U.S. patent application number 11/059117 was filed with the patent office on 2005-09-15 for methods of modulating a mammalian cytokine.
This patent application is currently assigned to Schering Corporation. Invention is credited to Kastelein, Robert A., Oft, Martin, Schmitz, Jochen.
Application Number | 20050203046 11/059117 |
Document ID | / |
Family ID | 34886190 |
Filed Date | 2005-09-15 |
United States Patent
Application |
20050203046 |
Kind Code |
A1 |
Schmitz, Jochen ; et
al. |
September 15, 2005 |
Methods of modulating a mammalian cytokine
Abstract
Provided are methods of modulating cytokine activity, e.g., for
the purpose of treating immune and inflammatory disorders,
including tumors and cancer. Also provided are methods of
administering agonists or antagonists of IL-33 and IL-33
receptor.
Inventors: |
Schmitz, Jochen; (Palo Alto,
CA) ; Oft, Martin; (Palo Alto, CA) ;
Kastelein, Robert A.; (Redwood City, CA) |
Correspondence
Address: |
DNAX RESEARCH, INC.
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation
Kenilworth
NJ
|
Family ID: |
34886190 |
Appl. No.: |
11/059117 |
Filed: |
February 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60545730 |
Feb 17, 2004 |
|
|
|
Current U.S.
Class: |
514/44A ;
424/85.2 |
Current CPC
Class: |
C07K 16/244 20130101;
A61P 31/06 20180101; A61P 31/00 20180101; A61P 31/12 20180101; A61K
2039/505 20130101; A61P 31/22 20180101; A61P 7/00 20180101; A61P
17/06 20180101; A61P 29/00 20180101; A61P 33/02 20180101; A61P
37/08 20180101; A61P 35/02 20180101; A61P 19/02 20180101; A61K
38/20 20130101; A61P 17/00 20180101; A61P 37/00 20180101; A61P
31/16 20180101; A61P 37/02 20180101; A61P 11/06 20180101; C07K
2317/76 20130101; Y02A 50/423 20180101; A61K 38/00 20130101; A61P
25/00 20180101; A61P 33/00 20180101; A61P 33/06 20180101; A61P
31/04 20180101; Y02A 50/41 20180101; A61P 1/00 20180101; A61P 1/04
20180101; A61P 25/28 20180101; A61P 35/00 20180101; A61P 43/00
20180101; Y02A 50/30 20180101; A61P 31/18 20180101; G01N 33/6869
20130101; A61P 25/02 20180101; C07K 16/2866 20130101 |
Class at
Publication: |
514/044 ;
424/085.2 |
International
Class: |
A61K 048/00; A61K
038/20 |
Claims
What is claimed is:
1. A method of modulating an immune disorder or condition,
comprising administering an effective amount of an agonist or
antagonist of IL-33 of IL-33 Receptor complex (IL-33R).
2. The method of claim 1, wherein the disorder or condition
comprises: a) innate response; b) asthma or allergy; c) multiple
sclerosis; d) an inflammatory bowel disorder; e) arthritis; f)
infection; g) a cancer or tumor.
3. The method of claim 2, wherein the infection comprises: a) an
intracellular pathogen; b) a bacterium; c) a parasite; or d) a
virus.
4. The method of claim 3, wherein the intracellular pathogen is: a)
Leishmania sp.; b) Mycobacterium sp.; c) Listeria sp.; d)
Toxoplasma sp.; e) Schistosoma; or f) a respiratory virus;
5. The method of claim 1, wherein the immune disorder or conditions
comprises: a) TH1-type response; or b) TH2-type response.
6. The method of claim 5, wherein the TH2-type response comprises
an early event in TH2-type response.
7. The method of claim 1, wherein the arthritis comprises: a)
rheumatoid arthritis; b) osteoarthritis; or c) psoriatic
arthritis.
8. The method of claim 1, wherein the agonist comprises: a) IL-33
or; b) a nucleic acid.
9. The method of claim 8, wherein the nucleic acid encodes
IL-33.
10. The method of claim 1, wherein the antagonist comprises a
binding composition from an antibody that specifically binds: a)
IL-33; b) an IL-33R complex; or c) a complex of IL-33 and
IL-33R.
11. The method of claim 10, wherein the binding composition from an
antibody comprises: a) a polyclonal antibody; b) a monoclonal
antibody; c) a humanized antibody, or a fragment thereof; d) an
Fab, Fv, or F(ab').sub.2 fragment; e) a peptide mimetic of an
antibody; or f) a detectable label.
12. The method of claim 1, wherein the antagonist comprises: a) a
soluble L-33R; b) a small molecule; or c) a nucleic acid.
13. The method of claim 12, wherein the nucleic acid specifically
hybridizes with a polynucleotide encoding IL-33.
14. The method of claim 13, wherein the nucleic acid comprises: a)
anti-sense nucleic acid; or b) small interference RNA (siRNA).
15. A method of modulating blood cell counts comprising
administering an effective amount of an agonist or antagonist of
IL-33.
16. The method of claim 15, wherein the IL-33 agonist increases the
counts of: a) total white blood cells; b) neutrophils; c)
lymphocytes; or d) eosinophils.
17. The method of claim 15, wherein the IL-33 antagonist increases
the count of platelets.
18. The method of claim 16, wherein the IL-33 antagonist decreases
the counts of: a) total white blood cells; b) neutrophils; c)
lymphocytes; or d) eosinophils.
19. A method of diagnosing the immune condition or disorder of
claim 1, comprising contacting a binding composition to a
biological sample, wherein the binding composition specifically
binds to IL-33, and measuring or determining the specific binding
of the binding composition to the biological sample.
20. A kit for the diagnosis of the immune condition or disorder of
claim 1, comprising a compartment and a binding composition that
specifically binds to: a) IL-33; b) an IL-33R complex; c) a complex
of IL-33 and IL-33R; or d) or a nucleic acid encoding IL-33.
Description
[0001] This filing is a U.S. patent application which claims
benefit of U.S. Provisional Patent Application No. 60/545,730,
filed Feb. 17, 2004, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to uses of mammalian
cytokines. More specifically, the invention discloses methods of
using IL-33, and a receptor for IL-33.
BACKGROUND OF THE INVENTION
[0003] 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, Crohn's disease, multiple sclerosis, and atherosclerosis
(see, e.g., Abbas, et al. (eds.) (2000) Cellular and Molecular
Immunology, W. B. Saunders Co., Philadelphia, Pa. Oppenheim and
Feldmann (eds.) (2001) Cytokine Reference, Academic Press, San
Diego, Calif.; Kaufmann, et al. (2001) Immunobiol. 204:603-613;
Saurez and Schultz-Cheery (2000) Dev. Comp. Immunol. 24:269-283;
van Reeth and Nauwynck (2000) Vet. Res. 31:187-213; Garcia-Sastre
(2001) Virology 279:375-384; Katze, et al. (2002) Nat. Rev.
Immunol. 2:675-687; van Reeth (2000) Vet. Microbiol. 74:109-116;
Tripp (2003) Curr. Pharm. Des. 9:51-59).
[0004] The interleukin-1 (IL-1) family of cytokines contributes to
the pathology of inflammatory disorders and proliferative
conditions, e.g., arthritis and cancer. Cytokines of the IL-1
family include IL-1alpha, IL-1beta, IL-1delta, IL-1epsilon, basic
fibroblast growth factor, IL-18, CREG and CREG2. IL-1alpha and
IL-1beta are biosynthesized as 31 kDa polypeptides that are further
processed to mature 17 kDa forms, while IL-1delta and IL-1epsilon
appear not to possess a distinct pro-form (see, e.g., Debets, et
al. (2001) J. Immunol. 167:1440-1446; McMahon, et al. (1997) J.
Biol. Chem. 272:28202-28205; Irikura, et al. (2002) New Engl. J.
Med. 169:393-398; Kim, et al. (2002) J. Biol. Chem.
277:10998-11003).
[0005] The IL-1 family also includes IL-1 receptors, i.e., IL-1RI,
IL-1RII, and IL-1R accessory protein (a.k.a. IL-1R1, IL-1R2, and
IL-1R3, respectively). IL-1alpha and IL-1beta trigger cell
signaling by binding to IL-1R1, while IL-1RII can function as a
molecule that absorbs circulating ligand. IL-1 receptor antagonist
(IL-1Ra), another IL-1 family protein, binds to IL-1 receptor
without transmitting a signal and serves as an inhibitor of IL-1.
IL-1ra and IL-1delta play similar roles in antagonizing signaling
through receptors, i.e., IL-1ra antagonizes IL-1alpha-mediated
signaling via IL-1R1, while IL-1delta antagonizes
IL-1epsilon-mediated signaling via IL-1R6 (see, e.g., You, et al.
(2001) New Engl. J. Med. 193:101-109). Debets, et al. (2001) J.
Immunol. 167:1440-1446; Apte and Voronov (2002) Sem. Cancer Biol.
12:277-290; Wong, et al. (1997) Proc. Natl. Acad. Sci. USA
94:227-232).
[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. The disorder strikes about 1% of the
population and cannot be cured. 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 (see,
e.g., (Debets, et al. (1997) J. Immunol. 158:2955-2963; Lacey, et
al. (2003) Arthritis Rheum. 48: 103-109; Chung (2001) Eur. Resp. J.
Suppl. 34: 50s-59s; Freeman and Buchman (2001) Expert Opin. Biol.
Ther. 1:301-308; Dinarello (2000) Chest 118:503-508). Krause, et
al. (2002) J. Immunol. 169:6610-6616; Choy and Panayi (2001) New
Engl. J. Med. 344:907-916; Woolley (2003) New Engl. J. Med.
348:1709-1711; Williams, et al. (2000) New Engl. J. Med. 164:
7240-7245; Feldmann and Maini (2001) Annu. Rev. Immunol.
19:163-196; Lacey, et al., supra; Niki, et al. (2001) J. Clin.
Invest. 107:1127-1135; Attur, et al. (2000) J. Biol. Chem.
51:40307-40315).
[0007] Proliferative disorders are the second most common cause of
death in the United States (Anderson (2002) National Vital
Statistics Reports 50:1-86; Toribara and Sleisenger (2003) New
Engl. J. Med. 332:861-867; Janne and Mayer (2000) New Engl. J. Med.
342:1960-1968; Fuchs and Mayer (1995) New Engl. J. Med. 333:32-41).
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-1beta promote tumor invasiveness,
while low doses can promote immune eradication of tumors (see,
e.g., Zeisler, et al. (1998) Eur. J. Cancer 34:931-933; Yoshida, et
al. (2002) Brit. J. Cancer 86:1396-1400; Nesbit, et al. (1999)
Oncogene 18:6469-6476; Dinarello, et al. (1998) J. Leuko. Biol.
63:658-664; Apte and Voronov, supra; Saijo, et al. (2002) New Engl.
J. Med. 169: 469-475; Murai, et al. (2001) J. Biol. Chem.
276:6797-6806; Koudssi, et al. (1998) J. Biol. Chem. 273:
25796-25803; Zeki, et al. (1999) J. Endocrinol. 160:67-73; Osawa,
et al. (2000) J. Biochem. 127:883-893).
[0008] There is an unmet need to treat inflammatory and immune
disorders. The present invention fulfils this need by providing
methods of using agonists and antagonists of IL-33 or IL-33
receptor.
SUMMARY OF THE INVENTION
[0009] The present invention is based, in part, upon the discovery
that an agonist or antagonist of IL-33 or IL-33 receptor
(previously known as IL-100 and IL-100 receptor) modulates response
to a number of immune and inflammatory conditions.
[0010] The present invention provides a method of modulating an
immune disorder or condition, comprising administering an effective
amount of an agonist or antagonist of IL-33 or IL-33R complex. Also
provided is the above method wherein the disorder or condition
comprises: a) innate response; b) asthma or allergy; c) multiple
sclerosis; d) an inflammatory bowel disorder; e) arthritis; f)
infection; g) a cancer or tumor. Further provided is the above
method wherein the infection comprises: a) an intracellular
pathogen; b) a bacterium; c) a parasite; or d) a virus; and the
above method wherein the intracellular pathogen is: a) Leishmania
sp.; b) Mycobacterium sp.; c) Listeria sp.; d) Toxoplasma sp.; e)
Schistosoma; or f) a respiratory virus. Moreover, the present
invention provides the above method wherein the immune disorder or
conditions comprises. TH1-type response or TH2-type response; and
the above method wherein the TH2-type response comprises an early
event in TH2-type response; as well as the above method wherein the
arthritis comprises rheumatoid arthritis; osteoarthritis; or
psoriatic arthritis.
[0011] In another embodiment, the present invention provides the
above method wherein the agonist comprises IL-33 or a nucleic acid;
as well as the above method wherein the nucleic acid encodes IL-33;
and the above method wherein the antagonist comprises a binding
composition from an antibody that specifically binds IL-33 or a
complex of IL-33, T1/ST2 and SIGIRR (IL-33R). In yet another
embodiment, the present invention provides the above method wherein
the binding composition from an antibody comprises a polyclonal
antibody; a monoclonal antibody; a humanized antibody, or a
fragment thereof; an Fab, Fv, or F(ab').sub.2 fragment; a peptide
mimetic of an antibody; or a detectable label. Also provided is the
above method, wherein wherein the antagonist comprises: a) a
soluble IL-33R; b) a small molecule; or c) a nucleic acid; and the
above method wherein the nucleic acid specifically hybridizes with
a polynucleotide encoding IL-33; as well as the above method
wherein the nucleic acid comprises anti-sense nucleic acid or small
interference RNA (siRNA).
[0012] In another aspect, the present invention provides a method
of modulating blood cell counts comprising administering an
effective amount of an agonist or antagonist of IL-33; and the
above method wherein the IL-33 agonist increases the counts of
total white blood cells; neutrophils; lymphocytes; or eosinophils;
as well as the above method wherein the IL-33 antagonist increases
the count of platelets; and the above method wherein the IL-33
antagonist decreases the counts of total white blood cells;
neutrophils; lymphocytes; or eosinophils.
[0013] Yet another aspect of the present invention provides a
method of diagnosing the immune condition or disorder noted above,
comprising contacting a binding composition to a biological sample,
wherein the binding composition specifically binds to IL-33, and
measuring or determining the specific binding of the binding
composition to the biological sample. Also provided is a kit for
the diagnosis of the immune condition or disorder of claim 1,
comprising a compartment and a binding composition that
specifically binds to: IL-33; an IL-33R complex; a complex of IL-33
and IL-33R; or a nucleic acid encoding IL-33.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows IL-5 production in IL-33+anti-IL-33 antibody
treated mice versus IL-33 alone and isotype control antibody
treated mice.
[0015] FIG. 2 shows CIA disease scores for anti-IL-33 and isotype
control treated mice.
[0016] FIG. 3 shows the incidence of CIA in anti-IL-33 and isotype
control treated mice.
