U.S. patent application number 13/456071 was filed with the patent office on 2012-08-16 for methods for modulating il-33 activity.
This patent application is currently assigned to Schering Corporation. Invention is credited to Alissa A. Chackerian, Robert A. Kastelein.
Application Number | 20120207752 13/456071 |
Document ID | / |
Family ID | 40846260 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120207752 |
Kind Code |
A1 |
Chackerian; Alissa A. ; et
al. |
August 16, 2012 |
METHODS FOR MODULATING IL-33 ACTIVITY
Abstract
Provided herein are methods of modulating IL-33 activity, e.g.,
for the purpose of treating immune diseases and conditions, as well
as methods of screening for compounds capable antagonizing IL-33
signaling.
Inventors: |
Chackerian; Alissa A.;
(Sunnyvale, CA) ; Kastelein; Robert A.; (Portola
Valley, CA) |
Assignee: |
Schering Corporation
Rahway
NJ
|
Family ID: |
40846260 |
Appl. No.: |
13/456071 |
Filed: |
April 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12501870 |
Jul 13, 2009 |
8187596 |
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13456071 |
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11779755 |
Jul 18, 2007 |
7560530 |
|
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12501870 |
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60887257 |
Jan 30, 2007 |
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60835250 |
Aug 3, 2006 |
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60832256 |
Jul 20, 2006 |
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Current U.S.
Class: |
424/133.1 ;
424/158.1; 424/172.1; 435/7.1; 436/501; 530/350; 530/351 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61P 33/02 20180101; Y10S 514/885 20130101; A61P 29/00 20180101;
G01N 2500/00 20130101; A61P 27/02 20180101; A61P 37/08 20180101;
A61P 19/02 20180101; A61P 25/00 20180101; A61P 31/12 20180101; G01N
33/6869 20130101; A61P 35/00 20180101; C07K 14/7155 20130101; C12Q
1/6897 20130101; A61P 11/06 20180101; A61P 31/04 20180101; A61P
33/00 20180101; A61P 33/12 20180101 |
Class at
Publication: |
424/133.1 ;
424/158.1; 530/350; 530/351; 424/172.1; 435/7.1; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; A61P 35/00 20060101
A61P035/00; A61P 25/00 20060101 A61P025/00; A61P 19/02 20060101
A61P019/02; A61P 11/06 20060101 A61P011/06; A61P 37/08 20060101
A61P037/08; A61P 27/02 20060101 A61P027/02; A61P 29/00 20060101
A61P029/00; A61P 31/04 20060101 A61P031/04; A61P 33/00 20060101
A61P033/00; A61P 31/12 20060101 A61P031/12; A61P 33/02 20060101
A61P033/02; A61P 33/12 20060101 A61P033/12; C07K 17/02 20060101
C07K017/02 |
Claims
1. A method of modulating an immune disorder or condition,
comprising inhibiting IL-33 signal transduction through ST2 and
IL-1RAcP by administering to a subject in need thereof an effective
amount of: a) an antagonist of IL-33 binding to a complex of ST2
and IL-1RAcP; or b) an antagonist of IL-1RAcP binding to a complex
of IL-33 and ST2.
2. The method of claim 1, wherein the immune disorder or condition
is selected from the group consisting of an innate response,
asthma, an allergy, multiple sclerosis, inflammatory bowel
disorder, arthritis, an infection, cancer, and a tumor.
3. The method of claim 2, wherein the infection is selected from
the group consisting of an intracellular pathogen, a bacterium, a
parasite and a virus.
4. The method of claim 3, wherein the infection is an intracellular
pathogen selected from the group consisting of Leishmania sp.,
Mycobacterium sp., Listeria sp., Toxoplasma sp., Schistosoma
sp.
5. The method of claim 1, wherein the immune disorder or condition
comprises: a) a T.sub.H1-type response; or b) a T.sub.H2-type
response.
6. The method of claim 2, wherein the arthritis is selected from
the group consisting of rheumatoid arthritis, osteoarthritis, and
psoriatic arthritis.
7. The method of claim 1, wherein the antagonist comprises an
antibody or a fragment thereof that specifically binds to: a)
IL-33; b) IL-1RAcP; c) ST2; d) a complex of ST2 and IL-1RAcP; e) a
complex of IL-33 and ST2; or f) a complex of IL-33, ST2 and
IL-1RAcP.
8. The method of claim 7, wherein the antibody or fragment thereof
does not bind to ST2 alone and does not bind to IL-33 alone.
9. The method of claim 7, wherein the antibody or fragment thereof
is a humanized antibody or human antibody.
10. The method of claim 7, wherein the antibody or fragment thereof
is a fragment selected from the group consisting of a Fab, an Fv
fragment, and an F(ab').sub.2 fragment.
11. A method of modulating blood cell counts, comprising inhibiting
IL-33 signal transduction through ST2 and IL-1RAcP by administering
to a subject in need thereof an effective amount of: a) an
antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP; or b)
an antagonist of IL-1RAcP binding to a complex of IL-33 and
ST2.
12. The method of claim 11, wherein the antagonist increases the
count of platelets.
13. The method of claim 11, wherein the antagonist decreases the
count of one or more of total white blood cells, neutrophils,
lymphocytes, and eosinophils.
14. The method of claim 11, wherein the antagonist comprises an
antibody or a fragment thereof that specifically binds to: a)
IL-33; b) IL-1RAcP; c) ST2; d) a complex of ST2 and IL-1RAcP; e) a
complex of IL-33 and ST2; or f) a complex of IL-33, ST2 and
IL-1RAcP.
15. The method of claim 14, wherein the antibody or fragment
thereof is a humanized antibody or human antibody.
16. The method of claim 14, wherein the antibody or fragment
thereof is a fragment selected from the group consisting of a Fab,
an Fv fragment, and an F(ab').sub.2 fragment.
17. An isolated and purified complex of ST2 and IL-1RAcP.
18. The isolated and purified complex of claim 17, wherein ST2 and
IL-1RAcP are human ST2 and human IL-1RAcP, respectively.
19. The isolated and purified complex of claim 18, further
comprising human IL-33.
20. An in vitro method of determining whether a test compound is an
antagonist of i) IL-33 binding to a complex of ST2 and IL-1RAcP, or
ii) IL-1RAcP binding to a complex of IL-33 and ST2, comprising an
assay selected from the group consisting of: a) an
NF-.kappa.B-dependent reporter gene expression assay; b) an MyD88
IRAK, IRAK4 or TRAF6 recruitment assay; c) an Erk1/2, p38,
I.kappa.B.alpha. or JNK phosphorylation assay; d) an NF-.kappa.B,
Erk1/2 or p38 phosphorylation assay in cells that naturally express
ST2; e) an IL-13, IL-6 or IL-5 expression assay; and f) an IL-6
production assay in mouse mast cell line WTMC; wherein the test
compound is determined to be an antagonist if it reduces the
activity of IL-33 in the assay when compared to an assay run
without the test compound.
Description
[0001] This application is a Continuation of U.S. patent
application Ser. No. 12/501,870, filed on Jul. 13, 2009, which is a
Divisional of U.S. patent application Ser. No. 11/779,755, filed on
Jul. 18, 2007, now U.S. Pat. No. 7,560,530, issued Jul. 14, 2009,
which claims benefit of U.S. Provisional Patent Application Nos.
60/832,256 (filed Jul. 20, 2006), 60/835,250 (filed Aug. 3, 2006),
and 60/887,257 (filed Jan. 30, 2007), each of which is hereby
incorporated by reference in their entireties.
[0002] The Sequence Listing filed electronically herewith is also
hereby incorporated by reference in its entirety (File Name:
BP06497-US-CNT_SeqListing.txt; Date Created: Apr. 24, 2012; File
Size: 18.2 KB.)
FIELD OF THE INVENTION
[0003] The present invention relates to methods for modulating
IL-33 activity.
BACKGROUND OF THE INVENTION
[0004] The immune system protects individuals from infective
agents, e.g., bacteria, multi-cellular organisms, as well as
cancers. This system includes several types of lymphoid and myeloid
cells such as monocytes, macrophages, dendritic cells (DCs),
eosinophils, T cells, B cells, and neutrophils. These lymphoid and
myeloid cells often produce signaling proteins known as cytokines.
Immune response includes inflammation, i.e., the accumulation of
immune cells systemically or in a particular location of the body.
In response to an infective agent or foreign substance, immune
cells secrete cytokines which, in turn, modulate immune cell
proliferation, development, differentiation, or migration. Immune
response sometimes results in pathological consequences, that is,
inflammatory disorders. These inflammatory disorders, which involve
immune cells and cytokines, include, e.g., psoriasis, rheumatoid
arthritis (RA), Crohn's disease (CD), multiple sclerosis (MS), and
atherosclerosis.
