U.S. patent application number 11/848462 was filed with the patent office on 2008-03-06 for uses of il-23 agonists and antagonists; related reagents.
This patent application is currently assigned to Schering Corporation. Invention is credited to Terrill K. McClanahan, Martin Oft.
Application Number | 20080057058 11/848462 |
Document ID | / |
Family ID | 32990805 |
Filed Date | 2008-03-06 |
United States Patent
Application |
20080057058 |
Kind Code |
A1 |
Oft; Martin ; et
al. |
March 6, 2008 |
USES OF IL-23 AGONISTS AND ANTAGONISTS; RELATED REAGENTS
Abstract
Provided are methods of treatment for tumors. In particular,
methods are provided for modulating activity of a cytokine molecule
and its receptor.
Inventors: |
Oft; Martin; (Palo Alto,
CA) ; McClanahan; Terrill K.; (Sunnyvale,
CA) |
Correspondence
Address: |
DNAX RESEARCH INC.;LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation
Kenilworth
NJ
07033
|
Family ID: |
32990805 |
Appl. No.: |
11/848462 |
Filed: |
August 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10797157 |
Mar 9, 2004 |
7282204 |
|
|
11848462 |
Aug 31, 2007 |
|
|
|
60453672 |
Mar 10, 2003 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/158.1; 435/375 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 37/02 20180101; C07K 14/7155 20130101; A61P 9/14 20180101;
A61P 43/00 20180101; A61P 35/00 20180101; A61P 29/00 20180101; C07K
16/244 20130101; C07K 14/54 20130101; A61K 2039/505 20130101; A61K
39/00 20130101; A61P 35/02 20180101; A61P 1/14 20180101 |
Class at
Publication: |
424/133.1 ;
424/139.1; 424/158.1; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C12N 5/00 20060101
C12N005/00 |
Claims
1-18. (canceled)
19. A method of inhibiting tumor growth comprising contacting a
tumor cell with an effective amount of a binding composition
comprising an antigen-binding site of an antibody that specifically
binds to IL-23.
20. The method of claim 19, wherein the binding composition
comprises an antigen-binding site of an antibody that specifically
binds to a polypeptide consisting of residues 1-168 of SEQ ID NO:
2.
21. The method of claim 20, wherein the tumor cell expresses
IL-23.
22. The method of claim 20, wherein the binding composition
comprises a monoclonal antibody or an antigen-binding fragment
thereof.
23. The method of claim 22, wherein the binding composition
comprises a humanized antibody or an antigen-binding fragment
thereof.
24. The method of claim 20, wherein the binding composition
comprises an Fab, Fv, or F(ab').sub.2 antibody fragment.
25. The method of claim 24, wherein the binding composition
comprises an Fab antibody fragment.
26. The method of claim 20, wherein the tumor cell is a colon
cancer cell.
27. The method of claim 20, wherein the tumor cell is an ovarian
cancer cell.
28. The method of claim 20, wherein the tumor cell is a breast
cancer cell.
29. The method of claim 20, wherein the tumor cell is a melanoma
cell.
30. A method of treating a subject suffering from a cancer or tumor
comprising administering to the subject an effective amount of a
binding composition comprising an antigen-binding site of an
antibody that specifically binds to IL-23.
31. The method of claim 30, wherein the binding composition
comprises an antigen-binding site of an antibody that specifically
binds to a polypeptide consisting of residues 1-168 of SEQ ID NO:
2.
32. The method of claim 31, wherein the binding composition
inhibits cachexia.
33. The method of claim 31, wherein the binding composition
inhibits angiogenesis.
34. The method of claim 31, wherein the binding composition
comprises a monoclonal antibody or an antigen-binding fragment
thereof.
35. The method of claim 34, wherein the binding composition
comprises a humanized antibody or an antigen-binding fragment
thereof.
36. The method of claim 31, wherein the binding composition
comprises an Fab, Fv, or F(ab').sub.2 antibody fragment.
37. The method of claim 36, wherein the binding composition
comprises an Fab antibody fragment.
38. The method of claim 31, wherein the cancer or tumor is colon
cancer.
39. The method of claim 31, wherein the cancer or tumor is ovarian
cancer.
40. The method of claim 31, wherein the cancer or tumor is breast
cancer.
41. The method of claim 31, wherein the cancer or tumor is
melanoma.
42. A method of inhibiting tumor growth comprising contacting a
tumor cell with an effective amount of a binding composition
comprising an antigen-binding site of an antibody that specifically
binds to a polypeptide consisting of the sequence of SEQ ID NO:
2.
43. A method of treating a subject suffering from a cancer or tumor
comprising administering to the subject an effective amount of a
binding composition comprising an antigen-binding site of an
antibody that specifically binds to a polypeptide consisting of the
sequence of SEQ ID NO: 2.
Description
[0001] This application is a continuation of co-pending U.S. patent
application Ser. No. 10/797,157, filed Mar. 9, 2004, which claims
benefit from U.S. Provisional Patent Application No. 60/453,672,
filed Mar. 10, 2003, each of which is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention concerns uses of mammalian cytokine
molecules and related reagents. More specifically, the invention
relates to identification of mammalian cytokine-like proteins and
inhibitors thereof that can be used in the treatment of
proliferative disorders.
