stages of osteoclastogenesis (FIG. 4). After 48 h,
M-CSF-derived bone marrow macrophages were cultured with murine
M-CSF (40 ng/ml) and sRANKL (100 ng/ml) until the end of
experiment. TRAP staining was used to quantify the number of
differentiated osteoclasts. In the presence of mAb 7E (2 .mu.g/ml),
the number of TRAP+ osteoclasts was significantly (P<0.01) lower
than in the isotype controls (FIGS. 4b and 4c). No OC was detected
in the presence of mAb 7E. To clarify whether the mAb 7E affected
OC differentiation in early or later stages, bone marrow cells were
pre-incubated with mAb 7E or mIgG for 1 h and then M-CSF was added
for another 48 h. The cells were collected and cultured for 3 more
days in medium containing M-CSF and sRANKL without mAb 7E antibody
(FIG. 4d). Early incubation with mAb 7E efficiently inhibited
osteoclast differentiation (P<0.01 compared with the mIgG
controls) (FIGS. 4e and 4f). Thus, IL-20 antibody blocked both the
early and later stages of osteoclast differentiation.
[0142] Additionally, IL-20 induced TNF.alpha. and RANKL expression
in synovial fibroblasts from the CIA rat model of rheumatoid
arthritis but not in synovial fibroblasts from healthy rats.
Example 5
M-CSF Upregulated IL-20 in HSCs
[0143] IL-20 antibody mAb 7E blocked the differentiation of
osteoclasts from bone marrow-derived HSCs (FIG. 4). To test this
possibility that HSCs secreted IL-20 into culture medium, IL-20
expression in the bone marrow-derived HSCs that had been cultured
and treated with M-CSF for 48 h was examined. Real-time PCR
(RT-PCR) showed that IL-20 mRNA was higher in HSCs treated with
M-CSF than in controls (FIG. 5a), evidence that IL-20 had been
endogenously secreted in response to M-CSF stimulation. For RT-PCR,
SYBR Green I (Bio-Rad) chemistry using a fluorescence detection
system (DNA Engine Opticon 2; Bio-Rad). The fluorescence- and
time-dependent generation of signals was assessed using the
manufacturer's software.
[0144] IL-20 receptors were also expressed in the M-CSF-derived OC
precursor cells. These results suggested that IL-20 acted on the
HSC-derived osteoclast precursor cells in an autocrine manner.
Example 6
IL-20-Induced RANK Expression in M-CSF-Derived OC Precursors from
Bone Marrow Cells
[0145] The RANKL-RANK signal is critical for osteoclast
differentiation (Wada, T et al. (2006) Trends Mol Med 12:17-25).
RANK is expressed on the surface of osteoclasts. To investigate
whether IL-20 increased osteoclast differentiation by increasing
RANKL-RANK signaling, RANK expression was analyzed in M-CSF-derived
osteoclast precursors from bone marrow cells. The cells were
harvested by scraping, incubated for 30 min with 0.5 mg/ml
anti-murine RANK antibody (eBioscience) or isotype control
antibody, incubated with fluoroisothiocyanate (FITC)-conjugated
secondary antibody, and then analyzed using a flow cytometer
(FACSCalibur; BD Biosciences), with 20000 events acquired for each
sample. Flow cytometric analysis showed that, in IL-20-treated
M-CSF-derived OC precursors, the surface expression of RANK protein
(FIG. 5b) and of RANK mRNA (FIG. 5c) was upregulated in osteoclast
precursors.
[0146] Consistent with the inhibitory effect of mAb 7E on
osteoclast differentiation, mAb 7E treatment inhibited both the
expression of RANK transcripts (FIG. 5d) and the surface expression
of RANK protein. M-CSF-derived BMCs were cultured for 24 h with the
indicated concentrations of IL-20, mIgG, mAb 7E, or both IL-20 and
mAb 7E in .alpha.-MEM containing M-CSF (50 ng/ml) and sRANKL (100
ng/ml). To assay RANK production, the cells were stimulated with
IL-20 (200 ng/ml), trypsinized, and then stained with PE-conjugated
antibody against RANK (eBioscience) for flow cytometric analysis as
described above. These results are evidence that IL-20 acts on
osteoclast precursors as an osteoclastogenic cytokine by increasing
their RANK expression.
Example 7
IL-20 Targeted Osteoblasts and Upregulated RANKL Expression
[0147] Increased RANKL expression in osteoblasts is also a key
factor for osteoclastogenesis (Jordan, W J et al. (2005) Eur J
Immunol 35:1576-1582). RT-PCR analysis (FIG. 6a) and cytochemical
staining (FIG. 6b) were used to clarify the function of IL-20 in
osteoblasts. Both in vitro assays showed that IL-20 and its three
receptor subunits were expressed in MC3T3-E1 osteoblasts. To assess
the phosphorylation pattern of several signal transduction
proteins, MC3T3-E1 cells were stimulated with murine IL-20 (200
ng/ml) (R&D Systems, Minneapolis, Minn., USA) for the indicated
times. Western blotting was done with antibodies specific for
phosphorylated ERK, AKT, STAT3, p38, and JNK (Cell Signaling
Technology) using the manufacturer's instructions. As shown in FIG.
4c, JNK, ERK, AKT, and p38 were phosphorylated in IL-20-treated
MC3T3-E1 osteoblasts thus providing more evidence that IL-20 was
endogenously expressed in osteoblasts and triggered signal
transduction in them in an autocrine manner. It was recently
reported that Th17 is critical in the induction and progression of
RA. Th17 involvement in RA pathogenesis has been attributed to
IL-17-stimulated osteoclastogenesis (Kotake, S, et al. (1999) J.