[0005] The interleukin-1 (IL-1) family of cytokines contributes to
the pathology of inflammatory disorders and proliferative
conditions, e.g., arthritis and cancer. There are 11 members of the
IL-1 cytokine family. IL-1 cytokines bind to members of the IL-1
cytokine receptor family. There are ten members in the IL-1
receptor family, and IL-1 ligands typically require participation
of two different IL-1 receptors to make up the cell surface
complex. For example, IL-1.alpha. and IL-1.beta. bind first to the
cell surface receptor IL-1R1, and this IL-1/IL-1R1 complex
subsequently binds to a second cell surface IL-1R family member,
IL-1 Receptor accessory chain protein (IL-1RAcP) Binding of
IL-1.alpha. or IL-1.beta. to both receptor components is necessary
to transduce the IL-1 signal.
[0006] IL-1 family members play a role in inflammatory conditions,
e.g., rheumatoid arthritis, psoriasis, asthma, chronic obstructive
pulmonary disorder (COPD), sepsis, and inflammatory bowel disorder
(IBD). Rheumatoid arthritis (RA) is a common chronic inflammatory
disorder characterized by degradation of joints, e.g., the synovial
membrane, cartilage, and bone. IL-1 stimulates a number of cells
involved in arthritic inflammation, e.g., fibroblasts, osteoclasts,
chondrocytes, and neutrophils, which may show abnormal
proliferation and release enzymes causing joint destruction.
[0007] Proliferative disorders are the second most common cause of
death in the United States. Cytokines of the IL-1 family have been
implicated in the control and pathology of proliferative disorders,
i.e., cancer. IL-1 modulates progression through the cell cycle,
e.g., by changing expression of cyclin-dependent kinases and
cyclin-dependent kinase inhibitors. High doses of IL-1.beta.
promote tumor invasiveness, while low doses can promote immune
eradication of tumors.
[0008] IL-33 exerts its biological effects via the receptor protein
ST2 and induces T Helper Type 2-associated cytokines (Schmitz, J.
et al., Immunity 23: 479-490 (2005)). In cells stimulated with
IL-33, the signaling components MyD88, IRAK, IRAK4 and TRAF6 are
recruited to ST2. In addition, IL-33 mediated signaling involves
phosphorylation of I.kappa.B.alpha. and phosphorylation of the MAP
kinases Erk1/2, p38 and JNK. T.sub.H2 cells, but not T.sub.H1
cells, respond to IL-33 stimulation with increased production of
IL-5 and IL-13. IL-33 administration results in splenomegaly with
significantly higher numbers of spleen eosinophils, mononuclear
cells, and plasma cells, but not neutrophils. IL-33 administration
also results in increased levels of blood eosinophils, lymphocytes
and neutrophils. IL-33 administration also leads to induction of
IL-4, IL-5 and IL-13 gene and protein expression in vivo. IL-33
administration also results in airway epithelial lining hypertrophy
and mucous production, epithelial hyperplasia in the esophagus, and
inflammatory infiltrates of eosinophils, neutrophils and
mononuclear cells in the esophageal epithelium. In addition, IL-33
administration results in increased levels of serum IgE and
IgA.
[0009] There remains an unmet need to treat inflammatory and immune
disorders. Specifically, the need exists for improved compositions
and methods of treatment for immune disorders related to IL-33
signaling in which reduction of IL-33 activity would provide
therapeutic benefit.
SUMMARY OF THE INVENTION
[0010] We have previously shown that the orphan receptor ST2 is one
of the components of the IL-33 receptor complex (Schmitz, J. et
al., Immunity 23: 479-490 (2005)). It was thought that the receptor
complex that binds IL-33 is a complex of ST2 and SIGIRR. See WO
2005/079844. Here, we explain that the second component of the
IL-33 receptor complex is not SIGIRR, but is instead IL-1RAcP.
Although no receptor promiscuity has been observed for the major
IL-1 ligands (IL-1.alpha. and IL-1.beta. exclusively bind to IL-1R1
and IL-1RAcP, and IL-18 binds exclusively to IL-18R.alpha. and
IL-18R.beta.), we focused on IL-1RAcP after we were unsuccessful in
finding the second IL-33 receptor among the orphan IL-1 receptors.
Accordingly, the present invention provides that IL-33 signal
transduction occurs through ST2 and IL-1RAcP.
[0011] In one aspect, the present invention provides methods of
modulating (e.g. preventing, reducing or ameliorating) an immune
disorder or condition, comprising inhibiting IL-33 signal
transduction through ST2 and IL-1RAcP by administering to a subject
in need thereof an effective amount of: (a) an antagonist of IL-33
binding to a complex of ST2 and IL-1RAcP, and/or (b) an antagonist
of IL-1RAcP binding to a complex of IL-33 and ST2.
[0012] The present invention also provides methods of modulating
blood cell counts, comprising inhibiting IL-33 signal transduction
through ST2 and IL-1RAcP by administering to a subject in need
thereof an effective amount of: (a) an antagonist of IL-33 binding
to a complex of ST2 and IL-1RAcP, and/or (b) an antagonist of
IL-1RAcP binding to a complex of IL-33 and ST2. Such modulation,
e.g. may include increasing the count of platelets and/or
decreasing the counts of one or more of total white blood cells,
neutrophils, lymphocytes and eosinophils.
[0013] In various embodiments the antagonist comprises an antibody,
or antigen-binding fragment thereof, that binds to IL-33, ST2, or a
complex of IL-33 bound to ST2.
[0014] In other embodiments, the antagonist comprises an antibody,
or antigen-binding fragment thereof, that binds to IL-1RAcP, a
complex of IL-1RAcP and ST2, or a complex of IL-1RAcP, ST2 and
IL-33. In some embodiments the antibody or fragment does not bind
to IL-33 alone and/or does not bind to ST2 alone.
[0015] In yet another embodiment, the antibody or fragment does not
bind to IL-1RAcP alone.
[0016] In one embodiment, the antibody or fragment thereof is a
monoclonal, humanized, or fully human antibody. In another
embodiment, the antibody or fragment is an Fab, an Fv fragment, or
an F(ab').sub.2 fragment.
[0017] In some embodiments, the immune disorder or condition is
selected from the group consisting of an innate response, asthma,
an allergy, multiple sclerosis, inflammatory bowel disorder,
arthritis, an infection, cancer, and a tumor. Exemplary arthritic
conditions may be selected from the group consisting of rheumatoid
arthritis, osteoarthritis, and psoriatic arthritis. Exemplary
infections may be selected from the group consisting of an
intracellular pathogen, a bacterium, a parasite and a virus.
Exemplary intracellular pathogens may be selected from the group
consisting of Leishmania sp., Mycobacterium sp., Listeria sp.,
Toxoplasma sp., Schistosoma sp.
[0018] In other embodiments, the immune disorder or condition is a
T.sub.H1-type response or a T.sub.H2-type response.
[0019] In another aspect, the present invention provides methods
and kits for the diagnosis of an immune condition or disorder. In
one embodiment, the diagnostic method comprises determining the
presence or level of one or more of IL-33, ST2, IL-1RAcP, a complex
of IL-33 and ST2, a complex of ST2 and IL-1RAcP and a complex of
ST2, IL-1RAcP and IL-33 in a sample from a subject, and comparing
that level to the levels in non-diseased tissues, subjects that are
known to have the immune condition or disorder, and/or subjects
that are known not to have the immune condition or disorder. In one
embodiment, the presence or level of a complex of IL-33 and ST2 is
detected. In one embodiment the detection involves use of a binding
compound comprising a detectable label, e.g. an antibody or
antigen-binding fragment thereof. In another embodiment, the
invention involves kits to perform the diagnostic methods of the
invention, comprising: a compartment; a detectably-labeled
molecule, such as an antibody, that binds one or more of IL-33,
ST2, IL-1RAcP, a complex of IL-33 and ST2, a complex of ST2 and
IL-1RAcP and a complex of ST2, IL-1RAcP and IL-33; and, optionally,
instructions for use. In one embodiment, the detectably-labeled
molecule binds to a complex of IL-33 and ST2.
[0020] In another aspect, the present invention provides an
isolated and purified complex of ST2 and IL-1RAcP, optionally
further comprising IL-33, and sequence variants thereof. In one
embodiment the ST2, IL-1RAcP and IL-33 are human proteins. In other
embodiments, the invention provides such complexes comprising
polypeptide having 70%, 80%, 90%, 95%, 98% or 100% sequence
identity with a naturally occurring human ST2, IL-1RAcP or IL-33
protein. In one aspect, the invention provides methods of making
these isolated and purified complexes, comprising mixing two or
more of the proteins and allowing said complex to form.
[0021] In yet another aspect, the present invention provides in
vitro methods of determining whether a test compound is an
antagonist of IL-33 binding to a complex of ST2 and IL-1RAcP,
and/or IL-1RAcP binding to a complex of IL-33 and ST2, comprising
an assay selected from the group consisting of a) an
NF-.kappa.B-dependent reporter gene expression assay, b) an MyD88
IRAK, IRAK4 or TRAF6 recruitment assay, c) an Erk1/2, p38,
I.kappa.B.alpha. or JNK phosphorylation assay, d) an NF-.kappa.B,
Erk1/2 or p38 phosphorylation assay in cells that naturally express
ST2, e) an IL-13, IL-6 or IL-5 expression assay, and f) an IL-6
production assay in mouse mast cell line WTMC. In some embodiments,
the test compound is determined to be an antagonist if it reduces
the activity of IL-33 in the assay when compared to an assay run
without the test compound.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows that IL-33 administration increases the
percentage of eosinophils found in the blood of wild-type (WT)
mice, but not IL-1RAcP deficient (KO) mice. Individual data bars
represent separate experimental animals, with one experiment
involving five animals (FIG. 1A) and another involving three
animals (FIG. 1B).