BACKGROUND OF THE INVENTION
[0003] Cancers and tumors can be controlled or eradicated by the
immune system. The immune system includes several types of lymphoid
and myeloid cells, e.g., monocytes, macrophages, dendritic cells
(DCs), eosinophils, T cells, B cells, and neutrophils. These
lymphoid and myeloid cells produce secreted signaling proteins
known as cytokines. The cytokines include, e.g., interleukin-10
(IL-10), interferon-gamma (IFNgamma), IL-12, and IL-23. 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 can produce pathological consequences, e.g., when it
involves excessive inflammation, as in the autoimmune disorders,
whereas impaired immune response may result in cancer. Anti-tumor
response by the immune system includes innate immunity, e.g., as
mediated by macrophages, NK cells, and neutrophils, and adaptive
immunity, e.g., as mediated by antigen presenting cells (APCs), T
cells, and B cells (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.; von Andrian and Mackay (2000) New Engl.
J. Med. 343:1020-1034; Davidson and Diamond (2001) New Engl. J.
Med. 345:340-350).
[0004] Methods of modulating immune response have been used in the
treatment of cancers, e.g., melanoma. These methods include
treatment with cytokines or anti-cytokine antibodies, such as IL-2,
IL-12, tumor necrosis factor-alpha (TNFalpha), IFNgamma,
granulocyte macrophage-colony stimulating factor (GM-CSF), and
transforming growth factor (TGF). Where a cancer cell can produces
a cytokine that enhance its own growth or its own survival, an
anti-cytokine antibody may be an appropriate therapeutic agent
(see, e.g., Ramirez-Montagut, et al. (2003) Oncogene 22:3180-3187;
Braun, et al. (2000) J. Immunol. 164:4025-4031; Shaw, et al. (1998)
J. Immunol. 161:2817-2824; Coussens and Werb (2002) Nature
420:860-867; Baxevanis, et al. (2000) J. Immunol. 164:3902-3912;
Shimizu, et al. (1999) J. Immunol. 163:5211-5218; Belardelli and
Ferrantini (2002) TRENDS Immunol. 23:201-208; Seki, et al. (2002)
J. Immunol. 168:3484-3492; Casares, et al. (2003) J. Immunol.
171:5931-5939; Oft, et al. (2002) Nature Cell Biol. 4:487-494)
[0005] Interleukin-23 (IL-23) is a heterodimeric cytokine comprised
of two subunits, i.e., p19 and p40. The p19 subunit is structurally
related to IL-6, granulocyte-colony stimulating factor (G-CSF), and
the p35 subunit of IL-12. The p40 subunit of IL-23 is also part of
IL-12, a heterodimeric cytokine comprising p35 and p40. IL-23
mediates signaling by binding to a heterodimeric receptor,
comprised of IL-23R and IL-12beta1. The IL-12beta1 subunit is
shared by the IL-12 receptor, which is composed of IL-12beta1 and
IL-12beta2. A number of early studies demonstrated that the
physiological consequences of a genetic deficiency in p40 (p40
knockout mouse; p40KO mouse; p40.sup.-/- mouse) were different
from, e.g., more severe or less severe, than those found in a p35KO
mouse. Some of these results were eventually explained by the
discovery of IL-23, and the finding that the p40KO prevents
expression of both IL-12 and IL-23 (Oppmann, et al. (2000) Immunity
13:715-725; Wiekowski, et al. (2001) J. Immunol. 166:7563-7570;
Parham, et al. (2002). J Immunol 168, 5699-708; Frucht (2002) Sci
STKE 2002, E1-E3; Elkins, et al. (2002) Infection Immunity
70:1936-1948; Cua, et al. (2003) Nature 421:744-748).
[0006] Present methods for treating cancer are not completely
effective, and cytokines, such as IL-12 or IFNgamma produce toxic
side effects (see, e.g., Naylor and Hadden (2003) Int.
Immunopharmacol. 3:1205-1215; Fernandez, et al. (1999) J. Immunol.
162:609-617). The present invention addresses these problems by
providing methods of using agonists and antagonists of IL-23.
SUMMARY OF THE INVENTION
[0007] The present invention is based upon the discovery that an
agonist or antagonist of IL-23 can modulate tumor growth.
[0008] The present invention provides a method of modulating tumor
growth comprising contacting a tumor cell with an effective amount
of an agonist or antagonist of IL-23. Also provided is the above
method, wherein the antagonist of IL-23 inhibits or prevents tumor
growth; as well as the above method wherein the tumor cell
expresses IL-23. In another aspect, the present invention provides
the above method wherein the agonist or antagonist of IL-23
comprises a binding composition that specifically binds a
polypeptide or nucleic acid of p19 (SEQ ID NOs:1, 2, 3, or 4); or
IL-23R (SEQ ID NOs:5 or 6); or the above method wherein the binding
composition comprises: an antigen-binding site of an antibody; an
extracellular region of IL-23R (SEQ ID NOs:5 or 6); a small
molecule; an anti-sense nucleic acid or small interference RNA
(siRNA); or a detectable label; and the above method wherein the
binding composition comprises: a polyclonal antibody; a monoclonal
antibody; a humanized antibody, or a fragment thereof, an Fab, Fv,
or F(ab').sub.2 fragment; or a peptide mimetic of an antibody.