[0017] FIG. 4 shows the mean number of arthritic paws in mice
treated with anti-IL-33 antibody or isotype control antibody.
[0018] FIG. 5 shows the EAE disease scores of anti-IL-33 and
isotype control treated mice.
[0019] FIG. 6 shows the incidence of EAE in anti-IL-33 and isotype
control treated mice.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] 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.
[0021] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0022] I. Definitions.
[0023] "Activation," "stimulation," and "treatment," as it applies
to cells or to receptors, may have the same meaning, e.g.,
activation, stimulation, or treatment of a cell or receptor with a
ligand, unless indicated otherwise by the context or explicitly.
"Ligand" encompasses natural and synthetic ligands, e.g.,
cytokines, cytokine variants, analogues, muteins, and binding
compositions derived from antibodies. "Ligand" also encompasses
small molecules, e.g., peptide mimetics of cytokines and peptide
mimetics of antibodies. "Activation" can refer to cell activation
as regulated by internal mechanisms as well as by external or
environmental factors. "Response," e.g., of a cell, tissue, organ,
or organism, encompasses a change in biochemical or physiological
behavior, e.g., concentration, density, adhesion, or migration
within a biological compartment, rate of gene expression, or state
of differentiation, where the change is correlated with activation,
stimulation, or treatment, or with internal mechanisms such as
genetic programming.
[0024] "Activity" of a molecule may describe or refer to the
binding of the molecule to a ligand or to a receptor, to catalytic
activity; to the ability to stimulate gene expression or cell
signaling, differentiation, or maturation; to antigenic activity,
to the modulation of activities of other molecules, and the like.
"Activity" of a molecule may also refer to activity in modulating
or maintaining cell-to-cell interactions, e.g., adhesion, or
activity in maintaining a structure of a cell, e.g., cell membranes
or cytoskeleton. "Activity" can also mean specific activity, e.g.,
[catalytic activity]/[mg protein], or [immunological activity]/[mg
protein], concentration in a biological compartment, or the like.
"Proliferative activity" encompasses an activity that promotes,
that is necessary for, or that is specifically associated with,
e.g., normal cell division, as well as cancer, tumors, dysplasia,
cell transformation, metastasis, and angiogenesis.
[0025] "Administration" and "treatment," as it applies to the
administration of an agonist or antagonist of IL-33, e.g., to an
animal, human, experimental subject, cell, tissue, organ, or
biological fluid, refers to contact of an exogenous pharmaceutical,
therapeutic, diagnostic agent, compound, or composition to the
animal, human, subject, cell, tissue, organ, or biological fluid.
"Administration" and "treatment" can refer, e.g., to therapeutic,
placebo, pharmacokinetic, diagnostic, research, and experimental
methods. "Treatment of a cell" encompasses contact of a reagent to
the cell, as well as contact of a reagent to a fluid, where the
fluid is in contact with the cell. "Administration" and "treatment"
also means in vitro and ex vivo treatments, e.g., of a cell, by a
reagent, diagnostic, binding composition, or by another cell.
"Treatment," as it applies to a human, veterinary, or research
subject, refers to therapeutic treatment, prophylactic or
preventative measures, to research and diagnostic applications.
"Treatment" as it applies to a human, veterinary, or research
subject, or cell, tissue, or organ, encompasses contact of an IL-33
agonist or IL-33 antagonist to a human or animal subject, a cell,
tissue, physiological compartment, or physiological fluid.
"Treatment of a cell" also encompasses situations where the IL-33
agonist or IL-33 antagonist contacts IL-33 receptor (T1/ST2), e.g.,
in the fluid phase or colloidal phase, as well as situations where
the agonist or antagonist contacts a fluid, e.g., where the fluid
is in contact with a cell or receptor, but where it has not been
demonstrated that the agonist or antagonist contacts the cell or
receptor.
[0026] "Binding composition" refers to a molecule, small molecule,
macromolecule, antibody, a fragment or analogue thereof, or soluble
receptor, capable of binding to a target, where the target is,
e.g., IL-33 or IL-33R. "Binding composition" also may refer to a
complex of molecules, e.g., a non-covalent complex, to an ionized
molecule, and to a covalently or non-covalently modified molecule,
e.g., modified by phosphorylation, acylation, cross-linking,
cyclization, or limited cleavage, which is capable of binding to a
target. "Binding composition" may also refer to a molecule in
combination with a stabilizer, excipient, salt, buffer, solvent, or
additive, capable of binding to a target. "Binding" may be defined
as an association of the binding composition with a target where
the association results in reduction in the normal Brownian motion
of the binding composition, in cases where the binding composition
can be dissolved or suspended in solution.
[0027] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences or, where the nucleic acid does not encode an amino
acid sequence, to essentially identical nucleic acid sequences.
Because of the degeneracy of the genetic code, a large number of
functionally identical nucleic acids may encode any given protein.
As to amino acid sequences, one of skill will recognize that an
individual substitution to a nucleic acid, peptide, polypeptide, or
protein sequence which substitutes an amino acid or a small
percentage of amino acids in the encoded sequence for a conserved
amino acid is a "conservatively modified variant." Conservative
substitution tables providing functionally similar amino acids are
well known in the art. An example of a conservative substitution is
the exchange of an amino acid in one of the following groups for
another amino acid of the same group (U.S. Pat. No. 5,767,063
issued to Lee, et al.; Kyte and Doolittle (1982) J. Mol. Biol. 157:
105-132):
[0028] (1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, or
Met;
[0029] (2) Neutral hydrophilic: Cys, Ser, Thr;
[0030] (3) Acidic: Asp, Glu;
[0031] (4) Basic: Asn, Gln, His, Lys, Arg;
[0032] (5) Residues that influence chain orientation: Gly, Pro;
[0033] (6) Aromatic: Trp, Tyr, Phe;
[0034] (7) Small amino acids: Gly, Ala, Ser.
[0035] "Derived" can be used to describe, e.g., deriving the
structure of a peptide, oligopeptide, or polypeptide from a parent
peptide, oligopeptide, or polypeptide, such as an antibody. In this
context, derived encompasses, e.g., peptide structures where the
peptide has the same sequence as a sequence found within the
parent, e.g., where the peptide is identical to the parent but with
a truncation at the N-terminus, C-terminus, or both N- and
C-termini of the parent, or with a truncation and a fusion, or with
a fusion only. Derived also means that the peptide has the same
sequence as found in the parent, but with conservative amino acid
changes, or with deletions or insertions, where the deletions or
insertions preserve a biological property in the peptide that is
inherent in the parent. "Derived" encompasses situations where the
peptide or polypeptide is synthesized using the parent as a
starting compound, and where the peptide or polypeptide is
synthesized de novo, using the structure of the parent as a
guide.
[0036] "Effective amount" or "therapeutically effective amount," of
the agonist or antagonist of the IL-33 of the present invention,
means an amount sufficient to ameliorate a symptom or sign of a
disorder or physiological condition or an amount sufficient to
permit or facilitate a diagnosis of the disorder or physiological
condition. An effective amount for a particular patient or
veterinary subject 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., U.S. Pat. No. 5,888,530 issued to Netti, et
al.). An effective amount can be 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%, usually by at least
10%, more usually at least 20%, most usually at least 30%,
preferably at least 40%, more preferably at least 50%, most
preferably at least 60%, ideally at least 70%, more ideally at
least 80%, and most ideally at least 90%, where 100% is defined as
the diagnostic parameter shown by a normal subject (see, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0037] "Exogenous" refers to substances that are produced outside
an organism, cell, or human body, depending on the context.
"Endogenous" refers to substances that are produced within a cell,
organism, or human body, depending on the context.
[0038] "Disorder" refers to a pathological state, or a condition
that is correlated with or predisposes to a pathological state.
"Infectious disorder" refers, e.g., to a disorder resulting from a
microbe, bacterium, parasite, virus, and the like, as well as to an
inappropriate, ineffective, or pathological immune response to the
disorder. "Oncogenic disorder" encompasses a cancer, transformed
cell, tumor, displasia, angiogenesis, metastasis, and the like, as
well as to an inappropriate, ineffective, or pathological immune
response to the disorder.
[0039] "Effective amount" means, e.g., an amount of an IL-33
agonist, IL-33 antagonist, binding compound or binding composition,
sufficient to ameliorate a symptom or sign of a disorder,
condition, or pathological state. "Effective amount" also relates
to an amount of an IL-33 agonist, antagonist, or binding compound
or composition, sufficient to allow or facilitate the diagnosis of
a symptom or sign of a disorder, condition, or pathological
state.
[0040] "Inhibitors" and "antagonists" or "activators" and
"agonists" refer to inhibitory or activating molecules,
respectively, e.g., for the activation of, e.g., a ligand,
receptor, cofactor, a gene, cell, tissue, or organ. A modulator of,
e.g., a gene, a receptor, a ligand, or a cell, is a molecule that
alters an activity of the gene, receptor, ligand, or cell, where
activity can be activated, inhibited, or altered in its regulatory
properties. The modulator may act alone, or it may use a cofactor,
e.g., a protein, metal ion, or small molecule. Inhibitors are
compounds that decrease, block, prevent, delay activation,
inactivate, desensitize, or down regulate, e.g., a gene, protein,
ligand, receptor, or cell. Activators are compounds that increase,
activate, facilitate, enhance activation, sensitize, or up
regulate, e.g., a gene, protein, ligand, receptor, or cell. An
inhibitor may also be defined as a composition that reduces,
blocks, or inactivates a constitutive activity. An "agonist" is a
compound that interacts with a target to cause or promote an
increase in the activation of the target. An "antagonist" is a
compound that opposes the actions of an agonist. An antagonist
prevents, reduces, inhibits, or neutralizes the activity of an
agonist. An antagonist can also prevent, inhibit, or reduce
constitutive activity of a target, e.g., a target receptor, even
where there is no identified agonist.
[0041] To examine the extent of inhibition, for example, samples or
assays comprising a given, e.g., protein, gene, cell, or organism,
are treated with a potential activator or inhibitor and are
compared to control samples 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% or less, typically 85% or
less, more typically 80% or less, most typically 75% or less,
generally 70% or less, more generally 65% or less, most generally
60% or less, typically 55% or less, usually 50% or less, more
usually 45% or less, most usually 40% or less, preferably 35% or
less, more preferably 30% or less, still more preferably 25% or
less, and most preferably less than 25%. Activation is achieved
when the activity value relative to the control is about 110%,
generally at least 120%, more generally at least 140%, more
generally at least 160%, often at least 180%, more often at least
2-fold, most often at least 2.5-fold, usually at least 5-fold, more
usually at least 10-fold, preferably at least 20-fold, more
preferably at least 40-fold, and most preferably over 40-fold
higher.
[0042] Endpoints in activation or inhibition can be monitored as
follows. Activation, inhibition, and response to treatment, e.g.,
of a cell, physiological fluid, tissue, organ, and animal or human
subject, can be monitored by an endpoint. The endpoint 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, toxic oxygen, or a protease. The
endpoint may comprise, e.g., a predetermined quantity of ion flux
or transport; cell migration; cell adhesion; cell proliferation;
potential for metastasis; cell differentiation; and change in
phenotype, e.g., change in expression of gene relating to
inflammation, apoptosis, transformation, cell cycle, or metastasis
(see, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30:145-158; Hood and
Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme, et al. (2003)
Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002) Med.
Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.
Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia
36:235-243; Stanimirovic and Satoh (2000) Brain Pathol.
10:113-126).
[0043] An endpoint of inhibition is generally 75% of the control or
less, preferably 50% of the control or less, more preferably 25% of
the control or less, and most preferably 10% of the control or
less. Generally, an endpoint of activation is at least 150% the
control, preferably at least two times the control, more preferably
at least four times the control, and most preferably at least 10
times the control.
[0044] "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.
[0045] "Hybridization" that is specific or selective typically
occurs when there is at least about 55% homology over a stretch of
at least about 30 nucleotides, preferably at least about 75% over a
stretch of about 25 nucleotides, and most preferably at least about
90% over about 20 nucleotides (see, e.g., Kanehisa (1984) Nucleic
Acids Res. 12:203-213). Hybridization under stringent conditions,
e.g., of a first nucleic acid to a second nucleic acid, are those
that: (1) Employ low ionic strength and high temperature for
washing, for example, 0.015 M sodium chloride/0.0015 M sodium
citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) Employ
during hybridization a denaturing agent, such as formamide, for
example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.
1% Ficoll.RTM. (Sigma-Aldrich, St. Louis, Mo.)/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; (3)
Employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm
DNA (50 ng/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C,
with washes at 42.degree. C. in 0.233 SSC and 0.1% SDS; or (4)
Employ a buffer of 10% dextran sulfate, 2.times.SSC (sodium
chloride/sodium citrate), and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C. (U.S. Pat. No. 6,387,657 issued to
Botstein, et al.).
[0046] Stringent conditions for hybridization of nucleic acids are
a function of salt, temperature, organic solvents, and chaotropic
agents. Stringent temperature conditions will usually include
temperatures in excess of about 30.degree. C., more usually in
excess of about 37.degree. C., typically in excess of about
45.degree. C., more typically in excess of about 50.degree. C.,
preferably in excess of about 65.degree. C., and more preferably in
excess of about 70.degree. C. Stringent salt conditions will
ordinarily be less than about 1 M, more ordinarily less than about
500 mM, usually less than about 400 mM, more usually less than
about 300 mM, typically less than about 200 mM, preferably less
than about 100 mM, and more preferably less than about 80 mM, even
down to less than about 20 mM. However, the combination of
parameters is more important than the measure of any single
parameter (Wetmur and Davidson (1968) J. Mol. Biol.
31:349-370).
[0047] "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, including infections,
tumors, and cancers that resist irradication by the immune system.
"Cancerous condition" includes, e.g., cancer, cancer cells, tumors,
angiogenesis, and precancerous conditions such as dysplasia.
[0048] "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.
[0049] "Inflammatory disorder" means a disorder or pathological
condition where the pathology results, in whole or in part, from an
increase in the number and/or increase in activation of cells of
the immune system, e.g., of T cells, B cells, monocytes or
macrophages, alveolar macrophages, dendritic cells, NK cells, NKT
cells, neutrophils, eosinophils, or mast cells.
[0050] "IL-33 Receptor", "IL-33R", or "IL-33R complex" as used
herein shall mean the association of two IL-1R family members,
T1/ST2 and SIGIRR to form receptor complex responsive to
stimulation with IL-33.
[0051] "Ligand" refers, e.g., to a small molecule, peptide,
polypeptide, and membrane associated or membrane-bound molecule, or
complex thereof, that can act as an agonist or antagonist of a
receptor. "Ligand" also encompasses an agent that is not an agonist
or antagonist, but that can bind to the receptor without
significantly influencing its biological properties, e.g.,
signaling or adhesion. Moreover, "ligand" includes a membrane-bound
ligand that has been changed, e.g., by chemical or recombinant
methods, to a soluble version of the membrane-bound ligand. By
convention, where a ligand is membrane-bound on a first cell, the
receptor usually occurs on a second cell. The second cell may have
the same or a different identity as the first cell. A ligand or
receptor may be entirely intracellular, that is, it may reside in
the cytosol, nucleus, or some other intracellular compartment. The
ligand or receptor may change its location, e.g., from an
intracellular compartment to the outer face of the plasma membrane.