[0023] FIG. 2 shows that IL-33 administration increases IL-5
production in serum (FIG. 2A) and bronchoalveolar lavage (BAL)
(FIG. 2B) in wild-type (WT) mice, but not in IL-1RAcP deficient
(KO) mice.
[0024] FIG. 3 shows that IL-33 administration increases serum IgE
in wild-type (WT) mice, but not in IL-1RAcP deficient (KO)
mice.
[0025] FIGS. 4A-4F show that, unlike wild-type (WT) mice, IL-1RAcP
deficient (KO) mice do not upregulate T.sub.H2 cytokine or cytokine
receptor genes in response to IL-33 administration
[0026] FIG. 5 shows that IL-33 induces IL-6 production from WTMC
cells in a dose dependent manner.
[0027] FIG. 6 shows that blocking ST2 prevents IL-33-induced IL-6
production in WTMC cells.
[0028] FIG. 7 shows that IL-1RAcP is necessary for IL-33-enhanced
IL-13 (FIG. 7A), IL-6 (FIG. 7B), and IL-5 (FIG. 7C) production from
T.sub.H2 cells
[0029] FIG. 8 shows the detection of the ST2/IL-33/IL-1RAcP
complex, in which ST2 is immobilized on the plate in FIG. 8A and
IL-1R1 is immobilized on the plate in FIG. 8B.
[0030] FIG. 9 shows that dominant negative IL-1RAcP inhibits IL-33
signaling.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] As used herein, including the appended claims, the singular
forms of words such as "a," "an," and "the," include their
corresponding plural references unless the context clearly dictates
otherwise.
[0032] "Administering" refers to the delivery to an animal, human,
subject, cell, tissue, organ, or biological fluid of an exogenous
composition, pharmaceutical, therapeutic, or diagnostic agent, e.g.
an agent containing an antagonist of IL-33 signal transduction
through ST2 and IL-1RAcP. In one embodiment, "administering" refers
to delivery to a subject. In another embodiment, "administering"
refers to delivery to a human subject.
[0033] An "antagonist of IL-33 binding to a complex of ST2 and
IL-1RAcP" is a molecule that inhibits IL-33 binding to a complex of
ST2 and IL-1RAcP. An "antagonist of IL-1RAcP binding to a complex
of IL-33 and ST2" is a molecule that inhibits binding of IL-1RAcP
to a complex of IL-33 and ST2. In one embodiment, the antagonist is
an antibody, such as a polyclonal antibody, a monoclonal antibody,
a humanized antibody, or a human antibody. In another embodiment,
the antagonist is an antibody fragment, such as a Fab, an Fv
fragment, or a F(ab').sub.2 fragment.
[0034] In another embodiment, the antibody or fragment thereof
specifically binds to (a) IL-33, (b) IL-1RAcP, (c) ST2, (d) a
complex of ST2 bound to IL-1RAcP, (e) a complex of IL-33 bound to
ST2, or (f) a complex of IL-33, ST2 and IL-1RAcP.
[0035] In one embodiment, an "effective amount" of the antagonist
of IL-33 signal transduction through ST2 and IL-1RAcP means an
amount sufficient to ameliorate a symptom or sign of a disorder or
physiological condition, or an amount sufficient to permit or
facilitate the diagnosis of a disorder or physiological condition.
An effective amount for a human or veterinary subject may vary
depending on factors such as the condition being treated, the
overall health of the subject, the method route and dose of
administration and the severity of side affects. In one embodiment,
an effective amount is the maximal dose or dosing protocol that
avoids significant side effects or toxic effects. The effect will
result in an improvement of a diagnostic measure, parameter, or
detectable signal by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, or 90%, where 100% is defined as the diagnostic parameter
shown by a normal subject (see, e.g., Maynard, et al., A Handbook
of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton,
Fla. (1996); Dent, Good Laboratory and Good Clinical Practice, Urch
Publ., London, UK (2001)).
[0036] In another embodiment, an "effective amount" of the
antagonist of IL-33 signal transduction through ST2 and IL-1RAcP
means an amount sufficient to inhibit IL-33 signal transduction
through ST2 and IL-1RAcP.
[0037] "Inhibiting IL-33 signal transduction through ST2 and
IL-1RAcP" means that the degree to which IL-33 stimulates signal
transduction through ST2 and IL-1RAcP is diminished in the presence
of an antagonist, relative to the degree to which IL-33 stimulates
signal transduction through ST2 and IL-1RAcP in the absence of the
antagonist.
[0038] To examine the extent of inhibition, a sample is treated
with a potential inhibitor and is compared to a control sample
without the inhibitor. Control samples, i.e., not treated with
antagonist, are assigned a relative activity value of 100%
Inhibition is achieved when the activity value relative to the
control is about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,
40%, 35%, 30%, 25%, or 20% or less.
[0039] An endpoint in inhibition may comprise a predetermined
quantity or percentage of, e.g., an indicia of inflammation,
oncogenicity, or cell degranulation or secretion, such as the
release of a cytokine. An endpoint of inhibition is generally 75%,
50%, 25%, or 10% of the control or less.
[0040] Inhibition of IL-33 signal transduction through ST2 and
IL-1RAcP can be determined by assaying for IL-33 signal
transduction in an in vitro assay. One in vitro assay that can be
used is an NF-.kappa.B-dependent reporter assay (Schmitz, J. et
al., Immunity 23: 479-490 (2005)). In this assay, cells, e.g.,
HEK293FT cells, are transfected with either a mock expression
vector or with an expression vector encoding ST2 in conjunction
with an NF-.kappa.B-driven reporter gene expression product, e.g.,
green fluorescent protein (GFP). Cells are incubated with IL-33
alone, or with IL-33 and a test molecule that is being tested for
antagonistic activity. Cells are then analyzed for reporter gene
expression product, e.g., by fluorescence activated cell sorting
(FACS.RTM.). If IL-33 treated cells exhibit reduced reporter gene
expression after incubation with the test molecule, the test
molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor
complex.
[0041] Another reporter gene expression product that can be used is
luciferase. In this assay, cellular extracts are assayed for
NF-.kappa.B-driven luciferase expression in response to incubation
with IL-33, and with or without a test molecule, i.e. a putative
inhibitor of IL-33 signal transduction.
[0042] Another in vitro assay that can be performed is to assay for
MyD88 IRAK, IRAK4, or TRAF6 recruitment (Schmitz, J. et al.,
Immunity 23: 479-490 (2005)). In this assay, cells, e.g., HEK293FT
cells, are transfected with either a mock expression vector or with
an expression vector encoding ST2, and incubated with IL-33. In the
control experiment, no test molecule is added to the incubation. In
the test experiment, the molecule to be tested for its ability to
inhibit IL-33 signal transduction is added. Cellular extracts are
immunoprecipitated with an anti-ST2 antibody, followed by Western
analysis with anti-MyD88, IRAK, IRAK4, or TRAF6 antibodies. A
decrease in expression of MyD88, IRAK, IRAK4, or TRAF6 in extracts
from IL-33 treated cells when incubated with the test molecule
indicates that the test molecule is an antagonist of IL-33 binding
to the IL-1RAcP receptor complex.
[0043] Another in vitro assay that can be performed is to assay for
phosphorylation of Erk1/2, p38, I.kappa.B.alpha., or JNK (Schmitz,
J. et al., Immunity 23: 479-490 (2005)). In this assay, cells,
e.g., HEK293FT cells, are transfected with either a mock expression
vector or with an expression vector encoding ST2, and incubated
with IL-33. In the control experiment, no test molecule is added to
the incubation. In the test experiment, the molecule to be tested
for its ability to inhibit IL-33 signal transduction is added.
Cellular extracts are prepared and blotted with
anti-phospho-Erk1/2, anti-phospho-p38,
anti-phospho-I.kappa.B.alpha., or anti-phospho-JNK antibodies. A
decrease in the level of phosphorylated Erk1/2, p38,
I.kappa.B.alpha., or JNK in extracts from IL-33 treated cells when
incubated with the test molecule indicates that the test molecule
is an antagonist of IL-33 binding to the IL-1RAcP receptor
complex.