Clin. Invest. 103:1345-1352). Transcripts of IL-17 were higher in
IL-20-treated MC3T3-E1 osteoblasts (FIG. 6d). To determine whether
IL-20 contributes to osteoclastogenesis by inducing RANKL
expression in osteoblasts, MC3T3-E1 cells with IL-20 and analyzed
RANKL expression using real-time-PCR and flow cytometry. RANKL
expression was time-dependently higher in IL-20-treated cells than
in untreated controls, and peaked 6 h after treatment (FIG. 6e).
The surface expression of RANKL protein was also higher in
IL-20-treated MC3T3-E1 cells (FIG. 6f). IL-20 acted on Th17 cells
and induced the release of RANKL. Moreover, IL-20 and IL-17
synergistically induce more RANKL expression, which in turn,
increases osteoclast differentiation and leads to bone erosion.
Example 8
IL-20 Antibody Inhibited IL-20-Induced RANKL Expression in
Osteoblasts
[0148] As discussed above, RANKL expression was higher in
IL-20-treated than in untreated MC3T3-E1 cells (FIGS. 6e and 6f).
To confirm that IL-20 antibody mAb 7E inhibits IL-20-induced RANKL
expression, cells were co-treated with IL-20 and mAb 7E.
Real-time-PCR showed that no RANKL transcripts were detected in
co-treated cells (FIG. 7). These results indicated that IL-20 is an
upstream activator for RANKL expression in osteoblasts, and that
mAb 7E inhibits IL-20-induced RANKL expression. The results
provided strong evidence that IL-20 is, in vitro, an upstream
inducer of RANKL in osteoblasts, and that this promotes
osteoclastogenesis.
Other Embodiments
[0149] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0150] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention.
Sequence CWU 1
1
61363DNAArtificial sequenceDNA encoding mAb 7E heavy chain variable
region 1gaattgaagc ttgaggagtc tggaggaggc ttggtgcagc ctggaggatc
catgaaactc 60tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggt
ccgccagtct 120ccagagaagg ggcttgagtg gattgctgaa attagaagca
aagctaataa ttatgcaaca 180tactttgctg agtctgtgaa agggaggttc
accatctcaa gagatgattc caaaagtggt 240gtctacctgc aaatgaacaa
cttaagagct gaggacactg gcatttattt ctgtaccaag 300ttatcactac
gttactggtt cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360tca
3632121PRTArtificial sequencemAb 7E heavy chain variable region
2Glu Leu Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp
Ala 20 25 30 Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu
Glu Trp Ile 35 40 45 Ala Glu Ile Arg Ser Lys Ala Asn Asn Tyr Ala
Thr Tyr Phe Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Gly 65 70 75 80 Val Tyr Leu Gln Met Asn Asn
Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Phe Cys Thr Lys Leu
Ser Leu Arg Tyr Trp Phe Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr
Thr Val Thr Val Ser Ser 115 120 3339DNAArtificial sequenceDNA
encoding mAb 7E light chain variable region 3gattttgtga tgacccagac
tccactcact ttgtcggtta ccattggaca accagcctcc 60atctcttgca agtcaagtca
gagcctcttg gatagtgatg gaaagacata tttgaattgg 120ttgttacaga
ggccaggcca gtctccaaag cacctcatct atctggtgtc taaactggac
180tctggagtcc ctgacaggtt cactggcagt ggatcaggga ccgatttcac
actgagaatc 240agcagagtgg aggctgagga tttgggagtt tattattgct
ggcaaagtac acattttccg 300tggacgttcg gtggaggcac caagctggaa atcaaacgg
3394112PRTArtificial sequencemAb 7E light chain variable region
4Asp Phe Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1
5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp
Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro
Gly Gln Ser 35 40 45 Pro Lys His Leu Ile Tyr Leu Val Ser Lys Leu
Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys Trp Gln Ser 85 90 95 Thr His Phe Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 5161PRTHomo
sapiens 5Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser
Thr Cys 1 5 10 15 Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
Cys Cys Ser Lys 20 25 30 Cys Ser Pro Gly Gln His Ala Lys Val Phe
Cys Thr Lys Thr Ser Asp 35 40 45 Thr Val Cys Asp Ser Cys Glu Asp
Ser Thr Tyr Thr Gln Leu Trp Asn 50 55 60 Trp Val Pro Glu Cys Leu
Ser Cys Gly Ser Arg Cys Ser Ser Asp Gln 65 70 75 80 Val Glu Thr Gln
Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys Thr Cys 85 90 95 Arg Pro
Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys Arg Leu 100 105 110
Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala Arg Pro 115
120 125 Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro Gly
Thr 130 135 140 Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro
His Gln Ile 145 150 155 160 Cys 6176PRTHomo sapiens 6Met Lys Ala
Ser Ser Leu Ala Phe Ser Leu Leu Ser Ala Ala Phe Tyr 1 5 10 15 Leu
Leu Trp Thr Pro Ser Thr Gly Leu Lys Thr Leu Asn Leu Gly Ser 20 25
30 Cys Val Ile Ala Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Glu
35 40 45 Ile Arg Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile
Arg Ile 50 55 60 Leu Arg Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro
Ala Asn Arg Cys 65 70 75 80 Cys Leu Leu Arg His Leu Leu Arg Leu Tyr
Leu Asp Arg Val Phe Lys 85 90 95 Asn Tyr Gln Thr Pro Asp His Tyr
Thr Leu Arg Lys Ile Ser Ser Leu 100 105 110 Ala Asn Ser Phe Leu Thr
Ile Lys Lys Asp Leu Arg Leu Cys His Ala 115 120 125 His Met Thr Cys
His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln 130 135 140 Ile Leu
Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys 145 150 155
160 Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln Trp Met Glu Glu Thr Glu
165 170 175
* * * * *
References