The complex of a ligand and receptor is termed a "ligand receptor
complex." Where a ligand and receptor are involved in a signaling
pathway, the ligand occurs at an upstream position and the receptor
occurs at a downstream position of the signaling pathway.
[0052] A "first polypeptide chain" and a "second polypeptide chain"
refers to two polypeptide chains not linked together by way of a
classical peptide bond. Typically, the first polypeptide chain
comprises an N-terminus and C-terminus, and the second polypeptide
chain comprises another N-terminus and another C-terminus, that is,
altogether there are two N-termini and two C-termini. The first
polypeptide chain can be encoded by a first vector, while the
second polypeptide chain can be encoded by a second vector. The
first polypeptide chain and second polypeptide chain can be encoded
by one vector, where a first promoter can be operably linked with
the first polypeptide chain and a second promoter can be operably
linked with the second polypeptide chain or, in another embodiment,
expression of both the first and second polypeptide chains can be
operably linked to the same promoter.
[0053] "Sensitivity," e.g., sensitivity of receptor to a ligand,
means that binding of a ligand to the receptor results in a
detectable change in the receptor, or in events or molecules
specifically associated with the receptor, e.g., conformational
change, phosphorylation, nature or quantity of proteins associated
with the receptor, or change in genetic expression mediated by or
associated with the receptor.
[0054] "Small molecules" are provided for the treatment of
physiology and disorders of tumors and cancers. "Small molecule" is
defined as a molecule with a molecular weight that is less than 10
kD, typically less than 2 kD, and preferably less than 1 kD. Small
molecules include, but are not limited to, inorganic molecules,
organic molecules, organic molecules containing an inorganic
component, molecules comprising a radioactive atom, synthetic
molecules, peptide mimetics, and antibody mimetics. As a
therapeutic, a small molecule may be more permeable to cells, less
susceptible to degradation, and less apt to elicit an immune
response than large molecules. Small molecules, such as peptide
mimetics of antibodies and cytokines, as well as small molecule
toxins are described (see, e.g., Casset, et al. (2003) Biochem.
Biophys. Res. Commun. 307:198-205; Muyldermans (2001) J.
Biotechnol. 74:277-302; Li (2000) Nat. Biotechnol. 18:1251-1256;
Apostolopoulos, et al. (2002) Curr. Med. Chem. 9:411-420;
Monfardini, et al. (2002) Curr. Pharm. Des. 8:2185-2199; Domingues,
et al. (1999) Nat. Struct. Biol. 6:652-656; Sato and Sone (2003)
Biochem. J. 371:603-608; U.S. Pat. No. 6,326,482 issued to Stewart,
et al).
[0055] "Soluble receptor" refers to receptors that are
water-soluble and occur, e.g., in extracellular fluids,
intracellular fluids, or weakly associated with a membrane. Soluble
receptor further refers to receptors that are engineered to be
water soluble. For T1/ST2, the soluble or extracellular domain is
defined as residues 1-337 of SEQ ID NO: 6 (human) and residues
1-342 of SEQ ID NO: 8 (mouse). For SIGIRR, the soluble or
extracellular domain is defined as residues 1-118 of SEQ ID NO: 10
(human) and residues 1-117 of SEQ ID NO: 12 (mouse).
[0056] "Specificity of binding," "selectivity of binding," and the
like, refer 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. "Specifically" or
"selectively" binds, when referring to a ligand/receptor,
antibody/antigen, or other binding pair, indicates a binding
reaction that is determinative of the presence of the protein in a
heterogeneous population of proteins and other biologics. Thus,
under designated conditions, a specified ligand binds to a
particular receptor and does not bind in a significant amount to
other proteins present in the sample. The antibody, or binding
composition derived from the antigen-binding site of an antibody,
binds to its antigen with an affinity that is at least two fold
greater, preferably at least ten times greater, more preferably at
least 20-times greater, and most preferably at least 100-times
greater than the affinity to any other antigen. In a preferred
embodiment the antibody will have an affinity that is greater than
about 10.sup.9 liters/mol (see, e.g., Munsen, et al. (1980) Analyt.
Biochem. 107:220-239).
[0057] II. General.
[0058] The present invention provides methods for the modulation or
treatment of a number of immune conditions and disorders. In
particular, the present invention provides agonists and antagonists
of IL-33 for the treatment and diagnosis of, e.g., asthma,
allergies, arthritis, and response to intracellular pathogens, such
as parasites, and response to disorders involving granulomas, e.g.,
tuberculosis, sarcoidosis, and Crohn's disease.
[0059] Nave T cells appear not to express T1/ST2 on their surface,
whereas expression is induced after contact with antigens on
differentiated TH2 effector cells. T1/ST2 has been used as a marker
for TH2-type T cells. T1/ST2 is also expressed on mast cells and
fibroblasts
[0060] Studies with T1/ST2 knockout mice seems to suggest that
T1/ST2 does not play a part in the differentiation of nave
CD4.sup.+ T cells to TH2-type T cells, though these results appear
to be a function of the nature of the assays used, e.g., which
pathogenic organism is used in challenge studies, or which phase of
TH2-response is studied. Evidence also suggests a role for T1/ST2
in early events in TH2-response (see, e.g., Kropf, et al. (2002)
Infect. Immunity 70:5512-5520; Hoshino, et al. (1999) J. Exp. Med.
190:1541-1547; Senn, et al. (2000) Eur. J. Immunol. 30:1929-1938;
Townsend, et al. (2000) J. Exp. Med. 191:1069-1075).
[0061] Anti-T1/ST2 antibodies have been used in a number of studies
addressing the role of T1/ST2 in immune function, while other
studies have examined T1/ST2 expression animal models for immune
response. Treatment with anti-T1/ST2 antibodies resulted in
decreased TH2-type immune responses. The antibody inhibited
eosinophil infiltration, IL-5 production, and IgE-production.
Infections by Schistosoma provoked an up-regulation of T1/ST2,
e.g., as determined by assessing expression in lung and liver
granulomas. Animal models for asthma, e.g., treatment with house
dust mite extract or with ovalbumin, resulted in increased
expression of T1/ST2 on CD4.sup.+ T cells, indicating a role for
T1/ST2 in allergic or asthmatic responses. Studies with BALB/c mice
revealed that treating with anti-T1/ST2 antibody induced higher
TH1-type response, enhancing the ability of CD4.sup.+ T cells to
respond to IL-12. Anti-T1/ST2 antibodies also reduce lesions due to
Leishmania major infections, and reduced expression of TH2-type
cytokines. An animal model of arthritis (collagen induced
arthritis; CIA) was exacerbated by anti-T1/ST2 antibodies. In
particular, T1/ST2 functions in early events in the generation of
TH2-type responses. Chronic exposure to various allergens resulted
in increased expression of T1/ST2 on CD4.sup.+ T cells. T1/ST2
plays a role in mediating innate response, as anti-T1/ST2
antibodies exacerbate the toxic effects of lipopolysaccharide
(LPS). Antibodies to T1/ST2 also modulated immune response to
viruses, e.g., respiratory syncytial virus (see, e.g., Xu, et al.
(1998) J. Exp. Med. 187:787-794; Lohning, et al. (1998) Proc. Natl.
Acad. Sci. USA 95:6930-6935; Coyle, et al. (1999) J. Exp. Med.
190:895-902; Lohning, et al. (1999) J. Immunol. 162:3882-3889;
Johnson, et al. (2003) Am. J. Respir. Crit. Care Med. 169:378-385;
Kropf, et al. (2003) Infect. Immunity 71:1961-1971; Xu, et al.
(1998) J. Exp. Med. 187:787-794; Kropf, et al. (2002) Eur. J.
Immunol. 32:2450-2459; Swirski, et al. (2002) J. Immunol.
169:3499-3506; Sweet, et al. (2001) J. Immunol. 166:6633-6639;
Walzl, et al. (2001) J. Exp. Med. 193:785-792.
[0062] IL-1 family members typically bind to a heterodimeric
members of the IL-1 receptor family. It was shown that another
known IL-1R family member, SIGIRR (single Ig IL-1 receptor related
protein), complexes with T1/ST2 to form the functional receptor
complex for IL-33. SIGIRR was originally found as an orphan IL-1R
member (see, e.g., Garlanda, et al. (2004) Proc. Natl. Acad. Sci.
101:3522-3526; Clark, et al. (2003) Genome Res. 13:2265-2270;
Thomassen et al. (1999) Cytokine 11:389-399; GenBank Accession No.
NP.sub.--068577; GenBank Accession No. NM.sub.--021805; GenBank
Accession No. NP.sub.--075546; and GenBank Accession No.
NM.sub.--0230459). SIGIRR is a widely expressed IL-1R member.
[0063] In precipitation experiments using biotinylated mature human
IL-33 (residues 112-270 of SEQ ID NO: 2), T1/ST2-Fc fusion, and
SIGIRR-Fc fusion, it was shown that IL-33 could bind both receptor
fusion proteins, however, the binding of IL-33 to SIGIRR was weaker
as compared to IL-33 and T1/ST2 binding. To test the signaling
capabilities of either or both receptors, an NF-.kappa.B-dependent
assay was run. Co-expression of both T1/ST2 and SIGIRR was both
necessary and sufficient to activate NF-.kappa.B signaling and MAP
kinase upon stimulation with IL-33. Activation of JNK kinases was
also observed.
[0064] II. Agonists, Antagonists, and Binding Compositions.
[0065] The present invention provides agonists and antagonists of
IL-33, including binding compositions that specifically bind to
IL-33 or to IL-33 receptor complex (T1/ST2 and SIGIRR). Binding
compositions include antibodies, antibody fragments, and soluble
receptors. The present invention contemplates blocking antibodies
that bind to IL-33 or to IL-33R, or agonistic antibodies that
stimulate signaling via the IL-33R complex. The binding
compositions of the present invention also include nucleic acids
that specifically hybridize to nucleic acids encoding IL-33 or
IL-33R, e.g., anti-sense nucleic acids and small interference RNA
(siRNA) Anti-idiotypic antibodies may also be used. Human IL-33 is
disclosed by GenBank NM.sub.--033439. Regions of increased
antigenicity, suitable for preparing anti-IL-33 antibodies, occur
at, e.g., amino acids 1-23; 30-38; 61-78; 84-93; 99-106; 127-133;
139-144; 148-158; 166-180; 196-204;231-237;and252-257,of GenBank
NM.sub.--033439, according to a Parker plot using Vector NTI.RTM.
Suite (Informax, Inc, Bethesda, Md.).
[0066] Receptors based on these extracellular regions are not
limited by these exact N-terminal and C-terminal amino acids, but
may be longer or shorter, e.g., by one, two, three, or more amino
acids, as long as the ligand binding properties are substantially
maintained. Fusion proteins based on the soluble receptors are also
contemplated, e.g., for facilitating purification or stability or
for providing a functional domain, e.g., a toxic polypeptide.
[0067] Monoclonal, polyclonal, and humanized antibodies can be
prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal
Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and
Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New
York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp.
139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al.
(1998) J. Immunol. 160:1029; Tang, et al. (1999) J. Biol. Chem.
274:27371-27378; Baca, et al. (1997) J. Biol. Chem.
272:10678-10684; Chothia, et al. (1989) Nature 342:877-883; Foote
and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No.
6,329,511 issued to Vasquez, et al.). Muteins and variants of
antibodies and soluble receptors are contemplated, e.g., pegylation
or mutagenesis to remove or replace deamidating Asn residues.
[0068] 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. (1997) Virology 228:278-284.
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. (1997) Immunity
7:283-290; Wright, et al. (2000) Immunity 13:233-242; Preston, et
al. (1997) Eur. J. Immunol. 27:1911-1918). 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. (1999) J. Immunol.
163:5157-5164).
[0069] Antibodies will usually bind with at least a K.sub.D of
about 10.sup.-3 M, more usually at least 10.sup.-6 M, typically at
least 10.sup.-7 M, more typically at least 10.sup.-8 M, preferably
at least about 10.sup.-9 M, and more preferably at least 10.sup.-10
M, and most preferably at least 10.sup.-11 M (see, e.g., Presta, et
al. (2001) Thromb. Haemost. 85:379-389; Yang, et al. (2001) Crit.
Rev. Oncol. Hematol. 38:17-23; Carnahan, et al. (2003) Clin. Cancer
Res. (Suppl.) 9:3982s-3990s).
[0070] Soluble receptors comprising the extracellular domains of
the IL-33 receptor complex (T1/ST2 and SIGIRR) can be prepared, as
the cytoplasmic, transmembrane, and extracellular regions of each
of the subunits have been identified (see, e.g., Lecart, et al.
(2002) Eur. J. Immunol. 32:2979-2987; Mitcham, et al. (1996) J.
Biol. Chem. 271:5777-5783; and the Sequence Listing below).
[0071] Soluble receptors can be prepared and used according to
standard methods (see, e.g., Jones, et al. (2002) Biochim. Biophys.
Acta 1592:251-263; Prudhomme, et al. (2001) Expert Opinion Biol.
Ther. 1:359-373; Fernandez-Botran (1999) Crit. Rev. Clin. Lab Sci.
36:165-224). Also provided are compositions for siRNA interference
(see, e.g., Arenz and Schepers (2003) Naturwissenschaften
90:345-359; Sazani and Kole (2003) J. Clin. Invest. 112:481-486;
Pirollo, et al. (2003) Pharmacol. Therapeutics 99:55-77; Wang, et
al. (2003) Antisense Nucl. Acid Drug Devel. 13:169-189).
[0072] IV. Therapeutic Compositions, Methods.
[0073] The present invention provides methods for treating and
diagnosing innate response, asthma, allergies, and arthritis.
[0074] To prepare pharmaceutical or sterile compositions including
an agonist or antagonist of IL-33, the reagent is mixed with a
pharmaceutically acceptable carrier or excipient. Formulations of
therapeutic and diagnostic agents can be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous
solutions, lotions, or suspensions (see, e.g., Hardman, et al.
(2001) Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y.).
[0075] 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, cytokines, and small molecules are
available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios
Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991)
Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New
York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide
Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.;
Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, et al.
(1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New
Engl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl.
J. Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med.
348:24-32; Lipsky, et al. (2000) New Engl. J. Med.
343:1594-1602).