[0044] Another in vitro assay that can be performed is to assay for
phosphorylation of NF-.kappa.B, Erk1/2 or p38 in cells that
naturally express ST2, e.g., mouse mast cells (WTMC) (Schmitz, J.
et al., Immunity 23: 479-490 (2005)). In the control experiment,
WTMC cells are incubated with IL-33 but no test molecule is added
to the incubation. In the test experiment, cells are incubated with
IL-33 and the molecule to be tested for its ability to inhibit
IL-33 signal transduction. Cell lysates are separated by SDS-PAGE
and electroblotted. Cellular extracts are blotted with
anti-NF-.kappa.B, anti-phospho-Erk1/2, or anti-phospho-p38
antibodies. A decrease in the level of phosphorylated NF-.kappa.B,
Erk1/2, or p38 in extracts from IL-33 treated cells when incubated
with the test molecule indicates that the test molecule is an
antagonist of IL-33 binding to the IL-1RAcP receptor complex.
[0045] Another in vitro assay that can be performed is to assay
expression of IL-5 or IL-13 in T.sub.H2 cells (Schmitz, J. et al.,
Immunity 23: 479-490 (2005)). In the control experiment, T.sub.H2
cells are incubated with IL-33 but no test molecule is added to the
incubation. In the test experiment, T.sub.H2 cells are incubated
with IL-33 and the molecule to be tested for its ability to inhibit
IL-33 signal transduction. Supernatants are analyzed for IL-5 and
IL-13 production. A decrease in the level of IL-5 or IL-13 in
supernatant from IL-33 treated cells when incubated with the test
molecule indicates that the test molecule is an antagonist of IL-33
binding to the IL-1RAcP receptor complex.
[0046] Another in vitro assay that can be used to determine whether
a molecule is an antagonist of IL-33 binding is to determine the
effect of a test molecule on IL-33--induced production of IL-6 in
the mouse mast cell line WTMC. A decrease in the level of IL-6
production by IL-33 treated cells when incubated with the test
molecule indicates that the test molecule is an antagonist of IL-33
binding to the IL-1RAcP receptor complex.
[0047] In vivo assays that can be used to determine whether a
molecule is an antagonist of IL-33 binding to the IL-1RAcP receptor
complex is to determine the effect of a test molecule on the
influence of IL-33 administration on spleen size, spleen cells
(e.g., mononuclear cells, eosinophils, and plasma cells), blood
cell count (e.g., eosinophils, lymphocytes or neutrophils)
(Schmitz, J. et al., Immunity 23: 479-490 (2005)). A decrease in
the effect of IL-33 after administration of IL-33 and the test
molecule on an in vivo endpoint, relative to the effect of IL-33
alone, indicates that the test molecule is an antagonist of IL-33
binding to the IL-1RAcP receptor complex.
[0048] In a one embodiment, the antagonist that is assayed for in
the above-described assays is an antibody.
[0049] "Expression" refers to a measure of mRNA or polypeptide
encoded by a specific gene. Units of expression may be a measure
of, e.g., the number of molecules of mRNA or polypeptide/mg
protein, the number of molecules of mRNA or polypeptide/cell, in
measurements of expression by cell, tissue, cell extract, or tissue
extract. The units of expression may be relative, e.g., a
comparison of signal from control and experimental mammals or a
comparison of signals with a reagent that is specific for the mRNA
or polypeptide versus with a reagent that is non-specific.
[0050] "Immune condition" or "immune disorder" encompasses, e.g.,
pathological inflammation, an inflammatory disorder, and an
autoimmune disorder or disease. "Immune condition" also refers to
infections, persistent infections, and proliferative conditions,
such as cancer, tumors, and angiogenesis.
[0051] In one embodiment, the immune disorder or immune condition
is selected from the group consisting of an innate response,
asthma, allergy, multiple sclerosis, inflammatory bowel disorder,
arthritis, an infection, cancer, and a tumor. In another
embodiment, the arthritis is selected from the group consisting of
rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. In
another embodiment, the immune disorder or condition comprises a
T.sub.H1-type response or a T.sub.H2-type response.
[0052] "Infection" refers to a disorder resulting from a pathogen,
microbe, bacterium, parasite, virus, and the like. In one
embodiment, the infection is caused by an organism selected from
the group consisting of a bacterium, a parasite, and a virus. In
another embodiment, the infection is caused by an organism selected
from the group consisting of Leishmania sp., Mycobacterium sp.,
Listeria sp., Toxoplasma sp., Schistosoma sp., and a respiratory
virus.
[0053] "Inflammatory disorder" means a disorder or pathological
condition where the pathology results, in whole or in part, from,
e.g., a change in number, change in rate of migration, or change in
activation, of cells of the immune system. Cells of the immune
system include, e.g., T cells, B cells, monocytes or macrophages,
antigen presenting cells (APCs), dendritic cells, microglia, NK
cells, NKT cells, neutrophils, eosinophils, mast cells, or any
other cell specifically associated with the immunology, for
example, cytokine-producing endothelial or epithelial cells.
[0054] "IL-1RAcP" means the Interleukin-1 Receptor Accessory
Protein. IL-1RAcP is further described at online Mendelian
Inheritance in Man (OMIM) entry 602626 and Gene ID No. 3556, and
exemplary naturally occurring sequences for human IL-1RAcP are
found at GenBank Accession Nos. NP.sub.--608273 and
NP.sub.--002173, all of which are available through the National
Center for Biotechnology Information (NCBI) website.
[0055] "IL-33 receptor complex" refers to the specific binding of
ST2 to IL-1RAcP to form a protein complex that itself specifically
binds to IL-33 and transduces the IL-33 signal from the cell
surface to the cell interior.
[0056] "IL-33/ST2 complex" refers to the specific binding of IL-33
and ST2 to form a protein complex that itself specifically binds to
IL-1RAcP. IL-33 is further described at OMIM entry 608678 and Gene
ID No. 90865, and an exemplary naturally occurring sequence for
human IL-33 is found at GenBank Accession No. AY905581, all of
which are available through the NCBI website. See also Schmitz et
al. (2005) Immunity 23: 479-490.
[0057] An "isolated and purified" complex, such as an isolated and
purified complex of ST2 and IL-1RAcP, optionally further comprising
IL-33, is an in vitro complex that is isolated and purified from
its natural source. Specifically, an isolated and purified complex
does not encompass complexes as they form in nature. Such complexes
may be formed, e.g., by mixture of two or more purified proteins,
or by mixture of one purified protein with a cell extract or
biological sample, or by the purification of a complex from a
natural source.
[0058] "IL-33 signal transduction through ST2 and IL-1RAcP" refers
to the molecular events that occur when IL-33, ST2 and IL-1RAcP
form a protein complex. In one embodiment, the IL-33 signal
transduction refers to recruitment of one or more of MyD88, IRAK,
IRAK4, and TRAF6 to ST2 in response to the formation of a complex
of IL-33, ST2, and IL-1RAcP. In another embodiment, IL-33 signal
transduction refers to phosphorylation of one or more of
I.kappa.B.kappa., Erk1/2, p38 and JNK in response to the formation
of a complex of IL-33, ST2, and IL-1RAcP. In another embodiment,
IL-33 signal transduction refers to IL-5 or IL-13 secretion by
T.sub.H2 cells.
[0059] Specificity of binding refers to a binding interaction
between a predetermined ligand and a predetermined receptor that
enables one to distinguish between the predetermined ligand and
other ligands, or between the predetermined receptor and other
receptors. In one embodiment, the ligand is IL-33, and the receptor
is ST2. In another embodiment, the ligand is IL-33, and the
receptor is a protein complex of ST2 and IL-1RAcP. In another
embodiment, the ligand is a protein complex of IL-33 and ST2, and
the receptor is IL-1RAcP. In another embodiment, the ligand is an
antibody or a fragment thereof that specifically binds to IL-33,
IL-1RAcP, ST2, a complex of ST2 bound to IL-1RAcP, a complex of
IL-33 bound to ST2, or a complex of IL-33, ST2 and IL-1RAcP.
[0060] A "pharmaceutical composition" comprises the active drug
ingredient and one or more pharmaceutically acceptable excipients,
carriers and/or diluents.
[0061] "Specifically binds" means a binding reaction that is
determinative of the presence of a protein in a heterogeneous
population of proteins and other biologics. For example, under
designated conditions, one protein binds to another protein and
does not bind in a significant amount to other proteins present in
the sample. In various embodiments, an antibody or fragment thereof
binds to its antigen with an affinity that is at least two fold
greater, at least ten times greater, at least 20-times greater, or
at least 100-times greater than the affinity to any other
protein.
[0062] In various embodiments, an antibody, or fragment thereof,
that specifically binds to IL-33, IL-1RAcP, ST2, a complex of ST2
bound to IL-1RAcP, a complex of IL-33 bound to ST2, or a complex of
IL-33, ST2 and IL-1RAcP will bind with a K.sub.D of at least about
10.sup.-6 M, 10.sup.-7M, 10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M, or
10.sup.-11 M or better (lower K.sub.D).
[0063] "ST2" as used herein is synonymous with the T1/ST2 protein
(also known as IL-1RL1). ST2 is further described at OMIM entry
601203 and Gene ID No. 9173, and exemplary naturally occurring
sequences for human ST2 are found at GenBank Accession Nos.
NP.sub.--003847 and NP.sub.--057316, all of which are available
through the NCBI website.