[0009] Yet another embodiment of the present invention provides a
method of modulating tumor growth comprising contacting a tumor
cell with an effective amount of an agonist or antagonist of IL-23;
wherein the tumor cell is a colon cancer cell; an ovarian cancer
cell; a breast cancer cell; or a melanoma cell.
[0010] In another aspect, the invention provides a method of
treating a subject suffering from a cancer or tumor comprising
administering to the subject an effective amount of an agonist or
antagonist of IL-23; and the above method wherein the antagonist of
IL-23 inhibits: growth of the cancer or tumor; cachexia; anorexia;
or angiogenesis. Also provided is the above method wherein the
antagonist of IL-23 comprises a binding composition that
specifically binds a polypeptide or nucleic acid of: p19 (SEQ ID
NOs:1, 2, 3, or 4) or IL-23R (SEQ ID NOs:5 or 6). Yet another
embodiment of the present invention provides the above method
wherein the binding composition comprises: an antigen-binding site
of an antibody; an extracellular region of IL-23R (SEQ ID NOs:5 or
6); an anti-sense nucleic acid or small interference RNA (siRNA); a
small molecule; or a detectable label; and the above method wherein
the binding composition comprises: a polyclonal antibody; a
monoclonal antibody; a humanized antibody, or a fragment thereof,
an Fab, Fv, or F(ab').sub.2 fragment; or a peptide mimetic of an
antibody.
[0011] In another embodiment, the invention provides the above
method wherein the cancer or tumor is of the: gastrointestinal
tract; respiratory tract; reproductive system; or endocrine system;
as well as the above method wherein the cancer or tumor is: colon
cancer; ovarian cancer; a melanoma; or breast cancer.
[0012] In other aspect of the present invention provides a method
of diagnosis of a cancer or tumor comprising contacting a sample
from a subject with the binding compositions of the above method,
as well as the above method of diagnosis, wherein the binding
composition comprises a nucleic acid probe or primer that
specifically binds or hybridizes to the polynucleotide of SEQ ID
NOs:1, 2, or 5.
[0013] Yet another embodiment of the present invention provides a
kit for the diagnosis of a cancer or tumor comprising the binding
composition of the above method and a compartment or instructions
for use or disposal. Also provided is the above kit wherein the
binding composition comprises an antibody that specifically binds
to p19 (SEQ ID NOs: 1, 2, 3, or 4) or IL-23R (SEQ ID NOs:5 or
6).
DETAILED DESCRIPTION
[0014] 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. All references cited herein are incorporated 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.
I. Definitions.
[0015] "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.
[0016] "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.
[0017] "Administration" and "treatment," as it applies 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-23
agonist or IL-23 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-23
agonist or IL-23 antagonist contacts IL-23 receptor (heterodimer of
IL-23R and IL-12Rbeta1), 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.
[0018] "Binding composition" refers to a molecule, small molecule,
macromolecule, antibody, a fragment or analogue thereof, or soluble
receptor, capable of binding to a target. "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.
"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.
[0019] "Cachexia" is a wasting syndrome involving loss of muscle
(muscle wasting) and fat, resulting from a disorder in metabolism.
Cachexia occurs in various cancers, chronic pulmonary obstructive
disorder (COPD), advanced organ failure, and AIDS. "Cancer
cachexia" is the cachexia that occurs with cancer. Cancer cachexia
is characterized by, e.g., marked weight loss, anorexia, asthenia,
and anemia. Anorexia is a disorder resulting from lack of
motivation to eat, e.g., food aversion (see, e.g., MacDonald, et
al. (2003) J. Am. Coll. Surg. 197:143-161; Rubin (2003) Proc. Natl.
Acad. Sci. USA 100:5384-5389; Tisdale (2002) Nature Reviews Cancer
2:862-871; Argiles, et al. (2003) Drug Discovery Today 8:838-844;
Lelli, et al. (2003) J. Chemother. 15:220-225; Argiles, et al.
(2003) Curr. Opin. Clin. Nutr. Metab. Care 6:401-406).
[0020] "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.
[0021] 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):
(1) Hydrophobic: Norleucine, Ile, Val, Leu, Phe, Cys, or Met;
(2) Neutral hydrophilic: Cys, Ser, Thr;
(3) Acidic: Asp, Glu;
(4) Basic: Asn, Gln, H is, Lys, Arg;
(5) Residues that influence chain orientation: Gly, Pro;
(6) Aromatic: Trp, Tyr, Phe;
(7) Small amino acids: Gly, Ala, Ser.
[0022] "Effective amount" encompasses an amount sufficient to
ameliorate or prevent a symptom or sign of the medical condition.
Effective amount also means an amount sufficient to allow or
facilitate diagnosis. 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 or
parameter 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).
[0023] "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.
[0024] "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.
[0025] "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.
[0026] "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, 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.
[0027] 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.
[0028] 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 Ttzkowitz (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).
[0029] 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.
[0030] 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 (see,
e.g., Rozinov and Nolan (1998) Chem. Biol. 5:713-728).
[0031] "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.
[0032] "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).