[0076] Antibodies, antibody fragments, and cytokines can be
provided by continuous infusion, or by doses at intervals of, e.g.,
one day, one week, or 1-7 times per week. Doses may be provided
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, or by inhalation. A
preferred dose protocol is one involving the maximal dose or dose
frequency that avoids significant undesirable side effects. A total
weekly dose is generally at least 0.05 .mu.g/kg body weight, more
generally at least 0.2 .mu.g/kg, most generally at least 0.5
.mu.g/kg, typically at least 1 .mu.g/kg, more typically at least 10
.mu.g/kg, most typically at least 100 .mu.g/kg, preferably at least
0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at
least 2.0 mg/kg, optimally at least 10 mg/kg, more optimally at
least 25 mg/kg, and most optimally at least 50 mg/kg (see, e.g.,
Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al.
(2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J.
Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003)
Cancer Immunol. Immunother. 52:133-144). The desired dose of a
small molecule therapeutic, e.g., a peptide mimetic, natural
product, or organic chemical, is about the same as for an antibody
or polypeptide, on a moles/kg body weight basis. The desired plasma
concentration of a small molecule therapeutic is about the same as
for an antibody, on a moles/kg body weight basis.
[0077] 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. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0078] Typical veterinary, experimental, or research subjects
include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs,
horses, and humans.
[0079] 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.
[0080] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art (see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice:A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.). An effective amount of
therapeutic will decrease the symptoms typically by at least 10%;
usually by at least 20%; preferably at least about 30%; more
preferably at least 40%, and most preferably by at least 50%.
[0081] The route of administration is by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or pulmonary
routes, or by sustained release systems or an implant (see, e.g.,
Sidman et al. (1983) Biopolymers 22:547-556; Langer, et al. (1981)
J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech.
12:98-105; Epstein, et al. (1985) Proc. Natl. Acad. Sci. USA
82:3688-3692; Hwang, et al. (1980) Proc. Natl. Acad. Sci. USA
77:4030-4034; U.S. Pat. Nos. 6,350466 and 6,316,024).
[0082] V. Kits and Diagnostic Reagents.
[0083] Diagnostic methods for inflammatory disorders, e.g.,
psoriasis, Crohn's disease, rheumatoid arthritis, asthma or
allergy, atherosclerosis, and cancers, based on antibodies, nucleic
acid hybridization, and the PCR method, are available.
[0084] This invention provides polypeptides of IL-33, fragments
thereof, nucleic acids of IL-33, and fragments thereof, in a
diagnostic kit, e.g., for the diagnosis of viral disorders,
including of influenza A, and viral disorders of the respiratory
tract and of mucosal tissues. Also provided are binding
compositions, including antibodies or antibody fragments, for the
detection of IL-33, and metabolites and breakdown products thereof.
Typically, the kit will have a compartment containing either a
IL-33 polypeptide, or an antigenic fragment thereof, a binding
composition thereto, or a nucleic acid, such as a nucleic acid
probe, primer, or molecular beacon (see, e.g., Rajendran, et al.
(2003) Nucleic Acids Res. 31:5700-5713; Cockerill (2003) Arch.
Pathol. Lab. Med. 127:1112-1120; Zammatteo, et al. (2002) Biotech.
Annu. Rev. 8:85-101; Klein (2002) Trends Mol. Med. 8:257-260).
[0085] A method of diagnosis can comprise contacting a sample from
a subject, e.g., a test subject, with a binding composition that
specifically binds to a polypeptide or nucleic acid of IL-33 or
IL-33 receptor. The method can further comprise contacting a sample
from a control subject, normal subject, or normal tissue or fluid
from the test subject, with the binding composition. Moreover, the
method can additionally comprise comparing the specific binding of
the composition 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.
[0086] The kit may comprise, e.g., a reagent and a compartment, a
reagent and instructions for use, or a reagent with a compartment
and instructions for use. The reagent may comprise an agonist or
antagonist of IL-33, or an antigenic fragment thereof, a binding
composition, or a nucleic acid in a sense and/or anti-sense
orientation. A kit for determining the binding of a test compound,
e.g., acquired from a biological sample or from a chemical library,
can comprise a control compound, a labeled compound, and a method
for separating free labeled compound from bound labeled compound.
The control compound can comprise a segment of the polypeptide of
IL-33 or IL-33 receptor or a nucleic acid encoding IL-33 or IL-33
receptor. The segment can comprise zero, one, two, or more
antigenic fragments.
[0087] A composition that is "labeled" is detectable, either
directly or indirectly, by spectroscopic, photochemical,
biochemical, immunochemical, isotopic, or chemical methods. For
example, useful labels include .sup.32P, .sup.33P, .sup.35S,
.sup.14C, .sup.3H, .sup.125I, stable isotopes, fluorescent dyes,
electron-dense reagents, substrates, epitope tags, or enzymes,
e.g., as used in enzyme-linked immunoassays, or fluorettes (Rozinov
and Nolan (1998) Chem. Biol. 5:713-728).
[0088] Diagnostic assays can be used with biological matrices such
as live cells, cell extracts, cell lysates, fixed cells, cell
cultures, bodily fluids, or forensic samples. Conjugated antibodies
useful for diagnostic or kit purposes, include antibodies coupled
to dyes, isotopes, enzymes, and metals, see, e.g., Le Doussal, et
al. (1991) New Engl. J. Med. 146:169-175; Gibellini, et al. (1998)
J. Immunol. 160:3891-3898; Hsing and Bishop (1999) New Engl. J.
Med. 162:2804-2811; Everts, et al. (2002) New Engl. J. Med.
168:883-889. Various assay formats exist, such as radioimmunoassays
(RIA), ELISA, and lab on a chip (U.S. Pat. Nos. 6,176,962 and
6,517,234).
[0089] Gene expression data is useful tool in the diagnosis and
treatment of diseases and pathological conditions (see, e.g., Li
and Wong (2001) Genome Informatics 12:3-13; Lockhart, et al. (1996)
Nature Biotechnol. 14:1675-1680; Homey, et al. (2000) J. Immunol.
164:3465-3470; Debets, et al. (2000) J. Immunol. 165:49504956).
[0090] VI. Uses.
[0091] The present invention provides methods for the treatment and
diagnosis of inflammatory and immune disorders, including
inappropriate or ineffective response to infection. Provided are
methods relating to, e.g., asthma, allergies, arthritis, disorders
involving eosinophilic inflammation, and disorders involving
pathogenic or ineffective TH2-type response.
[0092] The present invention provides methods for stimulating
immune defense against bacteria, parasites, and viruses,
intracellular pathogens, and cancers and tumors. Provided are
methods for the treatment of intracellular bacteria. Intracellular
bacterial species include Salmonella sp., Shigella sp., Listeria
sp., Francisella sp., Mycobacteria sp. (tuberculosis; leprosy),
Legionella sp., Rickettsia sp., Orienta sp., Ehrlichia sp.,
Anaplasma sp., Neorickettsia sp., Chlamydia sp., and Coxiella sp.
Additionally, IFNgamma mediates response to parasites, e.g.,
Plasmodia sp. (malaria), Toxoplasma sp., Leishmania sp.,
Trypanosoma sp., and Cryptosporidium sp. Provided are methods for
treating viruses, e.g., HIV, orthopoxviruses, such as variola virus
and vaccinia virus (smallpox), and herpesviruses, including
alphaherpesviruses, e.g., Herpes Simplex virus, and
betaherpesviruses, e.g., Cytomegalovirus. Also provided are methods
for the treatment of chronic inflammatory disorders (see, e.g.,
Kent, et al. (2000) Vaccine 18:2250-2256; Ismail, et al. (2002)
FEMS Microbiol. Lett. 207:111-120; Kaufmann (2001) Nature Revs.
Immunol. 1:20-30; Goebel and Gross (2001) TRENDS Microbiol.
9:267-273; Heussler, et al. (2001) Int. J. Parasitol. 31:1166-1176;
Luder, et al. (2001) Carsten, et al. (2001) TRENDS Parasitol.
17:480-486; Rook, et al. (2001) Eur. Resp. J. 17:537-557; Stenger
and Rollinghoff (2001) Ann. Rheum. Dis. 60:iii43-iii46; Haas, et
al. (2002) Am. J. Dermatopathol. 24:319-323; Dorman and Holland
(2000) Cytokine Growth Factor Revs. 11:321-333; Smith, et al.
(2002) J. Gen. Virol. 83 (Pt. 12) 2915-2931; Cohrs and Gilden
(2001) Brain Pathol. 11:465474; Tannenbaum and Hamilton (2002) Sem.
Cancer Biol. 10:113-123; Ikeda, et al. (2002) Cytokine Growth
Factor Revs. 13:95-109; Klimp, et al. (2002) Crit. Rev. Oncol.
Hematol. 44:143-161; Frucht, et al. (2001) TRENDS Immunol.
22:556-560).
[0093] The present invention provides methods of treating or
diagnosing a proliferative condition or disorder, e.g., cancer of
the uterus, cervix, breast, prostate, testes, penis,
gastrointestinal tract, e.g., esophagus, oropharynx, stomach, small
or large intestines, colon, or rectum, kidney, renal cell, bladder,
bone, bone marrow, skin, head or neck, skin, liver, gall bladder,
heart, lung, pancreas, salivary gland, adrenal gland, thyroid,
brain, ganglia, central nervous system (CNS) and peripheral nervous
system (PNS), and immune system, e.g., spleen or thymus. The
present invention provides methods of treating, e.g., immunogenic
tumors, non-immunogenetic tumors, dormant tumors, virus-induced
cancers, e.g., epithelial cell cancers, endothelial cell cancers,
squamous cell carcinomas, papillomavirus, adenocarcinomas,
lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas,
teratocarcinomas, chemically-induced cancers, metastasis, and
angiogenesis. The invention also contemplates reducing tolerance to
a tumor cell or cancer cell antigen, e.g., by modulating activity
of a regulatory T cell (Treg) (see, e.g., Ramirez-Montagut, et al.
(2003) Oncogene 22:3180-3187; Sawaya, et al. (2003) New Engl. J.
Med. 349:1501-1509; Farrar, et al. (1999) J. Immunol.
162:2842-2849; Le, et al. (2001) J. Immunol. 167:6765-6772;
Cannistra and Niloff (1996) New Engl. J. Med. 334:1030-1038;
Osborne (1998) New Engl. J. Med. 339:1609-1618; Lynch and Chapelle
(2003) New Engl. J. Med. 348:919-932; Enzinger and Mayer (2003) New
Engl. J. Med. 349:2241-2252; Forastiere, et al. (2001) New Engl. J.
Med. 345:1890-1900; Izbicki, et al. (1997) New Engl. J. Med.
337:1188-1194; Holland, et al. (eds.) (1996) Cancer Medicine
Encyclopedia of Cancer, 4.sup.th ed., Academic Press, San Diego,
Calif.).
[0094] The present invention provides methods for treating a
proliferative condition, cancer, tumor, or precancerous condition
such as a dysplasia, with an agonist or antagonist of IL-33, with
at least one additional therapeutic or diagnostic agent. The at
least one additional therapeutic or diagnostic agent can be, e.g.,
a cytokine or cytokine antagonist, such as interferon-alpha, or
anti-epidermal growth factor receptor, doxorubicin, epirubicin, an
anti-folate, e.g., methotrexate or fluoruracil, irinotecan,
cyclophosphamide, radiotherapy, hormone or anti-hormone therapy,
e.g., androgen, estrogen, anti-estrogen, flutamide, or
diethylstilbestrol, surgery, tamoxifen, ifosfamide, mitolactol, an
alkylating agent, e.g., melphalan or cis-platin, etoposide,
vinorelbine, vinblastine, vindesine, a glucocorticoid, a histamine
receptor antagonist, an angiogenesis inhibitor, radiation, a
radiation sensitizer, anthracycline, vinca alkaloid, taxane, e.g.,
paclitaxel and docetaxel, a cell cycle inhibitor, e.g., a
cyclin-dependent kinase inhibitor, a monoclonal antibody, a complex
of monoclonal antibody and toxin, a T cell adjuvant, bone marrow
transplant, or antigen presenting cells, e.g., dendritic cell
therapy. Vaccines can be provided, e.g., as a soluble protein or as
a nucleic acid encoding the protein (see, e.g., Le, et al., supra;
Greco and Zellefsky (eds.) (2000) Radiotherapy of Prostate Cancer,
Harwood Academic, Amsterdam; Shapiro and Recht (2001) New Engl. J.
Med. 344:1997-2008; Hortobagyi (1998) New Engl. J. Med.
339:974-984; Catalona (1994) New Engl. J. Med. 331:996-1004; Naylor
and Hadden (2003) Int. Immunopharmacol. 3:1205-1215; The Int.
Adjuvant Lung Cancer Trial Collaborative Group (2004) New Engl. J.
Med. 350:351-360; Slamon, et al. (2001) New Engl. J. Med.
344:783-792; Kudelka, et al. (1998) New Engl. J. Med. 338:991-992;
van Netten, et al. (1996) New Engl. J. Med. 334:920-921).
[0095] A number of biomarkers and methods for scoring inflammatory
disorders, e.g., psoriasis, Crohn's disease, and rheumatoid
arthritis are available (see, e.g., Bresnihan (2003) Arthritis Res.
Ther. 5:271-278; Barnero and Delmas (2003) Curr. Opin. Rheumatol.
15:641-646; Gionchetti, et al. (2003) Dig. Dis. 21:157-167; Wiik
(2002) Autoimmune Rev. 1:67-72; Sostegni, etal. (2003) Aliment
Pharmacol. Ther. 17 (Suppl.2):11-17).
[0096] Biomarkers and methods for scoring cancer are also described
(see, e.g., Alison (ed.) (2001) The Cancer Handbook, Grove's
Dictionaries, Inc., St. Louis, Mo.; Oldham (ed.) (1998) Principles
of Cancer Biotherapy, 3.sup.rd. ed., Kluwer Academic Publ.,
Hingham, Mass.; Thompson, et al. (eds.) (2001) Textbook of
Melanoma, Martin Dunitz, Ltd., London, UK; Devita, et al. (eds.)
(2001) Cancer: Principles and Practice of Oncology, 6.sup.th ed.,
Lippincott, Phila, Pa.; Holland, et al. (eds.) (2000) Holland-Frei
Cancer Medicine, BC Decker, Phila., Pa.; Garrett and Sell (eds.)
(1995) Cellular Cancer Markers, Humana Press, Totowa, N.J.; MacKie
(1996) Skin Cancer, 2.sup.nd ed., Mosby, St. Louis; Moertel (1994)
New Engl. J. Med. 330:1136-1142; Engleman (2003) Semin. Oncol. 30(3
Suppi. 8):23-29; Mohr, et al. (2003) Onkologie 26:227-233).
[0097] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
[0098] I. General Methods.
[0099] Standard methods in biochemistry and molecular biology are
described (see, e.g., Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning,
3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press,
San Diego, Calif.). Standard methods also appear in Ausbel, et al.