[0064] "ST2/IL-1RAcP complex" refers to the specific binding of ST2
and IL-1RAcP to form a protein complex that itself specifically
binds IL-33.
[0065] Purification of antigen is not necessary for the generation
of antibodies. Immunization can be performed by DNA vector
immunization, see, e.g., Wang, et al. Virology 228:278-284 (1997).
Alternatively, animals can be immunized with cells bearing the
antigen of interest. Splenocytes can then be isolated from the
immunized animals, and the splenocytes can fused with a myeloma
cell line to produce a hybridoma (Meyaard et al., Immunity
7:283-290 (1997); Wright et al., Immunity 13:233-242 (2000);
Preston et al., Eur. J. Immunol. 27:1911-1918 (1997)). Resultant
hybridomas can be screened for production of the desired antibody
by functional assays or biological assays, that is, assays not
dependent on possession of the purified antigen. Immunization with
cells may prove superior for antibody generation than immunization
with purified antigen (Kaithamana et al., J. Immunol. 163:5157-5164
(1999)).
[0066] Any suitable method can be used to elicit an antagonist
antibody with the desired biologic properties. It may be desirable
to prepare monoclonal antibodies (mAbs) from mammalian hosts such
as mice, rodents, primates, humans, etc. Techniques for preparing
such monoclonal antibodies may be found in, e.g., Stites et al.
(eds.) BASIC AND CLINICAL IMMUNOLOGY (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) ANTIBODIES: A LABORATORY MANUAL CSH Press;
Goding (1986) MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d
ed.) Academic Press, New York, N.Y. Thus, monoclonal antibodies may
be obtained by a variety of techniques familiar to researchers
skilled in the art. Typically, spleen cells from an animal
immunized with a desired antigen are immortalized, commonly by
fusion with a myeloma cell. See Kohler and Milstein (1976) Eur. J.
Immunol. 6:511-519. Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. See, e.g., Doyle et al. (eds.
1994 and periodic supplements) CELL AND TISSUE CULTURE: LABORATORY
PROCEDURES, John Wiley and Sons, New York, N.Y. Colonies arising
from single immortalized cells are screened for production of
antibodies of the desired specificity and affinity for the antigen,
and yield of the monoclonal antibodies produced by such cells may
be enhanced by various techniques, including injection into the
peritoneal cavity of a vertebrate host. Alternatively, one may
isolate DNA sequences which encode a monoclonal antibody or a
antigen binding fragment thereof by screening a DNA library from
human B cells according, e.g., to the general protocol outlined by
Huse et al. (1989) Science 246:1275-1281.
[0067] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., Huse et al.
supra; and Ward et al. (1989) Nature 341:544-546. The polypeptides
and antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033; or made in transgenic mice, see Mendez et al.
(1997) Nature Genetics 15:146-156. See also Abgenix and Medarex
technologies.
[0068] Any suitable non-human antibody can be used as a source for
the hypervariable region of a humanized antibody. Sources for
non-human antibodies include, but are not limited to, murine,
Lagomorphs (including rabbits), bovine, and primates. For the most
part, humanized antibodies are human immunoglobulins (recipient
antibody) in which hypervariable region residues of the recipient
are replaced by hypervariable region residues from a non-human
species (donor antibody) such as mouse, rat, rabbit or nonhuman
primate 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, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance of
the desired biological activity. For further details, see Jones et
al. (1986) Nature 321:522-525; Reichmann et al. (1988) Nature
332:323-329; and Presta (1992) Curr. Op. Struct. Biol.
2:593-596.
[0069] Methods for recombinantly engineering antibodies have been
described, e.g., by Boss et al. (U.S. Pat. No. 4,816,397), Cabilly
et al. (U.S. Pat. No. 4,816,567), Law et al. (European Patent
Application Publication No. 438310) and Winter (European Patent
Application Publication No. 239400).
[0070] In one embodiment, the antagonist of the present invention
is administered in a pharmaceutical composition. In another
embodiment, the antagonist of the present invention is administered
in a sterile composition.
[0071] To prepare pharmaceutical or sterile compositions, an active
drug ingredient is mixed with one or more pharmaceutically
acceptable carriers, excipients and/or diluents. A Pharmaceutical
formulation can be prepared in the form of, e.g., a lyophilized
powder, an aqueous solution, or a suspension. See, e.g., Hardman,
et al., Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y. (2001); Gennaro,
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y. (2000); Avis, et al. (eds.),
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
NY (1993); Excipient Toxicity and Safety, Marcel Dekker, Inc., New
York, N.Y. (2000).
[0072] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. Preferably, an administration regimen maximizes
the amount of therapeutic delivered to the patient consistent with
an acceptable level of side effects. Accordingly, the amount of
biologic delivered depends in part on the particular entity and the
severity of the condition being treated. Guidance in selecting
appropriate doses of antibodies is available. See, e.g.,
Wawrzynczak, Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK (1996); Kresina (ed.), Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y. (1991); Bach
(ed.), Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y. (1993).
[0073] An antibody or fragment thereof can be provided by
continuous infusion, or by doses at intervals. An antibody or
fragment thereof may be administered intravenously,
intramuscularly, or subcutaneously.
[0074] A preferred dose protocol is one involving the maximal dose
or dose frequency that avoids significant undesirable side effects.
In various embodiments, the total weekly dose is 0.05 .mu.g/kg, 0.2
.mu.g/kg, 0.5 .mu.g/kg, 1 .mu.g/kg, 10 .mu.g/kg, 100 .mu.g/kg, 0.2
mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg or
more.
[0075] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side affects (see, e.g.,
Maynard, et al., A Handbook of SOPs for Good Clinical Practice,
Interpharm Press, Boca Raton, Fla. (1996); Dent, Good Laboratory
and Good Clinical Practice, Urch Publ., London, UK (2001)).
[0076] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced. Preferably, a biologic that will be used is
derived from the same species as the animal targeted for treatment,
thereby minimizing a humoral response to the reagent.
[0077] The present invention also provides a method of modulating
blood cell counts, the method comprising administering an effective
amount of an antagonist of IL-33 signal transduction through ST2
and IL-1RAcP. In one embodiment, the antagonist increases the count
of platelets. In another embodiment, the antagonist decreases the
counts of total white blood cells, neutrophils, lymphocytes, and/or
eosinophils.
[0078] The present invention also provides methods and kits for the
diagnosis of an immune condition or disorder. A method of diagnosis
can comprise contacting a sample from a subject, e.g., a test
subject, with an antibody or fragment thereof that specifically
binds to (a) IL-33, (b) IL-1RAcP, (c) ST2, (d) a complex of ST2 and
IL-1RAcP, (e) a complex of IL-33 and ST2, or (f) a complex of
IL-33, ST2 and IL-1RAcP. The method can further comprise contacting
a sample from a control subject, normal subject, or normal tissue
or fluid from the test subject, with an antibody or fragment
thereof. Moreover, the method can additionally comprise comparing
the specific binding of the antibody or fragment thereof to the
test subject with the specific binding of the composition to the
normal subject, control subject, or normal tissue or fluid from the
test subject. Expression or activity of a test sample or test
subject can be compared with that from a control sample or control
subject. A control sample can comprise, e.g., a sample of
non-affected or non-inflamed tissue in a patient suffering from an
immune disorder. Expression or activity from a control subject or
control sample can be provided as a predetermined value, e.g.,
acquired from a statistically appropriate group of control
subjects.
[0079] Kits for the diagnosis of an immune condition or disorder
may comprise a compartment and a binding compound, e.g. an antibody
or antigen-binding fragment thereof, that specifically binds to one
or more components of IL-33 signal transduction through ST2 and
IL-1RAcP. Such kits may optionally include instructions for use. In
some embodiments the binding compound is detectably labeled.
[0080] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to specific embodiments.
[0081] Many modifications and variations of this invention, as will
be apparent to one of ordinary skill in the art can be made to
adapt to a particular situation, material, composition of matter,
process, process step or steps, to preserve the objective, spirit
and scope of the invention. All such modifications are intended to
be within the scope of the claims appended hereto without departing
from the spirit and scope of the invention. The specific
embodiments described herein are offered by way of example only,
and the invention includes the full scope of equivalents to which
such claims are entitled; and the invention is not to be limited by
the specific embodiments that have been presented herein.
[0082] All citations herein are incorporated herein by reference to
the same extent as if each individual publication, patent
application, patent, database entry or other document was
specifically and individually indicated to be incorporated by
reference including all figures and drawings.
Example 1
Animal Experiments
[0083] In order to determine whether IL-1RAcP is the second member
of the receptor complex, wild-type (WT) and IL-1RAcP deficient (KO)
mice were used. The wild-type (WT) mice used were strain
B6;129SF2/J (Jackson Laboratories). The IL-1RAcP deficient (KO)
mice used were strain B6;129S1-Il1rap.sup.tm1/Roml/J (Jackson
Laboratories). The IL-1RAcP deficient mice have been genetically
altered and do not have a functional IL-1RAcP receptor protein.