[0033] "Specifically" or "selectively" binds, when referring to a
ligand/receptor, antibody/antigen, or other binding pair, indicates
a binding reaction which 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, of the contemplated method binds to its antigen, or a
variant or mutein thereof, 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 with any other antibody, or
binding composition derived thereof. In a preferred embodiment the
antibody will have an affinity that is greater than about 10.sup.9
liters/mol, as determined, e.g., by Scatchard analysis (Munsen, et
al. (1980) Analyt. Biochem. 107:220-239).
II. General.
[0034] The present invention provides methods of using
polypeptides, nucleic acids, variants, muteins, and mimetics of the
IL-23 heterodimer, p19 subunit, p40 subunit, the IL-23 receptor
heterodimer, IL-23R subunit, or IL-12Rbeta1 subunit. Also provided
are methods for using a hyperkine, i.e., a fusion protein
comprising, e.g., the p19 subunit linked to the p40 subunit, as
well as nucleic acids encoding the hyperkine (see, e.g., SEQ ID
NOs:10 or 11) (Oppmann, et al., supra; Fischer, et al. (1997)
Nature Biotechnol. 15:142-145; Rakemann, et al. (1999) J. Biol.
Chem. 274:1257-1266; and Peters, et al. (1998) J. Immunol.
161:3575-3581).
[0035] Interleukin-23 (IL-23; a.k.a. IL-B30) is a heterodimeric
cytokine composed of a novel p19 subunit (SEQ ID NOs: 2 or 4) and
the p40 subunit (SEQ ID NOs: 8 or 9) of IL-12 (Oppmann, et al,
supra). Like p35, p19 requires co-expression of p40 for biological
activity (Wiekowski, et al., supra). The IL-23 receptor comprises a
novel receptor subunit (IL-23R; SEQ ID NO: 6) that binds p19 and
IL-12Rbeta1 (SEQ ID NO: 7) that binds p40 (see, e.g., Parham, et
al. (2002) J. Immunol. 168:5699-5708). These two receptor subunits
form the functional signaling complex and are expressed on
CD4.sup.+CD45Rb.sup.lo memory T cells as well as IFNgamma activated
bone marrow macrophages (Parham, et al., supra).
[0036] Antibodies can be raised to various cytokine proteins,
including individual, polymorphic, allelic, strain, or species
variants, and fragments thereof, both in their naturally occurring
(full-length) forms or in their recombinant forms (see, e.g., SEQ
ID NO: 2, 4, 10, or 11). Additionally, antibodies can be raised to
receptor proteins (see, e.g., SEQ ID NO: 6) in both their native
(or active) forms or in their inactive, e.g., denatured, forms.
Anti-idiotypic antibodies may also be used.
[0037] Administration of an IL-23 agonist, i.e., IL-23 or IL-23
hyperkine, can induce, e.g., proliferation of memory T cells, PHA
blasts, CD45RO T cells, CD45RO T cells; enhance production of
interferon-gamma (IFNgamma) by PHA blasts or CD45RO T cells. In
contrast to IL-12, IL-23 preferentially stimulates memory as
opposed to naive T cell populations in both human and mouse. IL-23
activates a number of intracellular cell-signaling molecules, e.g.,
Jak2, Tyk2, Stat1, Stat2, Stat3, and Stat4. IL-12 activates this
same group of molecules, but Stat4 response to IL-23 is relatively
weak, while Stat4 response to IL-12 is strong (Oppmann, et al.,
supra; Parham, et al. (2002) J. Immunol. 168:5699-5708).
[0038] IL-12 and IL-23 engage similar signal transduction
mechanisms. IL-23 engaging its receptor complex, activates Jak2,
Tyk2, and Stat-1, -3, -4, and -5, as does IL-12. However Stat-4
activation is significantly weaker in response to IL-23 than IL-12.
Also, in contrast to IL-12, the most prominent Stat induced by
IL-23 is Stat-3 (see, e.g., Parham, et al., supra).
[0039] Administration of the p19 subunit of IL-23 can result in,
e.g., stunted growth, infertility, and death of animals, as well as
inflammatory infiltrates, e.g., in the gastrointestinal tract,
lungs, skin, and liver, and epithelial cell hyperplasia, microcytic
anemia, increased neutrophil count, increased serum TNFalpha; and
increased expression of acute phase genes in liver; (Wiekowski, et
al., supra). Enhanced IL-23 expression occurred in immortalized not
transformed epithelial cell lines. Thus, IL-23 may provide an early
signal of tumor potential in vivo.
[0040] Other studies have demonstrated that IL-23 modulates immune
response to infection (see, e.g., Pirhonen, et al. (2002) J.
Immunol. 169:5673-5678; Broberg, et al. (2002) J. Interferon
Cytokine Res. 22:641-651; Elkins, et al. (2002) Infection Immunity
70:1936-1948; Cooper, et al. (2002) J. Immunol. 168:1322-1327).
[0041] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual
indicates a predisposition for the development of the disease, or
can provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. 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. 165:4950-4956).
III. Agonists, Antagonists, and Binding Compositions.
[0042] The present invention provides methods of using agonists and
antagonist of IL-23. An agonist of IL-23 encompasses, e.g., IL-23,
an IL-23 variant, mutein, hyperkine, or peptide mimetic, agonistic
antibodies to IL-23R, and nucleic acids encoding these agonists.
Antagonists of IL-23 include, e.g., antibodies to IL-23, blocking
antibodies to IL-23R, a soluble receptor based on the extracellular
region of a subunit of the IL-23R, peptide mimetics thereto, and
nucleic acids encoding these antagonists.