(2001) Current Protocols in Molecular Biology, Vols. 1-4, John
Wiley and Sons, Inc. New York, N.Y., which describes cloning in
bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian
cells and yeast (Vol. 2), glycoconjugates and protein expression
(Vol. 3), and bioinformatics (Vol. 4).
[0100] Methods for protein purification including
immunoprecipitation, chromatography, electrophoresis,
centrifugation, and crystallization are described (Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 1, John Wiley and
Sons, Inc., New York). Chemical analysis, chemical modification,
post-translational modification, production of fusion proteins,
glycosylation of proteins are described (see, e.g., Coligan, et al.
(2000) Current Protocols in Protein Science, Vol. 2, John Wiley and
Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in
Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, N.Y., pp.
16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life
Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia
Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391).
Methods for the production, purification, and fragmentation of
polyclonal and monoclonal antibodies are described (Coligan, et al.
(2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons,
Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow
and Lane, supra). Standard techniques for characterizing
ligand/receptor interactions are available (see, e.g., Coligan, et
al. (2001) Current Protcols in Immunology, Vol. 4, John Wiley,
Inc., New York).
[0101] Methods for flow cytometry, including fluorescence activated
cell sorting (FACS), are available (see, e.g., Owens, et al. (1994)
Flow Cytometry Principles for Clinical Laboratory Practice, John
Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry,
2.sup.nd ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical
Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent
reagents suitable for modifying nucleic acids, including nucleic
acid primers and probes, polypeptides, and antibodies, for use,
e.g., as diagnostic reagents, are available (see, e.g., Molecular
Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0102] Standard methods of histology of the immune system are
described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus:
Histopathology and Pathology, Springer Verlag, New York, N.Y.;
Hiatt, et al. (2000) Color Atlas of Histology, Lippincott,
Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic
Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).
[0103] Methods for using animal models, e.g., knockout mice, and
cell-based assays for the testing, evaluation, and screening of
diagnostic, therapeutic, and pharmaceutical agents are available
(see, e.g., Car and Eng (2001) Vet. Pathol. 38:20-30; Kenyon, et
al. (2003) Toxicol. Appl. Pharmacol. 186:90-100; Deurloo, et al.
(2001) Am. J. Respir. Cell Mol. Biol. 25:751-760; Zuberi, et al.
(2000) J. Immunol. 164:2667-2673; Temelkovski, et al. (1998) Thorax
53:849-856; Horrocks, et al. (2003) Curr. Opin. Drug Discov. Devel.
6:570-575; Johnston, et al. (2002) Drug Discov. Today
7:353-363).
[0104] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence aligrunents, are
available (see, e.g., GenBank, Vector NTI.RTM. Suite (Informax,
Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San
Diego, Calif.); DeCypher.RTM. (TimeLogic Corp., Crystal Bay, Nev.);
Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al.
(2000) Bioinformatics Applications Note 16:741-742; Wren, et al.
(2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne
(1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids
Res. 14:4683-4690).
[0105] II. Interleukin-100.
[0106] Similarity of the amino acid sequence of human IL-33 to
other members of the IL-1 family is as follows. Similarity with
IL-1Ra is 34% similarity; IL-1delta is 36%; IL-1F10 is 36%;
IL-1zeta is 9%; IL-1F8 is 32%; IL-1epsilon is 40%; and IL-1F9 is
31%.
[0107] Interleukin-100 was discovered by using computational
sequence analyses and identified as a new IL-1 family member by
secondary structure comparison with members of the IL-1 family,
respectively IL-1beta and IL-18. IL-1 family members are highly
inflammatory regulators of the immune system, released in response
to pathogenic challenges. Human and mouse homologs of IL-33 were
identified as well as a rat and dog IL-33. Gene expression analysis
showed that human IL-33 was expressed in epithelial cells, smooth
muscle cells, and mesangial cells. Upon stimulation with IL-1.beta.
and TNF-.alpha., human IL-33 mRNA levels are highly induced in
primary normal human dermal and lung fibroblasts as well as in
bronchial smooth muscle cells. Expression of IL-33 mRNA in
psoriatic skin samples as well as in lung pulmonary alveolar
proteinosis was significantly elevated.
[0108] Similar to IL-1 and IL-18, IL-33 has no signal peptide. In
stead, IL-33 is made and secreted as a large preproprotein that
requires extensive processing to release the mature, biologically
active form. It is likely that prepro IL-33 is processed by a
caspase. We identified a caspase cleavage site in the protein
sequence and showed that in vitro translated human IL-33 is cleaved
by recombinant constitutively active caspase-1. To investigate the
putative biological role of IL-33, recombinant proteins were
expressed and purified in E.coli. Therefore, the IL-33 gene was
cloned into a pET3a bacterial expression vector. The N-terminus of
the recombinant protein was selected by comparing the mature
sequence of IL-33 to IL-1.beta. and IL-18 so that the mature,
biologically active protein would lack the pro-domain. Amino acid
112 of the full-length protein was selected as the N-terminal amino
acid. Recombinant protein was expressed and purified from E.coli.
In vivo studies were carried out. The intraperitoneal (IP)
injection of recombinant human IL-33 into mice (C57BL/6J), with a
dosage of either 5 ug/day or 50 ug/day, led to severe eosinophilia
and splenomegaly after 7 days. Serum levels of several cytokines
were tested on day 3 and day 7. An induction of IL-5 up to 10000
pg/ml in the 50 ug rhIL-33/day group and up to 1000 pg/ml in the 5
ug rhIL-33/day group was observed on day 3. The serum levels of
IL-5 decreased to 1100 pg/ml in the 50 ug rhIL-33/day group and in
the 5 ug rhIL-33/day group to 500 pg/ml. IL-13 serum levels were
also detectable at day 7 after treatment with either 5 ug or 50 ug
IL-33/day at 30 pg/ml and 100 pg/ml, respectively. No IL-5 and
IL-13 could be detected in the PBS-treated control group.
Furthermore, no elevated levels for IFN-.gamma., TNF-.alpha.,
IL-12, IL-10, IL-6, IL4, IL-2 or MCP-1 could be detected in IL-33
treated mice or in the control group.
[0109] After IP injection of 50 ug rhIL-33/day for 2 days liver
lymphocytes were harvested. Cells were plated into culture dishes
with 2.times.10e6/ml of culture medium and were stimulated with 50
ng/ml PMA and 1 uM ionomycin for 4 hr. During the last 2 hr,
Brefelding A, a secretion inhibitor, was added. Cells were surface
stained for CD3 and NK1.1 and intracellularly stained either for
IL-5 or IL-4 and analyzed by FACS. CD3/NK1.1 positive liver
lymphocytes cells derived from mice treated for 2 days with IL-33
showed an accumulation of IL-5 and IL-4. These results suggest that
IL-33 activates NKT cells to secrete IL4 and IL-5.
[0110] To test if IL-33 binds NKT cells, biotinylated IL-33 was
used for a binding experiment. Human NKT cells, derived from PBMCs,
were incubated either with Strepdavidin-PE or biotinylated rhIL-33
and Strepdavidin-PE. The stained cells were analyzed by FACS.
Binding of rhIL-33 to human NKT cells was observed. This binding
could be competed with unbiotinylated rhIL-33.
[0111] IL-1 family members exert their biological response by
interacting with cell surface receptors. We have identified the
orphan IL-1 receptor ST2/T1 as a cellular receptor for IL-33. FACS
staining of a mouse mast cell line with a ST2/T1-specific
monoclonal antibody showed that this mast cell line expresses the
ST2/T1 receptor. This staining could be specifically and
dose-dependently competed by incubating the mast cell line with
IL-33 protein.
[0112] NKT cells are essential for airway inflammation and the
production of IL-4 and IL-13 in allergen-induced
airway-hyper-reactivity in mouse models of asthma. The induction of
IL-5 and IL-13 by IL-33 in NKT cells suggests that IL-33 can play a
role in the induction of these diseases. The generation of
therapeutic antibodies neutralizing IL-33 may be beneficial in the
treatment of these diseases.
[0113] NKT cells have been implicated in many diseases.
Identification of IL-33 as a modulator of NKT cells suggest that
IL-33 can affect other diseases, such as lupus, multiple sclerosis,
malignancies, airway inflammation and infectious diseases. The
induction of Th2 cytokines by IL-33, such as IL-5 and IL-13, may
help in the protection against microbial infection or in the
protection against tumors.
[0114] IL-1 family members typically bind to two different members
of the IL-1 receptor family to form a complete receptor complex.
The identification of IL-33 and ST2/T1 as one of the subunits that
make up the receptor for IL-33, makes it possible to identify the
second IL-33 receptor subunit. Identification of the complete IL-33
receptor will allow detailed identification of the biological
responses induced by IL-33.
[0115] Real time PCR analysis using Taqman revealed that IL-33 was
expressed by a number of cells and tissues (Table 1). The present
invention provides agonists and antagonists of IL-33 for the
modulation of inflammatory and autoimmune disorders and conditions,
e.g., psoriasis, asthma, allergies, and inflammatory bowel disease,
e.g., gastric inflammation, ulcerative colitis, Crohn's disease,
celiac disease, and irritable bowel syndrome.
1TABLE 1 Real time PCR analysis of IL-33 expression, relative to
ubiquitin (1.0). Human skin psoriasis vulgaris Human skin, normal
adjacent Skin sample no. Expression Skin sample no. Expression
PS-034 757 PS-034 261 PS-037 731 PS-037 267 PS-028 443 PS-028 285
PS-025 446 PS-025 261 PS-023 602 PS-023 235 Colon control 0.5 Colon
Crohn's 87 no. 4003197A Colon Crohn's 125 no. 9609C144 Colon
Crohn's 114 no. 403242A Expression of cytokines with
Nippostrongylus brasiliensis infection, as determined by real time
PCR Taqman analysis. Cytokine tested sample IL-25 IL-13 IL-4 IL-5
IL-33 Untreated stomach 0.86 0.03 0.51 0.01 9.43 stomach 2 day 1.46
0.69 0.09 0.02 56.4 stomach 4 day 0.73 0.67 0.39 0.09 80.32 stomach
8 day 1.04 3.30 0.33 0.44 84.9 stomach 11 days 0.27 1.22 1.77 0.01
8.86 stomach 16 days 0.35 0.38 1.31 0.12 3.43 Untreated control
lung 0.29 0.01 0.57 0.66 56.8 Lung nippo 2 days 0.91 0.37 0.61 0.65
90.37 Lung nippo 4 days 0.59 13.34 4.65 3.78 356.49 Lung nippo 8
days 0.36 55.88 8.78 1.14 48.76 Lung nippo 11 days 0.35 33.93 16.05
2.11 116.79 Lung nippo 16 days 0.04 23.77 25.48 0.84 30.65
[0116] IL-33 induction by IL-1.beta. plus TNF-.alpha. (8 hours) was
compared with IL-33 induction with medium alone. Induction was
studied in the indicated cell type (Table 2).
2TABLE 2 IL-33 induction. ND means not detectable. Numbers are
relative to ubiquitin (1.0). Cell IL-1.beta. plus TNF-.alpha.
Medium only NHDF 3250 50 NHEK ND ND NHBE 25 25 PAEC 100 200 NHLF 25
ND BSMC 2025 350
[0117] In vitro translated human IL-33 was found to be cleaved by
caspase-1. Without caspase treatment, analysis by SDS PAGE revealed
a band at about 32 kDa, corresponding to pro-human IL-33. Treatment
with caspase-1 for 1 hour at 37.degree. resulted in two bands of
about equal intensity, one corresponding to pro-IL-33, and the
other migrating at about 20-22 kDa (mature human IL-33). Treatment
for 2 hours at 37.degree. resulted in the same two bands, but with
about two thirds of the protein migrating at about 22 kDa. Similar
studies demonstrated that in vitro translated human IL-33 could
also be cleaved by elastase or by cathepsin G, to species migrating
at about 20-22 kDa, whereas MMP-3 treatment did not result in
cleavage under the conditions used. Amino acid 112 of IL-33 is
believed to be the position of cleavage, producing the mature
IL-33, due to homology with other IL-1 family members.
[0118] T1/ST2 was identified as at least one subunit of the
receptor for human IL-33. Expression of T1/ST2 was as follows
(Table 3).
3TABLE 3 Expression of T1/ST2 by human or mouse cells. Cell type
Human cells Mouse cells TH1-type T cells -/+ -/+ TH2-type T cells
-/+ +++ Mast cells +++ ++ monocytes/PBMC treated ++ not determined
with LPS Dendritic cells ex BM (not determined) +
[0119] Human IL-33 was cloned in a pET3a vector, and expressed in
E. coli. The cloned protein began with amino acid 112, and was 158
amino acids long (18 kDa). IPTG was used to induced expression, and
the protein was found to be water-soluble. The expressed protein
was purified using a A-column and Sephadex gel filtration. The
purified preparation was tested for endotoxin, where the results
demonstrated about 0.023 EU per microgram protein. Analysis by SDS
PAGE using a non-reducing conditions revealed that at least 95% of
the protein migrated at a single molecular weight of 18 kDa.
[0120] IL-33 was injected intraperitoneally (i.p.) into B6/Balb/c
mice. Three groups of mice were used: ( 1) Injection with phosphate
buffered saline (PBS); ( 2 ) hIL-33 (5 micrograms/day); and ( 3 )
hIL-33 (50 micrograms/day). The protocol also involved injections
(i.p.) for 3 days, with sacrifice after three days of treatment, or
injections (i.p.) for 7 days, with sacrifice after seven days of
treatment. Blood, serum, blood smears, white blood cell
differentials, histology, was performed. Thymus/spleen cells
suspensions were analyzed by FACS analysis.
[0121] IL-33 treatment induces IL-5 and IL-13, as determined by
measuring serum levels of IL-5 and IL-13 (Table 4). The cytokines
IL-4, IL-5, and IL-13 were also measured in various organs with
IL-33 administration. The organs tested were thymus, lung, spleen,
and liver. These three cytokines were all found to be induced, as
determined after 7 days treatment with IL-33. Increases were found
at both levels of IL-33 (5 and 50 microgram IL-33). For example, in
lung, IL-4, IL-5, and IL-13 expression with saline treatment was
about 1.0, or less. But with IL-33 (50 micrograms), expression of
IL-4 was 8.0; of IL-5 was 11.0; and of IL-13 was 41.0. IL-33
treatment also provoked increases in serum IgE and IgA. With 7 days
treatment, IgE levels were 30,000 ng/ml (PBS) and 17,000 ng/ml (50
micrograms IL-33). With 7 days treatment, IgA levels were 90 ng/ml
(PBS) and 420 ng/ml (50 micrograms IL-33). IL-33 treatment also
resulted in splenomegaly, where spleen mass in the PBS (control)
treated mouse was about 80 mg, 5 micrograms with IL-33 for 7 days
(150 mg spleen), and 50 micrograms with IL-33 for 7 days (190 mg
spleen). IL-33 treatment also produced extramedullary hematopoiesis
in the spleen, and thymus hypoplasia (decrease in thymus size), and
hypoplasia of the cortex of the thymus.