[0084] Wild-type and IL-1RAcP deficient mice were given daily
intraperitoneal injections of saline or 2 micrograms of human IL-33
for six days. On day seven, the mice were sacrificed and analyzed
for IL-33 responsiveness by several endpoints, as discussed in the
following Examples. Blood eosinophilia was measured in blood
smears. Serum and bronchoalveolar lavage (BAL) fluid were collected
to quantitate cytokine levels and serum immunoglobulin (Ig) E
levels. Lung and spleen tissue was collected to quantitate
messenger RNA of key cytokines Lung and small intestine tissue was
analyzed for histological changes. Two replicate experiments were
performed with either three or five mice per treatment group.
[0085] The following data show that IL-1RAcP deficient mice do not
respond to IL-33 administration, indicating that IL-1RAcP is
necessary to mediate the in vivo effects of IL-33. These results
implicate IL-1RAcP as the second component of the IL-33
receptor.
Example 2
Lack of Blood Eosinophilia in IL-1RAcP Deficient Mice
[0086] Tail blood was smeared onto glass slides, fixed in 95%
ethanol, and stained by the Wright-Giemsa method. Cells were
enumerated by microscope, and at least 300 cells were counted per
mouse. The graphs in FIGS. 1A and 1B represent the two replicate
experiments performed, involving five and three animals,
respectively. IL-33 administration to wild-type mice dramatically
increased the percentage of eosinophils found in the blood. However
the percent of eosinophils in the blood of IL-33-treated IL-1RAcP
deficient mice was not increased, and was similar to saline treated
mice.
Example 3
Lack of Goblet Cell Hyperplasia and Mucous Production in IL-1RAcP
Deficient Mice
[0087] Formalin-fixed, paraffin-embedded small intestines were
stained by Periodic acid-Schiff. Wile-type mice, but not IL-1RAcP
deficient mice, responded to IL-33 with increased goblet cell
mucous production in the small intestine. A total of eight mice
were examined per treatment condition. A similar finding was noted
in the upper airways of the lung.
Example 4
IL-1RAcP Deficient Mice Fail to Respond to IL-33 with Increased
Production of IL-5
[0088] Serum and BAL fluid were analyzed for cytokine content by
multiplex immunoassay. In both the serum (FIG. 2A) and BAL fluid
(FIG. 2B), IL-33 treatment dramatically increased the production of
IL-5 in wild-type mice, but not in IL-1RAcP deficient mice. The
data are representative of two experiments.
Example 5
Serum Levels of IgE are Increased in Wild-Type but not IL-1RAcP
Deficient Mice
[0089] Serum IgE levels were measured by ELISA. IL-33 increased
serum IgE in wild-type, but not in IL-1RAcP deficient mice (FIG.
3). The data are representative of two experiments.
Example 6
IL-1RAcP Deficient Mice do not Upregulate T.sub.H2Cytokine Genes In
Response to IL-33 Administration
[0090] RT-PCR was performed on RNA obtained from snap-frozen spleen
and lung tissue, and gene expression is shown in FIG. 4, relative
to the levels of ubiquitin (ub) in the sample. IL-33 increased mRNA
expression of the T helper 2 (T.sub.H2) cytokines and cytokine
receptors IL-4 (FIG. 4A), IL-5 (FIG. 4C), IL-6 (FIG. 4B), IL-13
(FIG. 4D), IL-17RB (FIG. 4E), and ST2 (FIG. 4F), in the lung and
spleen of wild-type mice, but not in IL-1RAcP deficient ("KO")
mice.
Example 7
[0091] IL-33 Induces IL-6 Production in WTMC Cells
[0092] The mouse mast cell line WTMC (Wright, G. J., et al., J.
Immunol. 171: 3034-3046 (2003)), expresses ST2 and responds to both
human and mouse IL-33 in a dose dependent manner by producing IL-6.
IL-33 activity can be blocked by preincubating the cells with a
blocking rat anti-mouse ST2 antibody (MD Biosciences).
[0093] 1.times.10.sup.5 WTMCs were plated per well (96-well plate
format) in RPMI 1640 w/ L-glutamine containing 10% fetal calf
serum, 1 mM Na-Pyruvate, 0.05 mM 2-mercapto-ethanol, 0.2 mM
L-glutamine, 1.times.MEM non-essential amino acids, 1.times.
PenStrep, 2.5 ng/ml mouse IL-3, and 2.5 ng/ml mouse IL-4. Cells
were incubated for 24 hours with varying concentrations of mouse or
human IL-33. Supernatants were assessed for IL-6 production by
ELISA. As shown in FIG. 5, IL-33 induced IL-6 production in a dose
dependent manner.
[0094] As shown in FIG. 6, a blocking rat anti-mouse ST2 antibody
(MD Biosciences) prevented IL-33 induced production in WTMCs. Mast
cells were seeded with into 24 well plates at 1.times.10.sup.6
cells/well. Some cells were preincubated with anti-ST2 antibody (10
.mu.g/ml) prior to the addition of IL-33 (50 ng/ml). Supernatants
were collected after 6 and 24 hours and analyzed for cytokine
production by multiplex immunoassay.
Example 8
IL-1RAcP is Necessary for IL-33-Enhanced IL-5, IL-6, and IL-13
Production From T.sub.H2Cells
[0095] CD4+ T cells were isolated from wildtype (B6;129SF2/J) and
IL-1RAcP knock-out (B6;129S1-Il1rap.sup.tmI/RomI/J) mouse spleen
and lymph nodes using the CD4+ T cell isolation kit (Miltenyi
Biotec). To polarize towards a T.sub.H2 phenotype, cells were
cultured on anti-CD3 coated plates in the presence of 5 ng/ml IL-2,
10 ng/ml IL-4, 10 .mu.g/ml anti-IFN.gamma., and 1 .mu.g/ml
anti-CD28 in complete RPMI for 4 days. Cells were then washed, and
rested for 3 days in IL-2 (5 ng/ml) alone. Cells were then washed
and stimulated with media alone, human IL-33 (50 ng/ml), or mouse
IL-1.beta. (10 ng/ml). Some cells were preincubated with anti-ST2
blocking antibody prior to cytokine addition. Supernatants were
harvested after 24 hours and cytokine levels were measured using a
multiplexed immunoassay (22-plex mouse cytokine premixed beads,
Linco), and analyzed by Luminex.RTM. immunoassay.
[0096] As shown in FIG. 7, both IL-1.beta. and IL-33 enhanced the
production of IL-13, IL-6, and IL-5 in wildtype, but not
IL-1RAcP-/- T.sub.H2 cells. The increased production of these
cytokines in wildtype mice by IL-33 was blocked in the presence of
anti-ST2 antibodies. Anti-ST2 did not block IL-1.beta.-enhanced
cytokine production.
Example 9
Detection of the ST2/IL-33/IL-1RAcP Complex
[0097] Murine IL-113 or IL-33 was titrated into plates containing
soluble his6-tagged murine IL-1RAcP and plate-bound ST2-Fc (FIG.
8A) or IL-1R1--Fc (FIG. 8B). Peroxidase-conjugated anti-his6 was
used as a detection antibody. Data is representative of 2
experiments. Data represent the mean+/-SEM of duplicate wells.
[0098] Receptor-ligand complex ELISA: 3 .mu.g/ml murine ST2-Fc or
IL-1R1--Fc fusion proteins (R&D Systems) in PBS were coated
overnight onto MaxiSorp plates (Nunc). After washing, plates were
incubated with 3 .mu.g/ml recombinant his6-tagged murine IL-1RAcP
and increasing concentrations of murine IL-33 or IL-113 in PBS/1%
bovine serum albumin/0.05% Tween-20.RTM. polyoxyethylene sorbitan
monolaurate for 1.5 hours. Plates were washed and incubated with
anti-his6-peroxidase (Roche Diagnostics) for 1 hour, washed again,
developed with TMB Peroxidase substrate (KPL), stopped with
H.sub.2PO.sub.4, and read on a plate reader at 450-570 nm.
[0099] Although IL-1RAcP is required to mediate the effects of
IL-33 in vivo and on Th2 cells, the possibility exists that this is
due to an indirect rather than a direct interaction of IL-33 and
IL-1RAcP. Therefore, we wished to detect formation of the
ligand-receptor complex in vitro. In the IL-1 receptor complex,
IL-1RAcP cannot bind IL-1 directly, rather it is hypothesized to
interact with the complex of IL-1 and its primary receptor, IL-1R1.
We are also unable to see a direct interaction of IL-33 with
IL-1RAcP (not shown). However, we can detect a specific
ST2/IL-33/IL-1RAcP complex by ELISA. We titrated IL-33 or IL-113
into wells containing plate-bound ST2 and soluble his6-tagged
IL-1RAcP (FIG. 8A). We detect IL-33 receptor complex formation with
an anti-his6 antibody as the concentration of IL-33, but not
IL-1.beta., increases. Conversely, if the plates are coated with
IL-1R1 instead of ST2, we detect IL-1 receptor complex formation
with increasing amounts of IL-1.beta. but not IL-33 (FIG. 8B). This
result demonstrates that ST2, IL-33, and IL-1RAcP can form a
ligand/receptor complex.