[0043] The present invention provides methods of using agonists and
antagonists of p19, the complex of p19 and p40, IL-23R, and the
complex of IL-23R and IL-12Rbeta1, including binding compositions
that specifically bind to proteins and protein complexes of p19,
the complex of p19 and p40, IL-23R, and the complex of IL-23R and
IL-12Rbeta1.
[0044] An IL-23 hyperkine encompasses, e.g., a fusion protein
comprising the polypeptide sequence of p19 and p40, where p19 and
p40 occur in one continous polypeptide chain. The sequences of p19
and p40 may be in either order. The fusion protein may contain a
linker sequence, residing in between the sequences of p19 and p40,
in one continuous polypeptide chain.
[0045] Regions of increased antigenicity can be used for antibody
generation. Regions of increased antigenicity of human p19 occur,
e.g., at amino acids 16-28; 57-87; 110-114; 136-154; and 182-186 of
GenBank AAQ89442 (gi:37183284). Regions of increased antigenicity
of human IL-23R occur, e.g., at amino acids 22-33; 57-63; 68-74;
101-112; 117-133; 164-177; 244-264; 294-302; 315-326; 347-354;
444-473; 510-530; and 554-558 of GenBank AAM44229 (gi: 21239252).
Analysis was by a Parker plot using Vector NTI.RTM. Suite
(Informax, Inc, Bethesda, Md.). The present invention also provides
an IL-23 antagonist that is a soluble receptor, i.e., comprising an
extracellular region of IL-23R, e.g., amino acids 1-353 of
GenBankAAM44229, or a fragment thereof, where the extracellular
region or fragment thereof specifically binds to IL-23. Mouse
IL-23R is GenBank NP.sub.--653131 (gi:21362353). Muteins and
variants are contemplated, e.g., pegylation or mutagenesis to
remove or replace deamidating Asn residues.
[0046] 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.).
[0047] 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).
[0048] Antibody to antigen and ligand to receptor binding
properties can be measured, e.g., by surface plasmon resonance
(Karlsson, et al. (1991) J. Immunol. Methods 145:229-240; Neri, et
al. (1997) Nat. Biotechnol. 15:1271-1275; Jonsson, et al. (1991)
Biotechniques 11:620-627) or by competition ELISA (Friguet, et al.
(1985) J. Immunol. Methods 77:305-319; Hubble (1997) Immunol. Today
18:305-306). Antibodies can be used for affinity purification to
isolate the antibody's target antigen and associated bound
proteins, see, e.g., Wilchek, et al. (1984) Meth. Enzymol.
104:3-55.
[0049] 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).
[0050] Soluble receptors comprising the extracellular domains of
IL-23R or IL-12Rbeta1 receptor polypeptides are provided. 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).
IV. Therapeutic Compositions, Methods.
[0051] The present invention provides IL-23 and anti-IL-23R for
use, e.g., in the treatment of proliferative conditions and
disorders, including cancer, tumors, angiogenesis, cachexia, cancer
cachexia, anorexia, and pre-cancerous disorders, e.g., dysplasia.
Nucleic acids are also provided for these therapeutic uses, e.g.,
nucleic acids encoding IL-23 or IL-23R, or an antigenic fragment
thereof, the corresponding anti-sense nucleic acids, and
hybridization products thereof. The invention also provides
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).
[0052] To prepare pharmaceutical or sterile compositions including
an agonist or antagonist of IL-23, the cytokine analogue or mutein,
antibody thereto, or nucleic acid thereof, is admixed with a
pharmaceutically acceptable carrier or excipient, see, e.g.,
Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National
Formulary, Mack Publishing Company, Easton, Pa. (1984).
Formulations of therapeutic and diagnostic agents may be prepared
by mixing with physiologically acceptable carriers, excipients, or
stabilizers in the form of, e.g., lyophilized powders, slurries,
aqueous solutions 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.).
[0053] The route of administration is by, e.g., topical or
cutaneous application, subcutaneous injection, injection or
infusion by intravenous, intraperitoneal, intracerebral,
intramuscular, intraocular, intraarterial, intracerebrospinal,
intralesional, or pulmonary routes, or by sustained release systems
or an implant. Gene transfer vectors, e.g., for the central nervous
system, have been described (see, e.g., Cua, et al. (2001) J.
Immunol. 166:602-608; 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. 6,350,466 and 6,316,024).
[0054] 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).
[0055] 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, intraspinally, 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 basis.
[0056] 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).
[0057] Typical veterinary, experimental, or research subjects
include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs,
horses, and humans.
[0058] 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.
[0059] 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%.
V. Kits and Diagnostic Reagents.
[0060] This invention provides IL-23 proteins, fragments thereof,
nucleic acids, and fragments thereof, in a diagnostic kit. Also
provided are binding compositions, including antibodies or antibody
fragments, for the detection of IL-23 and IL-23 receptor, and
metabolites and breakdown products thereof. Typically, the kit will
have a compartment containing either a p19 polypeptide, or an
antigenic fragment thereof, a binding composition thereto, or a
nucleic acid, e.g., a nucleic acid probe or primer. The nucleic
acid probe or primer specifically hybridizes under stringent
conditions to a nucleic acid encoding p19 or IL-23R.