4TABLE 4 IL-33 treatment and white blood cell counts, platelet
counts, and cytokine levels, at days 3 and 7. 5 micrograms IL- 50
micrograms IL- PBS 33/.day 33/day White blood cell counts (white
blood cells/microliter blood) Day 3 12,000 12,000 14,000 Day 7
12,000 20,000 25,000 Platelets (platelets/microliter blood) Day 3
Day 7 1,300,000 1,000,000 600,000 Neutrophils Day 3 500 350 520 Day
7 750 700 1200 Lymphocytes Day 3 10,000 10,000 7,500 Day 7 11,000
15,500 18,000 Monocytes Day 3 230 130 200 Day 7 400 250 300
Eosinophils Day 3 420 220 130 Day 7 500 2000 1750 Serum levels of
IL-5 (pg/ml) Day 3 ND 500 7500 Day 7 ND 500 1100 Serum levels of
IL-7 (pg/ml) Day 7 ND 40 78
[0122] Transformed and non-transformed cells were injected into
mice, followed by an incubation period of the cells within the
mouse, and retrieval and purification of the injected cells, with
assessment of T1/ST2 expression (Table 5). Non-transformed mammary
gland cells and ras-transformed mammary gland cells were used. With
injection of the ras-transformed cells into a host immune-deficient
nude mouse, the ras-transformed cells expressed T1/ST2 at increased
levels, that is, expression was 103 (Table 5). With injection of
the ras-transformed cells into a host mouse having an intact immune
system, retrieval of the transformed cell, and Taqman.RTM.
analysis, revealed greater increases in T1/ST2 expression. It is
believed that these increases in T1/ST2 expression reflect
increases in soluble T1/ST2, where the soluble T1/ST2 acts as a
decoy. When soluble T1/ST2 acts as a decoy, it binds to IL-33, and
inhibits the host from mounting a TH2-type immune response against
the tumor cells. The host mice with intact immune systems that were
used, were Xtb mice and XBalb mice (Table 6). The soluble version
of T1/ST2 (also known as Fit 1) is described (see, e.g., Bergers,
et al. (1994) EMBO J. 13:1176-1188; Reikerstorfer, et al. (1995) J.
Biol. Chem. 270:17645-17648).
[0123] IL-33 was administered to mice harboring 4T1 breast cancer.
Administered IL-33 was effective in reducing tumor size (Table 6).
The present invention provides agonists of IL-33, e.g., IL-33 or a
nucleic acid encoding IL-33, for the treatment of proliferative
conditions, including cancers and tumors.
5TABLE 5 Injection of ras-transformed cells into nude mice and
immunocompetant mice, followed by Taqman analysis of T1/ST2
expression by recovered ras-transformed cells. Expression of T1/ST2
Nude mouse host 103 Xtb mouse host 421 XBalb mouse host 669
[0124]
6TABLE 6 Administered IL-33 reduces tumor size in mice. Tumor size
(mm.sup.3) Treatment 23 24 25 26 27 28 29 30 31 32 33 IL-33 175 125
125 200 180 170 205 200 200 240 240 no IL-33 175 200 225 240 280
300 370 405 430 470 500
[0125] Taqman analysis of human tissue samples revealed decreased
expression of IL-33 in various cancers, e.g., breast cancer and
ovarian cancer (Table 7). The results indicate that the tumor cells
refrain from producing IL-33, in order to avoid activating the
immune system to mount an anti-tumor response (Table 7).
7TABLE 7 Real time PCR analysis of IL-33 expression by human cancer
tissues. Cancerous tissue Normal adjacent tissue Human breast 6221
30.4 435.0 (infiltrating duct) Human breast 6612 59.7 54.7
(infiltrating duct) Human breast 6652 2.4 292.0 (infiltrating duct)
Human breast 7748/02 2.6 411.0 lobular Human breast 7156 lobular
77.4 189.1 Human ovary papillary 1872.7 609.4 serous
cystadenocarcinoma 1590 Human ovary papillary 206.6 793.9 serous
cystadenocarcinoma 3572/02 Human ovary papillary 235.1 1052.7
serous cystadenocarcinoma 246869
[0126] IL-33 treatment prolonged experimental autoimmune
encephalitis, an animal model for multiple sclerosis. EAE was
induced by proteolipid protein (PLP) (Kjellen, et al. (2001) J.
Neuroimmunol. 120:25-33; Laman, et al. (2001) J. Neuroimmunol.
119:124-130; Fife, et al. (2001) J. Immunol. 166:7617-7624). Three
groups of mice were used for injections of PBS, 0.5 micrograms
IL-33, or 2.0 micrograms of IL-33, with daily i.p. injections from
day 0 to day 12. Disease scores were assessed on days 7 to 23
(Table 8). The results demonstrated that in PBS-treated mice, the
disease spontaneously resolved. However, with treatment with either
dose of IL-33, the disease was prolonged, higher than that found
with PBS treatment, and maintained itself at a disease score of
between 2.5-3.0 (Table 8). The present invention provides an
antagonist of IL-33 for the treatment of autoimmune disorders,
including autoimmune disorders of the central nervous system, e.g.,
multiple sclerosis.
8TABLE 8 EAE disease score in mice at selected days after treatment
with PBS or with IL-33. Day 11 Day 13 Day 15 Day 17 Day 19 Day 21
PBS 0 2.05 1.75 0.45 0.20 0.0 0.5 micrograms 0 0.75 1.15 2.4 2.9
2.9 IL-33 2.0 micrograms 0 1.95 1.95 3.1 2.3 2.55 IL-33
[0127] IL-33 treatment led to IL-5 induction in NKT cells. NKT
cells were identified by the presence of both the NK1.1 marker and
the CD3 marker. Black-6 mice were treated for two days with PBS or
50 micrograms/day of IL-33. Liver lymphocytes were isolated, and
restimulated with PMA ionomycin for 3 hours and brefeldin for 1
hour. The results demonstrated that IL-33 induced IL-5 in NKT
cells.
[0128] Anti-T1/ST2 antibody was tested for its ability to bind wild
type mast cells (WTMC), and the influence of added IL-33 on binding
of this antibody to the mast cells. Adding IL-33 abolished the
ability of anti-T1/ST2 antibody to bind the mast cells,
demonstrating that the receptor of IL-33 is T1/ST2.
[0129] III. In vivo Effects of IL-33 Antibody Treatment
[0130] A mouse monoclonal antibody against human IL-33 was raised
using methods well known in the art (see above). To test the
ability of this antibody to antagonize IL-33 activity, Balb/c mice
were injected subcutaneously with 0.2 mg of anti-IL-33 antibody on
day 0. On day 1, the mice were injected intraperitonally with 100
ng of mIL-33. Serum was collected on day 2, and IL-5 levels
measured. Treatment with the anti-IL-33 antibody resulted in little
to no production of IL-5 when compared to mice treated with IL-33
alone and mice treated with isotype control antibody and IL-33 (See
FIG. 1). IV. Treatment of Collagen Induced Arthritis (CIA)
[0131] B10.RIII mice, known to be susceptible to developing CIA,
were injected with bovine collagen type II (bovine CII; Sigma) in
complete Freund's adjuvant (Difco). Mice were injected above the
tail base with 100 ul of a 1 mg/ml emulsion of bovine CII. A second
boost dose was administered at day 21. Mice were assessed by the
following clinical scale: 0=normal; 1=redness and/or swelling at
one joint/site; 2=redness and/or swelling at more than on
joint/site; and 3=redness and or swelling in the entire paw. CIA
induced mice have a percent disease onset of 70-90%.
[0132] Mice were treated at day 23 with 1 mg of anti-IL-33 antibody
or isotype control antibody. Antibodies were administered every 7
days for two more treatments. Anti-IL-33 treated mice showed
decreased disease scores as well as a lower percent incidence of
disease onset (see FIGS. 2 and 3). The anti-IL-33 treated mice also
had a lower mean number of arthritic paws (see FIG. 4).
[0133] V. Treatment of Experimental Autoimmune Encephilitis
(EAE)
[0134] C57BL/6 mice were immunized with 50 ug of myelin
oligodendrocyte glycoprotein (MOG) peptide to induce EAE. The
clinical assessment of the disease is scored as follows: 1=limp
tail; 2=hind limb weakness; 3=inability to right+single hind limb
weakness; 4=inability to right+single hind limb paralysis;
5=bilateral hind limb paralysis; 6=bilateral hind limb
paralysis+abdomen collapse; and 7=6+moribund. EAE mice were treated
subcutaneously with either 100 mg of anti-IL-33 antibody or isotype
control antibody. Anti-IL-33 treated mice showed lower disease
scores and lower disease incidence than the control group (see
FIGS. 5 and 6).
[0135] VI. Pull-down Assay to Identify IL-33R Complex
[0136] IL-33 was biotinylated with EX-LINK Supho-NHS-Biotin
(Pierce). Pull-down of 2 .mu.g biotinylated IL-33 was performed in
500 .mu.l RIPA-Lysis buffer (upstate cell singaling solution) with
50 .mu.l of a 50% Slurry of Agarose bound Avidin D (Vector
Laborities). 5 .mu.g of either recombinant extra-cellular ST2-Fc
(R&D Systems) or SIGIRR-Fc (R&D Systems) was used. After
incubation overnight at 4.degree. C. precipitates were washed
3.times. with 500 .mu.l RIPA-Lysis buffer. The precipitated
proteins were separated by SDS-Page, electroblotted, and visualized
by Western blot/ECL reaction with antibodies specific against ST2
(R&D Systems) or SIGIRR (R&D Systems). Pull-down of
biotinylated IL-33 with ST2-Fc or SIGIRR-Fc was performed in the
same manner as above only Protein G-Sepharose (Amersham Bioscience)
was used instead of Agarose bound Avidin D. IL-33 presents was
visualized via a Streptavidin-HRP conjugate (Pierce) and ECL
reaction.
[0137] VII. Phosphorylation of NF-.kappa.B and MAP Kinases
[0138] The mastcell line WTMC was described previously (see, e.g.,
Wright, et al. (2003). J. Immunol. 171:3034-3046.). Cells were
lysed in RIPA lysis Buffer (Upstate) containing Complete Mini
protease inhibitor cocktail (Roche) and 10 mM Na.sub.3VO.sub.4.
Proteins were separated by SDS-Page, transferred to Immobilon-P
membranes (Millipore) and immunoblotted using antibodies to
phosphorylated p65 NF-.kappa.B, p65 NF-.kappa.B, phosphorylated
p44/42 MAP kinases, p44/42 MAP kinases, phosphorylated p38 MAP
kinase and p38 MAP kinase (all Antibodies form Cell Signaling
Technology).
[0139] VIII. Transient Transfection and Reporter Gene Assays
[0140] HEK293FT cells were seeded before transfection with an
NF-.kappa.B-driven GFP reporter gene construct (pNF-.kappa.B-hrGFP;
Stratagene) and with a combination of plasmids encoding for ST2, or
SIGIRR or both, as indicated with Fugene-6 (Roche) according to
manufacturer's recommendations. Cells were split 24 hours after
transfection. After 24 hours cells were either left untreated or
stimulated with mouse IL-33 at the concentration of 50 ng/ml.
Sixteen hours after stimulation, cells were analyzed for
GFP-expression by FACS.
9TABLE 9 Sequence Identifiers SEQ ID NO: 1 Human IL-33 nucleic acid
sequence SEQ ID NO: 2 Human IL-33 polypeptide sequence SEQ ID NO: 3
Mouse IL-33 nucleic acid sequence SEQ ID NO: 4 Mouse IL-33 nucleic
acid sequence SEQ ID NO: 5 Human T1/ST2 nucleic acid sequence SEQ
ID NO: 6 Human T1/ST2 polypeptide sequence SEQ ID NO: 7 Mouse
T1/ST2 nucleic acid sequence SEQ ID NO: 8 Mouse T1/ST2 polypeptide
sequence SEQ ID NO: 9 Human SIGIRR nucleic acid sequence SEQ ID NO:
10 Human SIGIRR polypeptide sequence SEQ ID NO: 11 Mouse SIGIRR
nucleic acid sequence SEQ ID NO: 12 Mouse SIGIRR polypeptide
sequence
[0141] All citations herein are incorporated herein by reference to
the same extent as if each individual publication, patent
application, or patent was specifically and individually indicated
to be incorporated by reference including all figures and
drawings.
[0142] 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 modif ications 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 is to be limited by the terms of the appended
claims, along with 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 by way of
example.