Example 10
Dominant Negative IL-1RAcP Inhibits IL-33 Signaling
[0100] Signal transduction by IL-1RAcP in the IL-1 signaling
cascade is mediated by its cytoplasmic Toll/IL-1 receptor (TIR)
domain and involves recruitment of the adaptor MyD88 and activation
of NF-.kappa.B. IL-33 uses the same signaling components as IL-1,
including IRAK, IRAK4, MyD88, and TRAF6, leading to the activation
of NF-.kappa.B and MAP kinases. MyD88 is required for IL-33
signaling, as MyD88-/- mice do not respond to IL-33 administration
(data not shown). In order to investigate the contribution of
IL-1RAcP to IL-33 signal transduction, we used a dominant negative
form of IL-1RAcP that contains a stop codon before the TIR
domain.
[0101] As a positive control, HEK293FT cells, which endogenously
express human IL-1R1 and IL-1RAcP, were transfected with 3 .mu.g of
a NF-.kappa.B-driven reporter gene construct (pNF-.kappa.B-hrGFP,
Stratagene), and with increasing amounts of cMyc-tagged murine
dominant negative (dn) IL-1RAcP construct. Cells were stimulated at
24 hours post-transfection with 20 ng/ml murine IL-1.alpha.
(R&D Systems) for 24 hours, and analyzed for GFP expression by
FACS.RTM. cell sorting. This control tests the ability of the dn
IL-1RAcP to interact with the endogenous IL-1R1 and block
IL-1.alpha. mediated downstream signaling. This interaction is
reflected by the diminution of GFP expression when increasing
amounts of the dn IL1-RAcP are transfected into control cells (FIG.
9, bottom row).
[0102] For experimental samples, HEK293FT cells were transfected
with 1 .mu.g of a plasmid encoding murine ST2, 3 .mu.g of the
NF-.kappa.B-driven reporter gene construct, and with increasing
amounts of cMyc-tagged murine dominant negative (dn) IL-1RAcP
construct. Cells were stimulated at 24 hours post-transfection with
20 ng/ml murine IL-33 for 24 hours, and analyzed for GFP expression
by FACS.RTM. cell sorting. Results of a typical experiment are
provided at FIG. 9 (top row). This experimental system tests the
ability of the dn IL-1RAcP to interact with murine ST2 and block
IL-33 signaling. The addition of the dn IL-1RAcP caused a roughly
dose-responsive decrease in IL-33-induced reporter gene expression,
suggesting that ST2 uses IL-1RAcP as a second receptor to mediate
IL-33 signal transduction.
Sequence CWU 1
1
51356PRTHomo sapiens 1Met Thr Leu Leu Trp Cys Val Val Ser Leu Tyr
Phe Tyr Gly Ile Leu1 5 10 15Gln Ser Asp Ala Ser Glu Arg Cys Asp Asp
Trp Gly Leu Asp Thr Met 20 25 30Arg Gln Ile Gln Val Phe Glu Asp Glu
Pro Ala Arg Ile Lys Cys Pro 35 40 45Leu Phe Glu His Phe Leu Lys Phe
Asn Tyr Ser Thr Ala His Ser Ala 50 55 60Gly Leu Thr Leu Ile Trp Tyr
Trp Thr Arg Gln Asp Arg Asp Leu Glu65 70 75 80Glu Pro Ile Asn Phe
Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys 85 90 95Asp Val Leu Trp
Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 100 105 110Thr Cys
Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro 115 120
125Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met Lys Leu
130 135 140Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln Arg Ile
Thr Cys145 150 155 160Pro Asn Val Asp Gly Tyr Phe Pro Ser Ser Val
Lys Pro Thr Ile Thr 165 170 175Trp Tyr Met Gly Cys Tyr Lys Ile Gln
Asn Phe Asn Asn Val Ile Pro 180 185 190Glu Gly Met Asn Leu Ser Phe
Leu Ile Ala Leu Ile Ser Asn Asn Gly 195 200 205Asn Tyr Thr Cys Val
Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His 210 215 220Leu Thr Arg
Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala225 230 235
240Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu Lys
245 250 255Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr Phe
Ser Phe 260 265 270Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr Ile
Asp Gly Lys Lys 275 280 285Pro Asp Asp Ile Thr Ile Asp Val Thr Ile
Asn Glu Ser Ile Ser His 290 295 300Ser Arg Thr Glu Asp Glu Thr Arg
Thr Gln Ile Leu Ser Ile Lys Lys305 310 315 320Val Thr Ser Glu Asp
Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser 325 330 335Ala Lys Gly
Glu Val Ala Lys Ala Ala Lys Val Lys Gln Lys Gly Asn 340 345 350Arg
Cys Gly Gln 3552570PRTHomo sapiens 2Met Thr Leu Leu Trp Cys Val Val
Ser Leu Tyr Phe Tyr Gly Ile Leu1 5 10 15Gln Ser Asp Ala Ser Glu Arg
Cys Asp Asp Trp Gly Leu Asp Thr Met 20 25 30Arg Gln Ile Gln Val Phe
Glu Asp Glu Pro Ala Arg Ile Lys Cys Pro 35 40 45Leu Phe Glu His Phe
Leu Lys Phe Asn Tyr Ser Thr Ala His Ser Ala 50 55 60Gly Leu Thr Leu
Ile Trp Tyr Trp Thr Arg Gln Asp Arg Asp Leu Glu65 70 75 80Glu Pro
Ile Asn Phe Arg Leu Pro Glu Asn Arg Ile Ser Lys Glu Lys 85 90 95Asp
Val Leu Trp Phe Arg Pro Thr Leu Leu Asn Asp Thr Gly Asn Tyr 100 105
110Thr Cys Met Leu Arg Asn Thr Thr Tyr Cys Ser Lys Val Ala Phe Pro
115 120 125Leu Glu Val Val Gln Lys Asp Ser Cys Phe Asn Ser Pro Met
Lys Leu 130 135 140Pro Val His Lys Leu Tyr Ile Glu Tyr Gly Ile Gln
Arg Ile Thr Cys145 150 155 160Pro Asn Val Asp Gly Tyr Phe Pro Ser
Ser Val Lys Pro Thr Ile Thr 165 170 175Trp Tyr Met Gly Cys Tyr Lys
Ile Gln Asn Phe Asn Asn Val Ile Pro 180 185 190Glu Gly Met Asn Leu
Ser Phe Leu Ile Ala Leu Ile Ser Asn Asn Gly 195 200 205Asn Tyr Thr
Cys Val Val Thr Tyr Pro Glu Asn Gly Arg Thr Phe His 210 215 220Leu
Thr Arg Thr Leu Thr Val Lys Val Val Gly Ser Pro Lys Asn Ala225 230
235 240Val Pro Pro Val Ile His Ser Pro Asn Asp His Val Val Tyr Glu
Lys 245 250 255Glu Pro Gly Glu Glu Leu Leu Ile Pro Cys Thr Val Tyr
Phe Ser Phe 260 265 270Leu Met Asp Ser Arg Asn Glu Val Trp Trp Thr
Ile Asp Gly Lys Lys 275 280 285Pro Asp Asp Ile Thr Ile Asp Val Thr
Ile Asn Glu Ser Ile Ser His 290 295 300Ser Arg Thr Glu Asp Glu Thr
Arg Thr Gln Ile Leu Ser Ile Lys Lys305 310 315 320Val Thr Ser Glu
Asp Leu Lys Arg Ser Tyr Val Cys His Ala Arg Ser 325 330 335Ala Lys
Gly Glu Val Ala Lys Ala Ala Lys Val Lys Gln Lys Val Pro 340 345
350Ala Pro Arg Tyr Thr Val Glu Leu Ala Cys Gly Phe Gly Ala Thr Val
355 360 365Leu Leu Val Val Ile Leu Ile Val Val Tyr His Val Tyr Trp
Leu Glu 370 375 380Met Val Leu Phe Tyr Arg Ala His Phe Gly Thr Asp
Glu Thr Ile Leu385 390 395 400Asp Gly Lys Glu Tyr Asp Ile Tyr Val
Ser Tyr Ala Arg Asn Ala Glu 405 410 415Glu Glu Glu Phe Val Leu Leu
Thr Leu Arg Gly Val Leu Glu Asn Glu 420 425 430Phe Gly Tyr Lys Leu