[0061] The kit can 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 can comprise p19, the complex
of p19 and p40, IL-23R, the complex of IL-23R and IL-12Rbeta1, or
an antigenic fragment thereof, a binding composition, or a nucleic
acid. 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.
[0062] 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).
[0063] This invention provides polypeptides and nucleic acids of
IL-23 and IL-23R, fragments thereof, in a diagnostic kit, e.g., for
the diagnosis of proliferative conditions, cancer, tumors, and
precancerous disorders, e.g., dysplasia.
[0064] Also provided are binding compositions, including antibodies
or antibody fragments, for the detection of p19, the complex of p19
and p40, IL-23R, the complex of IL-23R and IL-12Rbeta1, and
metabolites and breakdown products thereof. Typically, the kit will
have a compartment containing either a IL-23 or IL-23R 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).
[0065] 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 p19, the
complex of p19 and p40, IL-23R, and the complex of IL-23R and
IL-12Rbeta1. 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.
VI. Uses.
[0066] The present invention provides methods for using agonists
and antagonists of IL-23 for the treatment and diagnosis of
inflammatory disorders and conditions, e.g., neoplastic diseases,
cancers, tumors, angiogenesis, precancerous conditions such as
dysplasias, anorexia, cachexia, and cancer cachexia, by modulating
immune response.
[0067] 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; Tzbicki, 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.).
[0068] 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-23, 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 IL-12, 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).
[0069] The present invention provides methods for the treatment and
diagnosis of anorexia and cachexia, including cancer cachexia.
Cachexia is a wasting syndrome that occurs in a number of diseases,
including cancer, e.g., cancer of the lung and upper
gastrointestinal tract. Cachexia occurs in about half of all cancer
patients. Diagnosis of cachexia is by a history of substantial
weight loss, loss of appetite, and profound weakness, in the
context of advanced disease, and muscle wasting (loss of lean body
mass). Cytokines, e.g., IL-6, IL-1, TNFalpha, and IFNgamma, have
been associated with cachexia (see, e.g., MacDonald, et al., supra;
Rubin, supra; Tisdale, supra; Lelli, et al., supra; Argiles, et
al., supra).
[0070] Also provided are methods of treating extramedullary
hematopoiesis (EMH) of cancer. EMH is described (see, e.g., Rao, et
al (2003) Leuk. Lymphoma 44:715-718; Lane, et al. (2002) J. Cutan.
Pathol. 29:608-612).
[0071] The gastrointestinal tract comprises, e.g., the lips, mouth,
esophagus, stomach, small intestines, appendix, large intestines,
colon, anus, and rectum. The respiratory tract comprises, e.g., the
trachea, bronchioles, bronchi, lungs, alveoli. The reproductive
system includes, e.g., the testes, penis, ovaries, uterus,
fallopian tubes. The endocrine system includes, e.g., the
pituitary, hypothalamus, pineal gland, thyroid gland, parathyroid,
endocrine pancreas, islets, gonads, and adrenal gland.
[0072] 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.
[0073] All citations 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.
[0074] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. 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.
EXAMPLES
I. General Methods
[0075] Standard methods in molecular biology are described
(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).
[0076] 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).
Production, purification, and fragmentation of polyclonal and
monoclonal antibodies is 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 Protocols in Immunology, Vol 4, John Wiley, Inc., New
York).
[0077] 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 (Molecular Probes
(2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;
Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).
[0078] 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.).
[0079] Methods for the treatment and diagnosis of cancer are
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, B C 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
Suppl. 8):23-29; Mohr, et al. (2003) Onkologie 26:227-233).
[0080] Software packages and databases for determining, e.g.,
antigenic fragments, leader sequences, protein folding, functional
domains, glycosylation sites, and sequence alignments, are
available (see, e.g., GenBank, Vector NTI.TM. Suite sequence
analysis and data management software (Informax, Inc, Bethesda,
Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.);
DeCypher.RTM. software package (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).
II. Mice and Tumor Induction
[0081] IL-23 p19 deficient mice were generated as described in Cua,
et al., supra. Mice specifically lacking in IL-23 (p19KO mice; p19
knockout mice; p19.sup.-/- mice), p19.sup.+/- mice, and p19.sup.+/+
wild-type control mice, had a B6/129 F2 background.
[0082] Skin tumors were chemically induced in either wild-type (wt)
or IL-23 deficient mice (p19KO mice). Tumors were initiated using
50 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed by
a promotion steps consisting of two treatments of 30 micrograms
each of TPA per week (see, e.g., Oft, et al. (2002) Nat. Cell.
Biol. 4:487-494).
[0083] With tumor studies with Ep2X1B1-nu/nu mice, tumors
metastasize, while cachexia does not occur. The mice die, e.g.,
from extramedullary hematopoiesis (EMH). With tumor studies with
Ep2XB1-Balb/c mice, tumor metastasis does not occur, apparently
because of the intact immune system in these mice.
III. Expression of Subunits of p19 and IL-23R
[0084] Expression of the p19 subunit of IL-23 and the IL-23R
subunit of IL-23 receptor was elevated in a number of cancers,
tumors, and cell lines, e.g., cancer of the gastrointestinal tract,
reproductive tract, skin, and breast (Table 1). TABLE-US-00001
TABLE 1 Expression of subunits of p19 and IL-23R by Taqman .RTM.
real time quantitative PCR analysis, relative to ubiquitin (1.0).