Sequence CWU 1
1
12 1 813 DNA Homo sapiens 1 atgaagccta aaatgaagta ttcaaccaac
aaaatttcca cagcaaagtg gaagaacaca 60 gcaagcaaag ccttgtgttt
caagctggga aaatcccaac agaaggccaa agaagtttgc 120 cccatgtact
ttatgaagct ccgctctggc cttatgataa aaaaggaggc ctgttacttt 180
aggagagaaa ccaccaaaag gccttcactg aaaacaggta gaaagcacaa aagacatctg
240 gtactcgctg cctgtcaaca gcagtctact gtggagtgct ttgcctttgg
tatatcaggg 300 gtccagaaat atactagagc acttcatgat tcaagtatca
caggaatttc acctattaca 360 gagtatcttg cttctctaag cacatacaat
gatcaatcca ttacttttgc tttggaggat 420 gaaagttatg agatatatgt
tgaagacttg aaaaaagatg aaaagaaaga taaggtgtta 480 ctgagttact
atgagtctca acacccctca aatgaatcag gtgacggtgt tgatggtaag 540
atgttaatgg taaccctgag tcctacaaaa gacttctggt tgcatgccaa caacaaggaa
600 cactctgtgg agctccataa gtgtgaaaaa ccactgccag accaggcctt
ctttgtcctt 660 cataatatgc actccaactg tgtttcattt gaatgcaaga
ctgatcctgg agtgtttata 720 ggtgtaaagg ataatcatct tgctctgatt
aaagtagact cttctgagaa tttgtgtact 780 gaaaatatct tgtttaagct
ctctgaaact tag 813 2 270 PRT Homo sapiens 2 Met Lys Pro Lys Met Lys
Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys 1 5 10 15 Trp Lys Asn Thr
Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser 20 25 30 Gln Gln
Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 35 40 45
Ser Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50
55 60 Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His
Leu 65 70 75 80 Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys
Phe Ala Phe 85 90 95 Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala
Leu His Asp Ser Ser 100 105 110 Ile Thr Gly Ile Ser Pro Ile Thr Glu
Tyr Leu Ala Ser Leu Ser Thr 115 120 125 Tyr Asn Asp Gln Ser Ile Thr
Phe Ala Leu Glu Asp Glu Ser Tyr Glu 130 135 140 Ile Tyr Val Glu Asp
Leu Lys Lys Asp Glu Lys Lys Asp Lys Val Leu 145 150 155 160 Leu Ser
Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly 165 170 175
Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe 180
185 190 Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys
Cys 195 200 205 Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His
Asn Met His 210 215 220 Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp
Pro Gly Val Phe Ile 225 230 235 240 Gly Val Lys Asp Asn His Leu Ala
Leu Ile Lys Val Asp Ser Ser Glu 245 250 255 Asn Leu Cys Thr Glu Asn
Ile Leu Phe Lys Leu Ser Glu Thr 260 265 270 3 801 DNA Mus musculus
3 atgagaccta gaatgaagta ttccaactcc aagatttccc cggcaaagtt cagcagcacc
60 gcaggggaac gctcggtccc gccttgcaaa ataagaagat cccaacagaa
gaccaaagaa 120 ttctgccatg tctactgcat gagactccgt tctggcctca
ccataagaaa ggagactagt 180 tattttagga aagaacccac gaaaagatat
tcactaaaat cgggtaccaa gcatgaagag 240 aacttctctg cctatccacg
ggattctagg aagagatcct tgcttggcag tatccaagca 300 tttgctgcgt
ctgttgacac attgagcatc caaggaactt cacttttaac acagtctcct 360
gcctccctga gtacatacaa tgaccaatct gttagttttg ttttggagaa tggatgttat
420 gtgatcaatg ttgacgactc tggaaaagac caagagcaag accaggtgct
actacgctac 480 tatgagtctc cctgtcctgc aagtcaatca ggcgacggtg
tggatgggaa gaagctgatg 540 gtgaacatga gttccatcaa agacacagac
atctggctgc atgccaacga caaggactac 600 tccgtggagc ttcaaagggg
tgacgtctcg cctccggaac aggccttctt cgtccttcac 660 aaaaagtcct
cggactttgt ttcatttgaa tgcaagaatc ttcctggcac ttacatagga 720
gtgaaggaca accagctggc tctagtggag gaaaaagatg agagctgcaa caatattatg
780 tttaagctct cgaaaatcta a 801 4 266 PRT Mus musculus 4 Met Arg
Pro Arg Met Lys Tyr Ser Asn Ser Lys Ile Ser Pro Ala Lys 1 5 10 15
Phe Ser Ser Thr Ala Gly Glu Ala Leu Val Pro Pro Cys Lys Ile Arg 20
25 30 Arg Ser Gln Gln Lys Thr Lys Glu Phe Cys His Val Tyr Cys Met
Arg 35 40 45 Leu Arg Ser Gly Leu Thr Ile Arg Lys Glu Thr Ser Tyr
Phe Arg Lys 50 55 60 Glu Pro Thr Lys Arg Tyr Ser Leu Lys Ser Gly
Thr Lys His Glu Glu 65 70 75 80 Asn Phe Ser Ala Tyr Pro Arg Asp Ser
Arg Lys Arg Ser Leu Leu Gly 85 90 95 Ser Ile Gln Ala Phe Ala Ala
Ser Val Asp Thr Leu Ser Ile Gln Gly 100 105 110 Thr Ser Leu Leu Thr
Gln Ser Pro Ala Ser Leu Ser Thr Tyr Asn Asp 115 120 125 Gln Ser Val
Ser Phe Val Leu Glu Asn Gly Cys Tyr Val Ile Asn Val 130 135 140 Asp
Asp Ser Gly Lys Asp Gln Glu Gln Asp Gln Val Leu Leu Arg Tyr 145 150
155 160 Tyr Glu Ser Pro Cys Pro Ala Ser Gln Ser Gly Asp Gly Val Asp
Gly 165 170 175 Lys Lys Leu Met Val Asn Met Ser Pro Ile Lys Asp Thr
Asp Ile Trp 180 185 190 Leu His Ala Asn Asp Lys Asp Tyr Ser Val Glu
Leu Gln Arg Gly Asp 195 200 205 Val Ser Pro Pro Glu Gln Ala Phe Phe
Val Leu His Lys Lys Ser Ser 210 215 220 Asp Phe Val Ser Phe Glu Cys
Lys Asn Leu Pro Gly Thr Tyr Ile Gly 225 230 235 240 Val Lys Asp Asn
Gln Leu Ala Leu Val Glu Glu Lys Asp Glu Ser Cys 245 250 255 Asn Asn
Ile Met Phe Lys Leu Ser Lys Ile 260 265 5 1671 DNA Homo sapiens 5
atggggtttt ggatcttagc aattctcaca attctcatgt attccacagc agcaaagttt
60 agtaaacaat catggggcct ggaaaatgag gctttaattg taagatgtcc
tagacaagga 120 aaacctagtt acaccgtgga ttggtattac tcacaaacaa
acaaaagtat tcccactcag 180 gaaagaaatc gtgtgtttgc ctcaggccaa
cttctgaagt ttctaccagc tgaagttgct 240 gattctggta tttatacctg
tattgtcaga agtcccacat tcaataggac tggatatgcg 300 aatgtcacca
tatataaaaa acaatcagat tgcaatgttc cagattattt gatgtattca 360
acagtatctg gatcagaaaa aaattccaaa atttattgtc ctaccattga cctctacaac
420 tggacagcac ctcttgagtg gtttaagaat tgtcaggctc ttcaaggatc
aaggtacagg 480 gcgcacaagt catttttggt cattgataat gtgatgactg
aggacgcagg tgattacacc 540 tgtaaattta tacacaatga aaatggagcc
aattatagtg tgacggcgac caggtccttc 600 acggtcaagg atgagcaagg
cttttctctg tttccagtaa tcggagcccc tgcacaaaat 660 gaaataaagg
aagtggaaat tggaaaaaac gcaaacctaa cttgctctgc ttgttttgga 720
aaaggcactc agttcttggc tgccgtcctg tggcagctta atggaacaaa aattacagac
780 tttggtgaac caagaattca acaagaggaa gggcaaaatc aaagtttcag
caatgggctg 840 gcttgtctag acatggtttt aagaatagct gacgtgaagg
aagaggattt attgctgcag 900 tacgactgtc tggccctgaa tttgcatggc
ttgagaaggc acaccgtaag actaagtagg 960 aaaaatccaa ttgatcatca
tagcatctac tgcataattg cagtatgtag tgtattttta 1020 atgctaatca
atgtcctggt tatcatccta aaaatgttct ggattgaggc cactctgctc 1080
tggagagaca tagctaaacc ttacaagact aggaatgatg gaaagctcta tgatgcttat
1140 gttgtctacc cacggaacta caaatccagt acagatgggg ccagtcgtgt
agagcacttt 1200 gttcaccaga ttctgcctga tgttcttgaa aataaatgtg
gctatacctt atgcatttat 1260 gggagagata tgctacctgg agaagatgta
gtcactgcag tggaaaccaa catacgaaag 1320 agcaggcggc acattttcat
cctgacccct cagatcactc acaataagga gtttgcctac 1380 gagcaggagg
ttgccctgca ctgtgccctc atccagaacg acgccaaggt gatacttatt 1440
gagatggagg ctctgagcga gctggacatg ctgcaggctg aggcgcttca ggactccctc
1500 cagcatctta tgaaagtaca ggggaccatc aagtggaggg aggaccacat
tgccaataaa 1560 aggtccctga attccaaatt ctggaagcac gtgaggtacc
aaatgcctgt gccaagcaaa 1620 attcccagaa aggcctctag tttgactccc
ttggctgccc agaagcaata g 1671 6 556 PRT Homo sapiens 6 Met Gly Phe
Trp Ile Leu Ala Ile Leu Thr Ile Leu Met Tyr Ser Thr 1 5 10 15 Ala
Ala Lys Phe Ser Lys Gln Ser Trp Gly Leu Glu Asn Glu Ala Leu 20 25
30 Ile Val Arg Cys Pro Arg Gln Gly Lys Pro Ser Tyr Thr Val Asp Trp
35 40 45 Tyr Tyr Ser Gln Thr Asn Lys Ser Ile Pro Thr Gln Glu Arg
Asn Arg 50 55 60 Val Phe Ala Ser Gly Gln Leu Leu Lys Phe Leu Pro
Ala Glu Val Ala 65 70 75 80 Asp Ser Gly Ile Tyr Thr Cys Ile Val Arg
Ser Pro Thr Phe Asn Arg 85 90 95 Thr Gly Tyr Ala Asn Val Thr Ile
Tyr Lys Lys Gln Ser Asp Cys Asn 100 105 110 Val Pro Asp Tyr Leu Met
Tyr Ser Thr Val Ser Gly Ser Glu Lys Asn 115 120 125 Ser Lys Ile Tyr
Cys Pro Thr Ile Asp Leu Tyr Asn Trp Thr Ala Pro 130 135 140 Leu Glu
Trp Phe Lys Asn Cys Gln Ala Leu Gln Gly Ser Arg Tyr Arg 145 150 155
160 Ala His Lys Ser Phe Leu Val Ile Asp Asn Val Met Thr Glu Asp Ala
165 170 175 Gly Asp Tyr Thr Cys Lys Phe Ile His Asn Glu Asn Gly Ala
Asn Tyr 180 185 190 Ser Val Thr Ala Thr Arg Ser Phe Thr Val Lys Asp
Glu Gln Gly Phe 195 200 205 Ser Leu Phe Pro Val Ile Gly Ala Pro Ala
Gln Asn Glu Ile Lys Glu 210 215 220 Val Glu Ile Gly Lys Asn Ala Asn
Leu Thr Cys Ser Ala Cys Phe Gly 225 230 235 240 Lys Gly Thr Gln Phe
Leu Ala Ala Val Leu Trp Gln Leu Asn Gly Thr 245 250 255 Lys Ile Thr
Asp Phe Gly Glu Pro Arg Ile Gln Gln Glu Glu Gly Gln 260 265 270 Asn
Gln Ser Phe Ser Asn Gly Leu Ala Cys Leu Asp Met Val Leu Arg 275 280
285 Ile Ala Asp Val Lys Glu Glu Asp Leu Leu Leu Gln Tyr Asp Cys Leu
290 295 300 Ala Leu Asn Leu His Gly Leu Arg Arg His Thr Val Arg Leu
Ser Arg 305 310 315 320 Lys Asn Pro Ile Asp His His Ser Ile Tyr Cys
Ile Ile Ala Val Cys 325 330 335 Ser Val Phe Leu Met Leu Ile Asn Val
Leu Val Ile Ile Leu Lys Met 340 345 350 Phe Trp Ile Glu Ala Thr Leu
Leu Trp Arg Asp Ile Ala Lys Pro Tyr 355 360 365 Lys Thr Arg Asn Asp
Gly Lys Leu Tyr Asp Ala Tyr Val Val Tyr Pro 370 375 380 Arg Asn Tyr
Lys Ser Ser Thr Asp Gly Ala Ser Arg Val Glu His Phe 385 390 395 400
Val His Gln Ile Leu Pro Asp Val Leu Glu Asn Lys Cys Gly Tyr Thr 405
410 415 Leu Cys Ile Tyr Gly Arg Asp Met Leu Pro Gly Glu Asp Val Val
Thr 420 425 430 Ala Val Glu Thr Asn Ile Arg Lys Ser Arg Arg His Ile
Phe Ile Leu 435 440 445 Thr Pro Gln Ile Thr His Asn Lys Glu Phe Ala
Tyr Glu Gln Glu Val 450 455 460 Ala Leu His Cys Ala Leu Ile Gln Asn
Asp Ala Lys Val Ile Leu Ile 465 470 475 480 Glu Met Glu Ala Leu Ser
Glu Leu Asp Met Leu Gln Ala Glu Ala Leu 485 490 495 Gln Asp Ser Leu
Gln His Leu Met Lys Val Gln Gly Thr Ile Lys Trp 500 505 510 Arg Glu
Asp His Ile Ala Asn Lys Arg Ser Leu Asn Ser Lys Phe Trp 515 520 525
Lys His Val Arg Tyr Gln Met Pro Val Pro Ser Lys Ile Pro Arg Lys 530
535 540 Ala Ser Ser Leu Thr Pro Leu Ala Ala Gln Lys Gln 545 550 555
7 1704 DNA Mus musculus 7 atgattgaca gacagagaat gggactttgg
gctttggcaa ttctgacact tcccatgtat 60 ttgacagtta cggagggcag
taaatcgtcc tggggtctgg aaaatgaggc tttaattgtg 120 agatgccccc
aaagaggacg ctcgacttat cctgtggaat ggtattactc agatacaaat 180
gaaagtattc ctactcaaaa aagaaatcgg atctttgtct caagagatcg tctgaagttt
240 ctaccagcca gagtggaaga ctctgggatt tatgcttgtg ttatcagaag
ccccaacttg 300 aataagactg gatacttgaa tgtcaccata cataaaaagc
cgccaagctg caatatccct 360 gattatttga tgtactcgac agtacgtgga
tcagataaaa atttcaagat aacgtgtcca 420 acaattgacc tgtataattg
gacagcacct gttcagtggt ttaagaactg caaagctctc 480 caagagccaa
ggttcagggc acacaggtcc tacttgttca ttgacaacgt gactcatgat 540
gatgaaggtg actacacttg tcaattcaca cacgcggaga atggaaccaa ctacatcgtg
600 acggccacca gatcattcac agttgaagaa aaaggctttt ctatgtttcc
agtaattaca 660 aatcctccat acaaccacac aatggaagtg gaaataggaa
aaccagcaag tattgcctgt 720 tcagcttgct ttggcaaagg ctctcacttc
ttggctgatg tcctgtggca gattaacaaa 780 acagtagttg gaaattttgg
tgaagcaaga attcaagaag aggaaggtcg aaatgaaagt 840 tccagcaatg
acatggattg tttaacctca gtgttaagga taactggtgt gacagaaaag 900
gacctgtccc tggaatatga ctgtctggcc ctgaaccttc atggcatgat aaggcacacc
960 ataaggctga gaaggaaaca accaattgat caccgaagca tctactacat
agttgctgga 1020 tgtagtttat tgctaatgtt tatcaatgtc ttggtgatag