Cys Ile Phe Asp Arg Asp Ser Leu Pro Gly Gly 435 440 445Ile Val Thr
Asp Glu Thr Leu Ser Phe Ile Gln Lys Ser Arg Arg Leu 450 455 460Leu
Val Val Leu Ser Pro Asn Tyr Val Leu Gln Gly Thr Gln Ala Leu465 470
475 480Leu Glu Leu Lys Ala Gly Leu Glu Asn Met Ala Ser Arg Gly Asn
Ile 485 490 495Asn Val Ile Leu Val Gln Tyr Lys Ala Val Lys Glu Thr
Lys Val Lys 500 505 510Glu Leu Lys Arg Ala Lys Thr Val Leu Thr Val
Ile Lys Trp Lys Gly 515 520 525Glu Lys Ser Lys Tyr Pro Gln Gly Arg
Phe Trp Lys Gln Leu Gln Val 530 535 540Ala Met Pro Val Lys Lys Ser
Pro Arg Arg Ser Ser Ser Asp Glu Gln545 550 555 560Gly Leu Ser Tyr
Ser Ser Leu Lys Asn Val 565 5703270PRTHomo sapiens 3Met Lys Pro Lys
Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys1 5 10 15Trp Lys Asn
Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser 20 25 30Gln Gln
Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 35 40 45Ser
Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50 55
60Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu65
70 75 80Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala
Phe 85 90 95Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp
Ser Ser 100 105 110Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala
Ser Leu Ser Thr 115 120 125Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu
Glu Asp Glu Ser Tyr Glu 130 135 140Ile Tyr Val Glu Asp Leu Lys Lys
Asp Glu Lys Lys Asp Lys Val Leu145 150 155 160Leu Ser Tyr Tyr Glu
Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly 165 170 175Val Asp Gly
Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe 180 185 190Trp
Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys 195 200
205Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His
210 215 220Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val
Phe Ile225 230 235 240Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys
Val Asp Ser Ser Glu 245 250 255Asn Leu Cys Thr Glu Asn Ile Leu Phe
Lys Leu Ser Glu Thr 260 265 2704328PRTHomo
sapiensSIGNAL(1)..(18)CHAIN(19)..(328) 4Met Gly Phe Trp Ile Leu Ala
Ile Leu Thr Ile Leu Met Tyr Ser Thr1 5 10 15Ala Ala Lys Phe Ser Lys
Gln Ser Trp Gly Leu Glu Asn Glu Ala Leu 20 25 30Ile Val Arg Cys Pro
Arg Gln Gly Lys Pro Ser Tyr Thr Val Asp Trp 35 40 45Tyr Tyr Ser Gln
Thr Asn Lys Ser Ile Pro Thr Gln Glu Arg Asn Arg 50 55 60Val Phe Ala
Ser Gly Gln Leu Leu Lys Phe Leu Pro Ala Ala Val Ala65 70 75 80Asp
Ser Gly Ile Tyr Thr Cys Ile Val Arg Ser Pro Thr Phe Asn Arg 85 90
95Thr Gly Tyr Ala Asn Val Thr Ile Tyr Lys Lys Gln Ser Asp Cys Asn
100 105 110Val Pro Asp Tyr Leu Met Tyr Ser Thr Val Ser Gly Ser Glu
Lys Asn 115 120 125Ser Lys Ile Tyr Cys Pro Thr Ile Asp Leu Tyr Asn
Trp Thr Ala Pro 130 135 140Leu Glu Trp Phe Lys Asn Cys Gln Ala Leu
Gln Gly Ser Arg Tyr Arg145 150 155 160Ala His Lys Ser Phe Leu Val
Ile Asp Asn Val Met Thr Glu Asp Ala 165 170 175Gly Asp Tyr Thr Cys
Lys Phe Ile His Asn Glu Asn Gly Ala Asn Tyr 180 185 190Ser Val Thr
Ala Thr Arg Ser Phe Thr Val Lys Asp Glu Gln Gly Phe 195 200 205Ser
Leu Phe Pro Val Ile Gly Ala Pro Ala Gln Asn Glu Ile Lys Glu 210 215
220Val Glu Ile Gly Lys Asn Ala Asn Leu Thr Cys Ser Ala Cys Phe
Gly225 230 235 240Lys Gly Thr Gln Phe Leu Ala Ala Val Leu Trp Gln
Leu Asn Gly Thr 245 250 255Lys Ile Thr Asp Phe Gly Glu Pro Arg Ile
Gln Gln Glu Glu Gly Gln 260 265 270Asn Gln Ser Phe Ser Asn Gly Leu
Ala Cys Leu Asp Met Val Leu Arg 275 280 285Ile Ala Asp Val Lys Glu
Glu Asp Leu Leu Leu Gln Tyr Asp Cys Leu 290 295 300Ala Leu Asn Leu
His Gly Leu Arg Arg His Thr Val Arg Leu Ser Arg305 310 315 320Lys
Asn Pro Ser Lys Glu Cys Phe 3255556PRTHomo
sapiensSIGNAL(1)..(18)CHAIN(19)..(556) 5Met Gly Phe Trp Ile Leu Ala
Ile Leu Thr Ile Leu Met Tyr Ser Thr1 5 10 15Ala Ala Lys Phe Ser Lys
Gln Ser Trp Gly Leu Glu Asn Glu Ala Leu 20 25 30Ile Val Arg Cys Pro
Arg Gln Gly Lys Pro Ser Tyr Thr Val Asp Trp 35 40 45Tyr Tyr Ser Gln
Thr Asn Lys Ser Ile Pro Thr Gln Glu Arg Asn Arg 50 55 60Val Phe Ala
Ser Gly Gln Leu Leu Lys Phe Leu Pro Ala Ala Val Ala65 70 75 80Asp
Ser Gly Ile Tyr Thr Cys Ile Val Arg Ser Pro Thr Phe Asn Arg 85 90
95Thr Gly Tyr Ala Asn Val Thr Ile Tyr Lys Lys Gln Ser Asp Cys Asn
100 105 110Val Pro Asp Tyr Leu Met Tyr Ser Thr Val Ser Gly Ser Glu
Lys Asn 115 120 125Ser Lys Ile Tyr Cys Pro Thr Ile Asp Leu Tyr Asn
Trp Thr Ala Pro 130 135 140Leu Glu Trp Phe Lys Asn Cys Gln Ala Leu
Gln Gly Ser Arg Tyr Arg145 150 155 160Ala His Lys Ser Phe Leu Val
Ile Asp Asn Val Met Thr Glu Asp Ala 165 170 175Gly Asp Tyr Thr Cys
Lys Phe Ile His Asn Glu Asn Gly Ala Asn Tyr 180 185 190Ser Val Thr
Ala Thr Arg Ser Phe Thr Val Lys Asp Glu Gln Gly Phe 195 200 205Ser
Leu Phe Pro Val Ile Gly Ala Pro Ala Gln Asn Glu Ile Lys Glu 210 215
220Val Glu Ile Gly Lys Asn Ala Asn Leu Thr Cys Ser Ala Cys Phe
Gly225 230 235 240Lys Gly Thr Gln Phe Leu Ala Ala Val Leu Trp Gln
Leu Asn Gly Thr 245 250 255Lys Ile Thr Asp Phe Gly Glu Pro Arg Ile
Gln Gln Glu Glu Gly Gln 260 265 270Asn Gln Ser Phe Ser Asn Gly Leu
Ala Cys Leu Asp Met Val Leu Arg 275 280 285Ile Ala Asp Val Lys Glu
Glu Asp Leu Leu Leu Gln Tyr Asp Cys Leu 290 295 300Ala Leu Asn Leu
His Gly Leu Arg Arg His Thr Val Arg Leu Ser Arg305 310 315 320Lys
Asn Pro Ile Asp His His Ser Ile Tyr Cys Ile Ile Ala Val Cys 325 330
335Ser Val Phe Leu Met Leu Ile Asn Val Leu Val Ile Ile Leu Lys Met
340 345 350Phe Trp Ile Glu Ala Thr Leu Leu Trp Arg Asp Ile Ala Lys
Pro Tyr 355 360 365Lys Thr Arg Asn Asp Gly Lys Leu Tyr Asp Ala Tyr
Val Val Tyr Pro 370 375 380Arg Asn Tyr Lys Ser Ser Thr Asp Gly Ala
Ser Arg Val Glu His Phe385 390 395 400Val His Gln Ile Leu Pro Asp
Val Leu Glu Asn Lys Cys Gly Tyr Thr 405 410 415Leu Cys Ile Tyr Gly
Arg Asp Met Leu Pro Gly Glu Asp Val Val Thr 420 425 430Ala Val Glu
Thr Asn Ile Arg Lys Ser Arg Arg His Ile Phe Ile Leu 435 440 445Thr
Pro Gln Ile Thr His Asn Lys Glu Phe Ala Tyr Glu Gln Glu Val 450 455
460Ala Leu His Cys Ala Leu Ile Gln Asn Asp Ala Lys Val Ile Leu
Ile465 470 475 480Glu Met Glu Ala Leu Ser Glu Leu Asp Met Leu Gln
Ala Glu Ala Leu 485 490 495Gln Asp Ser Leu Gln His Leu Met Lys Val
Gln Gly Thr Ile Lys Trp 500 505 510Arg Glu Asp His Ile Ala Asn Lys
Arg Ser Leu Asn Ser Lys Phe Trp 515 520 525Lys His Val Arg Tyr Gln
Met Pro Val Pro Ser Lys Ile Pro Arg Lys 530 535 540Ala Ser Ser Leu
Thr Pro Leu Ala Ala Gln Lys Gln545 550 555
* * * * *