The values are from diseased and adjacent normal tissues, where
indicated. Expression of human p19 normal colon, 4.8 colon stage I,
30.5 adjacent adenocarcinoma normal colon, 2.0 colon stage II, 73.4
adjacent adenocarcinoma normal colon, 0.8 colon stage II, 18.1
adjacent adenocarcinoma normal colon, 0.21 colon stage III, 34.0
adjacent adenocarcinoma normal skin adjacent 2.2 human skin II 21.8
melanoma normal skin adjacent 6.7 human skin II 16.4 nodular
melanoma normal skin adjacent 8.4 human skin II 26.8 nodular
melanoma normal skin adjacent 9.3 human skin II 75.1 superficial
spreading melanoma uterus adjacent 1.6 ovary papillary 55.0 serous
cystadenocarcinoma ovary adjacent 1.9 ovary papillary 17.7 serous
cystadenocarcinoma breast adjacent 8.2 breast IIB carcinoma, 32.0
medullary breast adjacent 0.6 breast IIA 3.1 carcinoma,
infiltrating duct breast adjacent 0.2 breast IIA 3.9 carcinoma,
infiltrating duct Expression of human IL-23R monocyte/PBMC resting
10.0 leukocytes leukemia SR cell line 415.8 leukocytes leukemia
K562 cell line 396.7 leukocytes leukemia MOLT-4 cell line 0.0
leukocytes leukemia HL60 TB cell line 374.1
[0085] RNA from tissues or cell pellets was extracted using
RNeasy.RTM. columns (Qiagen, Valencia, Calif.) and treated with
Dnase I (Promega, Madison, Wis.). cDNA were prepared and used as
templates for quantitative real time PCR. cDNA (25 ng) was analysed
for expression of a range of genes using GeneAmp.RTM. 5700 Sequence
Detection System (Applied Biosystems, Foster City, Calif.).
Analysis of cDNA samples from normal and tumor colon and ovary
tissue was normalized to expression of the housekeeping gene,
ubiquitin.
IV. p19 Antagonists Prevent or Reduce Tumors
[0086] Tumors induced by injected tumor cells or by chemical
carcinogenesis, were eradicated or reduced in mice treated
antagonists to IL-23, e.g., by treatment with anti-p19 antibody, or
by genetic ablation of the p19 subunit (p19KO). p19 is a subunit of
IL-23 only, while p40 is a subunit of both IL-23 and IL-12. In
contrast, treatment with an IL-12, under some conditions,
exacerbated tumors, i.e., resulted in an increase in tumor volume,
relative to control mice.
[0087] Tumors in mice resulted in cancer, cancer cachexia,
extramedullary hematopoiesis, and death. Treatment of tumor-bearing
Balb/c mice with anti-p19 antibody resulted in a halt to increases
in tumor volume, while treatment with anti-p40 antibody provoked
weight gain of the animal, likely a reversal of cachexia, but an
increase in tumor volume (Table 2). TABLE-US-00002 TABLE 2 Tumor
growth in Balb/c mice inoculated with Ep2 (a.k.a. XTb cells) cancer
cells (ras-transformed mouse mammary cells). Tumor size (mm.sup.3)
Antibody treatment Day 1 Day 11 Day 21 Isotype antibody (8D5) 0
mm.sup.3 225 mm.sup.3 500 mm.sup.3 Anti-p19 antibody (29A2) 0 200
250 Anti-p40 antibody (C17.8) 0 250 1150
[0088] Cancer death and cancer cachexia were induced in mice, where
death and weight loss were prevented by anti-p40 antibody. Mice
were injected with 1.times.10.sup.6 EpXT tumor cells (s.c.). Tumor
bearing nude mice (Ep2XB1 nu/nu) died from lethal lung metastasis,
with deaths occurring at from days 22-42 after the injection. Tumor
bearing Exp2XB1 Balb/c mice died at about days 22-49 after the
injection, where the BalbC/c mice died in absence of lung
metastasis. Cachexia was indicated by the decrease in body weight
occurring (prior to death). Progressive weight loss occurred,
starting at about day 16. The initial weight, at day 1 was 22-23
grams, while the weight at death was in the range of 16-18
grams.
[0089] Antibody treatment was with C17.8 rat anti-p40 antibody (1
mg/week). With antibody treatment, the Ep2XB1-Balb/C mice
(immunocompetant mice), survived until about day 64, after which
deaths occurred until day 85. Anti-p40 antibody treatment also
resulted in a maintenance of body weight (at about 17 grams) in
half of the mice, with a progressive increase in body weight of the
remaining mice, to a maximum, within the time frame of the
experiment, of 22-23 grams. Thus, anti-p40 antibody resulted in
improvement in health, according to survival time and regain of
body weight, though anti-p40 could also result in a decline in
health, as shown by an increase in tumor size (Table 2).
[0090] Cancer was chemically induced by treatment with DMBA (50
micrograms) and 2.times.30 micrograms
tetradecanoylphorbol-13-acetate (TPA) per week (Gschwendt, et al.