tcttaaaagt gttctggatt 1080 gaggttgctc tgttctggag agatatagtg
acaccttaca aaacccggaa cgatggcaag 1140 ctctacgatg cgtacatcat
ttaccctcgg gtcttccggg gcagcgcggc gggaacccac 1200 tctgtggagt
actttgttca ccacactctg cccgacgttc ttgaaaataa atgtggctac 1260
aaattgtgca tttatgggag agacctgtta cctgggcaag atgcagccac cgtggtggaa
1320 agcagtatcc agaatagcag aagacaggtg tttgttctgg cccctcacat
gatgcacagc 1380 aaggaatttg cctacgagca ggagattgct ctgcacagcg
ccctcatcca gaacaactcc 1440 aaggtgattc ttattgaaat ggagcctctg
ggtgaggcaa gccgactaca ggttggggac 1500 ctgcaagatt ctctccagca
tcttgtgaaa attcagggga ccatcaagtg gagggaagat 1560 catgtggccg
acaagcagtc tctaagttcc aaattctgga agcatgtgag gtaccaaatg 1620
ccagtgccag aaagagcctc caagacggca tctgttgcgg ctccgttgag tggcaaggca
1680 tgcttagacc tgaaacactt ttga 1704 8 567 PRT Mus musculus 8 Met
Ile Asp Arg Gln Arg Met Gly Leu Trp Ala Leu Ala Ile Leu Thr 1 5 10
15 Leu Pro Met Tyr Leu Thr Val Thr Glu Gly Ser Lys Ser Ser Trp Gly
20 25 30 Leu Glu Asn Glu Ala Leu Ile Val Arg Cys Pro Gln Arg Gly
Arg Ser 35 40 45 Thr Tyr Pro Val Glu Trp Tyr Tyr Ser Asp Thr Asn
Glu Ser Ile Pro 50 55 60 Thr Gln Lys Arg Asn Arg Ile Phe Val Ser
Arg Asp Arg Leu Lys Phe 65 70 75 80 Leu Pro Ala Arg Val Glu Asp Ser
Gly Ile Tyr Ala Cys Val Ile Arg 85 90 95 Ser Pro Asn Leu Asn Lys
Thr Gly Tyr Leu Asn Val Thr Ile His Lys 100 105 110 Lys Pro Pro Ser
Cys Asn Ile Pro Asp Tyr Leu Met Tyr Ser Thr Val 115 120 125 Arg Gly
Ser Asp Lys Asn Phe Lys Ile Thr Cys Pro Thr Ile Asp Leu 130 135 140
Tyr Asn Trp Thr Ala Pro Val Gln Trp Phe Lys Asn Cys Lys Ala Leu 145
150 155 160 Gln Glu Pro Arg Phe Arg Ala His Arg Ser Tyr Leu Phe Ile
Asp Asn 165 170 175 Val Thr His Asp Asp Glu Gly Asp Tyr Thr Cys Gln
Phe Thr His Ala 180 185 190 Glu Asn Gly Thr Asn Tyr Ile Val Thr Ala
Thr Arg Ser Phe Thr Val 195 200 205 Glu Glu Lys Gly Phe Ser Met Phe
Pro Val Ile Thr Asn Pro Pro Tyr 210 215 220 Asn His Thr Met Glu Val
Glu Ile Gly Lys Pro Ala Ser Ile Ala Cys 225 230 235 240 Ser Ala Cys
Phe Gly Lys Gly Ser His Phe Leu Ala Asp Val Leu Trp 245 250 255 Gln
Ile Asn Lys Thr Val Val Gly Asn Phe Gly Glu Ala Arg Ile Gln 260 265
270 Glu Glu Glu Gly Arg Asn Glu Ser Ser Ser Asn Asp Met Asp Cys Leu
275 280 285 Thr Ser Val Leu Arg Ile Thr Gly Val Thr Glu Lys Asp Leu
Ser Leu 290 295 300 Glu Tyr Asp Cys Leu Ala Leu Asn Leu His Gly Met
Ile Arg His Thr 305 310 315 320 Ile Arg Leu Arg Arg Lys Gln Pro Ile
Asp His Arg Ser Ile Tyr Tyr 325 330 335 Ile Val Ala Gly Cys Ser Leu
Leu Leu Met Phe Ile Asn Val Leu Val 340 345 350 Ile Val Leu Lys Val
Phe Trp Ile Glu Val Ala Leu Phe Trp Arg Asp 355 360 365 Ile Val Thr
Pro Tyr Lys Thr Arg Asn Asp Gly Lys Leu Tyr Asp Ala 370 375 380 Tyr
Ile Ile Tyr Pro Arg Val Phe Arg Gly Ser Ala Ala Gly Thr His 385 390
395 400 Ser Val Glu Tyr Phe Val His His Thr Leu Pro Asp Val Leu Glu
Asn 405 410 415 Lys Cys Gly Tyr Lys Leu Cys Ile Tyr Gly Arg Asp Leu
Leu Pro Gly 420 425 430 Gln Asp Ala Ala Thr Val Val Glu Ser Ser Ile
Gln Asn Ser Arg Arg 435 440 445 Gln Val Phe Val Leu Ala Pro His Met
Met His Ser Lys Glu Phe Ala 450 455 460 Tyr Glu Gln Glu Ile Ala Leu
His Ser Ala Leu Ile Gln Asn Asn Ser 465 470 475 480 Lys Val Ile Leu
Ile Glu Met Glu Pro Leu Gly Glu Ala Ser Arg Leu 485 490
495 Gln Val Gly Asp Leu Gln Asp Ser Leu Gln His Leu Val Lys Ile Gln
500 505 510 Gly Thr Ile Lys Trp Arg Glu Asp His Val Ala Asp Lys Gln
Ser Leu 515 520 525 Ser Ser Lys Phe Trp Lys His Val Arg Tyr Gln Met
Pro Val Pro Glu 530 535 540 Arg Ala Ser Lys Thr Ala Ser Val Ala Ala
Pro Leu Ser Gly Lys Ala 545 550 555 560 Cys Leu Asp Leu Lys His Phe
565 9 1233 DNA Homo sapiens 9 atgccaggtg tctgtgatag ggcccctgac
ttcctctccc cgtctgaaga ccaggtgctg 60 aggcctgcct tgggcagctc
agtggctctg aactgcacgg cttgggtagt ctctgggccc 120 cactgctccc
tgccttcagt ccagtggctg aaagacgggc ttccattggg aattgggggc 180
cactacagcc tccacgagta ctcctgggtc aaggccaacc tgtcagaggt gcttgtgtcc
240 agtgtcctgg gggtcaacgt gaccagcact gaagtctatg gggccttcac
ctgctccatc 300 cagaacatca gcttctcctc cttcactctt cagagagctg
gccctacaag ccacgtggct 360 gcggtgctgg cctccctcct ggtcctgctg
gccctgctgc tggccgccct gctctatgtc 420 aagtgccgtc tcaacgtgct
gctctggtac caggacgcgt atggggaggt ggagataaac 480 gacgggaagc
tctacgacgc ctacgtctcc tacagcgact gccccgagga ccgcaagttc 540
gtgaacttca tcctaaagcc gcagctggag cggcgtcggg gctacaagct cttcctggac
600 gaccgcgacc tcctgccgcg cgctgagccc tccgccgacc tcttggtgaa
cctgagccgc 660 tgccgacgcc tcatcgtggt gctttcggac gccttcctga
gccgggcctg gtgcagccac 720 agcttccggg agggcctgtg ccggctgctg
gagctcaccc gcagacccat cttcatcacc 780 ttcgagggcc agaggcgcga
ccccgcgcac ccggcgctcc gcctgctgcg ccagcaccgc 840 cacctggtga
ccttgctgct ctggaggccc ggctccgtga ctccttcctc cgatttttgg 900
aaagaagtgc agctggcgct gccgcggaag gtgcggtaca ggccggtgga aggagacccc
960 cagacgcagc tgcaggacga caaggacccc atgctgattc ttcgaggccg
agtccctgag 1020 ggccgggccc tggactcaga ggtggacccg gaccctgagg
gcgacctggg tgtccggggg 1080 cctgtttttg gagagccatc agctccaccg
cacaccagtg gggtctcgct gggagagagc 1140 cggagcagcg aagtggacgt
ctcggatctc ggctcgcgaa actacagtgc ccgcacagac 1200 ttctactgcc
tggtgtccaa ggatgatatg tag 1233 10 410 PRT Homo sapiens 10 Met Pro
Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu 1 5 10 15
Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys 20
25 30 Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val
Gln 35 40 45 Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His
Tyr Ser Leu 50 55 60 His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser
Glu Val Leu Val Ser 65 70 75 80 Ser Val Leu Gly Val Asn Val Thr Ser
Thr Glu Val Tyr Gly Ala Phe 85 90 95 Thr Cys Ser Ile Gln Asn Ile
Ser Phe Ser Ser Phe Thr Leu Gln Arg 100 105 110 Ala Gly Pro Thr Ser
His Val Ala Ala Val Leu Ala Ser Leu Leu Val 115 120 125 Leu Leu Ala
Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu 130 135 140 Asn
Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn 145 150
155 160 Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro
Glu 165 170 175 Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu
Glu Arg Arg 180 185 190 Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp
Leu Leu Pro Arg Ala 195 200 205 Glu Pro Ser Ala Asp Leu Leu Val Asn
Leu Ser Arg Cys Arg Arg Leu 210 215 220 Ile Val Val Leu Ser Asp Ala
Phe Leu Ser Arg Ala Trp Cys Ser His 225 230 235 240 Ser Phe Arg Glu
Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro 245 250 255 Ile Phe
Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala 260 265 270
Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp 275
280 285 Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val
Gln 290 295 300 Leu Ala Leu Pro Arg Lys Val Arg Tyr Arg Pro Val Glu
Gly Asp Pro 305 310 315 320 Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro
Met Leu Ile Leu Arg Gly 325 330 335 Arg Val Pro Glu Gly Arg Ala Leu
Asp Ser Glu Val Asp Pro Asp Pro 340 345 350 Glu Gly Asp Leu Gly Val
Arg Gly Pro Val Phe Gly Glu Pro Ser Ala 355 360 365 Pro Pro His Thr
Ser Gly Val Ser Leu Gly Glu Ser Arg Ser Ser Glu 370 375 380 Val Asp
Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser Ala Arg Thr Asp 385 390 395
400 Phe Tyr Cys Leu Val Ser Lys Asp Asp Met 405 410 11 1229 DNA Mus
musculus 11 atggcaggtg tctgtgacat ggcccctaat ttcctttccc catctgaaga
ccaggccttg 60 ggtcttgccc ttggcagaga agttgctttg aattgcacag
cttgggtgtt ctctaggccc 120 cagtgtcccc agccatcagt gcagtggctg
aaagatggtc tggcattggg caatggaagc 180 cacttcagcc tccatgagga
cttctgggtc agcgccaact tctcagagat tgtgtccagt 240 gtcctggtgc
tcaacttgac caatgcagag gactatggaa ccttcacctg ttctgtctgg 300
aatgtcagct cccattcctt cactctttgg cgagctggcc ctgctggcca tgtggctgca
360 gtactggctt ccctcctggt cctggtggtt ctgctgctgg tggccctgct
ctatgttaag 420 tgtcggctga acatgctgct ttggtaccaa gacacttacg
gggaggtgga gatgaacgat 480 gggaagttat acgatgccta cgtgtcctat
agcgactgcc cagaggaccg caaatttgta 540 aattttattc tgaagcctca
gttggagcgg cgtcggggat acaaactctt cctagaggac 600 cgcgacctct
tgcctcgcgc ggagccctct gccgaccttt tggtgaacct gagtcgctgt 660
cggcgtctca tcgtggttct ttcagatgcc ttcctaagcc ggccctggtg tagccagagc
720 ttccgggagg gactgtgccg cctactggag ctcacccgca gacctatctt
catcaccttt 780 gagggccaga ggcgtgagcc catacaccct gctctccggc
tcctgcgcca gcaccgccac 840 ctcgtgaccc tggtgctttg gaagcctggc
tccgtgactc cttcctctga tttttggaaa 900 gagctacagc tagcactgcc
acgaaggtgc agtacaggcc ggtggaggga gaccctcaaa 960 cccgacttca
ggatgacaaa gatcccatgc taatcgtgag aggacgtgct gcccagggcc 1020
ggggcatgga gtcagagctg gatccagacc ctgagggaga cctgggtgtc cgtggacctg
1080 tctttgggga gccaccaact ccactgcagg aaaccaggat ctgcatagga
gagagccacg 1140 gcagtgaaat ggatgtctct gacctcggct ctcgaaacta
cagtgcacgg acagacttct 1200 actgcctcgt gtctgaggat gatgtgtag 1229 12
409 PRT Mus musculus 12 Met Ala Gly Val Cys Asp Met Ala Pro Asn Phe
Leu Ser Pro Ser Glu 1 5 10 15 Asp Gln Ala Leu Gly Leu Ala Leu Gly
Arg Glu Val Ala Leu Asn Cys 20 25 30 Thr Ala Trp Val Phe Ser Arg
Pro Gln Cys Pro Gln Pro Ser Val Gln 35 40 45 Trp Leu Lys Asp Gly
Leu Ala Leu Gly Asn Gly Ser His Phe Ser Leu 50 55 60 His Glu Asp
Phe Trp Val Ser Ala Asn Phe Ser Glu Ile Val Ser Ser 65 70 75 80 Val
Leu Val Leu Asn Leu Thr Asn Ala Glu Asp Tyr Gly Thr Phe Thr 85 90
95 Cys Ser Val Trp Asn Val Ser Ser His Ser Phe Thr Leu Trp Arg Ala
100 105 110 Gly Pro Ala Gly His Val Ala Ala Val Leu Ala Ser Leu Leu
Val Leu 115 120 125 Val Val Leu Leu Leu Val Ala Leu Leu Tyr Val Lys
Cys Arg Leu Asn 130 135 140 Met Leu Leu Trp Tyr Gln Asp Thr Tyr Gly
Glu Val Glu Met Asn Asp 145 150 155 160 Gly Lys Leu Tyr Asp Ala Tyr
Val Ser Tyr Ser Asp Cys Pro Glu Asp 165 170 175 Arg Lys Phe Val Asn
Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg Arg 180 185 190 Gly Tyr Lys
Leu Phe Leu Glu Asp Arg Asp Leu Leu Pro Arg Ala Glu 195 200 205 Pro
Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu Ile 210 215
220 Val Val Leu Ser Asp Ala Phe Leu Ser Arg Pro Trp Cys Ser Gln Ser
225 230 235 240 Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg
Arg Pro Ile 245 250 255 Phe Ile Thr Phe Glu Gly Gln Arg Arg Glu Pro
Ile His Pro Ala Leu 260 265 270 Arg Leu Leu Arg Gln His Arg His Leu
Val Thr Leu Val Leu Trp Lys 275 280 285 Pro Gly Ser Val Thr Pro Ser
Ser Asp Phe Trp Lys Glu Leu Gln Leu 290 295 300 Ala Leu Pro Arg Lys
Val Gln Tyr Arg Pro Val Glu Gly Asp Pro Gln 305 310 315 320 Thr Arg
Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Val Arg Gly Arg 325 330 335
Ala Ala Gln Gly Arg Gly Met Glu Ser Glu Leu Asp Pro Asp Pro Glu 340
345 350 Gly Asp Leu Gly Val Arg Gly Pro Val Phe Gly Glu Pro Pro Thr
Pro 355 360 365 Leu Gln Glu Thr Arg Ile Cys Ile Gly Glu Ser His Gly
Ser Glu Met 370 375 380 Asp Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser
Ala Arg Thr Asp Phe 385 390 395 400 Tyr Cys Leu Val Ser Glu Asp Asp
Val 405
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