(1991) Trends Biochem Sci. 16:167-169). Chemical carcinogenesis
treatments were applied to B6/129 wild type mice and to p19KO mice.
Wild type mice readily developed tumors but the p19KO mice did not
acquire tumors (Table 3). TABLE-US-00003 TABLE 3 p19KO Mice Resist
Chemical Carcinogenesis. Initiation with DMBA Initiation with DMBA
(50 micrograms); (50 micrograms); Promotion with TPA (2 .times. 30
Promotion with TPA (2 .times. 30 micrograms/week for 13 weeks).
micrograms/week for 20 weeks). First tumor First tumor occurrence
Tumor number occurrence Tumor number (after TPA) per mouse (after
TPA) per mouse B2/129 8 weeks 11 8 weeks 8 wild type mouse p19KO
None found in 0 None found in 0 mouse examined time examined time
frame. frame.
[0091] Separate studies demonstrated that the p19KO prevented tumor
formation while the p35KO exacerbated tumor formation (Table 4).
TABLE-US-00004 TABLE 4 Influence of p19KO versus p35KO on chemical
carcinogenesis. Average number of tumors per mouse C57/129 wild
type 10.0 p19KO (C57/129) 0.0 C57B/6 wild type 4.5 p35KO (C57/129)
11.0
[0092] Tissue and cell expression of the subunits of IL-23 and
subunits of IL-12 was determined, after carcinogen treatment. DMBA
alone, TPA alone, and DMBA with TPA, induced expression of the p19
subunit of IL-23, these chemicals was applied to the mouse's back.
For example, two days after treatment with DMBA resulted in an
increase in p19 expression from 1.5 (untreated) to 6.3 (at t=2
days). Expression of p40 increased, but was relatively low in this
time interval (0.1 untreated; 0.4 at t=2 days). Five hours after
treatment with TPA resulted in an increase in p19 expression (2.5
control; 15.5 with TPA treatment), but relatively little change in
p40 expression (2.0 control; 3.5 with TPA treatment). Five hours
after treatment with DMBA plus TPA resulted in large increases in
p19 expression (6.0 control; 32.0 DMBA+TPA), but moderate levels of
p40 expression (2.0 control; 4.0 DMBA+TPA).
[0093] Response of human keratinocytes to, e.g., DMBA, TPA, and
lipopolysaccharide (LPS), was also determined (Table 5). TPA
specifically induced p19, with little or no induction of p40, the
common subunit of IL-23 and IL-12. LPS induced p19, indicating a
role in IL-23 in innate response. Toll-like receptors that bind LPS
occur on keratinocytes (see, e.g., Song, et al. (2002) J. Invest.
Dermatol. 119:424-432). Etoposide is an anti-cancer agent that
inhibits topoisomerase II and induces apoptosis (see, e.g.,
Robertson, et al. (2000) J. Biol. Chem. 275:32438-32443; Karpinich,
et al. (2000) J. Biol. Chem. 277:16547-16552). TABLE-US-00005 TABLE
5 Response of Human Keratinocytes to Various Additives. Additive
EBI3 subunit of IL-27 p19 p40 p35 (p28 + EBI3) Control 1.1 N.D. 0.4
0.01 DMBA 1.0 N.D. N.D. N.D. TPA 1.9 N.D. 0.2 1.25 LPS 4.45 0.05
0.35 0.25 Etoposide 2.5 0.4 1.75 0.6 N.D. means not detected.
[0094] Anti-p19 antibodies were tested for their effect on the 4T1
mouse breast cancer cell model. Mice were treated with control
mIgG1 (27F11) antibody or with anti-p19 antibody (29A2). Tumor
growth was monitored on days 1, 3, 4, 5, 6, 7, 8, 9, 10, and 11.
Antibodies (1 mg/dose) were administered on days 2, 5, 8, and 10.
On day 4, the tumor size of the control antibody treated mouse was
about 175 mm.sup.3, while tumor size of the anti-p19 antibody
treated mouse was about 135 mm.sup.3. Thus, anti-p19 antibody is
effective in treating a model of breast cancer. After day 4, tumors
in both groups grew at about the same rate, indicating that the
antibody dose was not sufficient to counteract the IL-23 expressed
by the tumor at later periods in time.
[0095] Histology of the Ep2 mouse breast cancer model demonstrated
co-localization of IL-23R and NK cells, as determined by staining
for p19, which resides bound to IL-23R, and by staining for CD49B,
a marker for NK cells. This co-localization occurred in the central
part of the tumor, i.e., in the necrotic region. Histology of the
Ep2 mouse breast cancer also demonstrated co-localization of p19
and T cells. T cell location was determined by staining for CD3.
This co-localization occurred at the peripheral part of the
tumor.
V. Listing of Sequence Identifiers
[0096] SEQ ID NO: 1 is human IL-23p19 nucleic acid sequence.
[0097] SEQ ID NO: 2 is human IL-23p19 amino acid sequence.
[0098] SEQ ID NO: 3 is mouse IL-23p19 nucleic acid sequence.
[0099] SEQ ID NO: 4 is mouse IL-23p19 amino acid sequence.
[0100] SEQ ID NO: 5 is human IL-23 receptor nucleic acid
sequence.
[0101] SEQ ID NO: 6 is human IL-23 receptor amino acid
sequence.
[0102] SEQ ID NO: 7 is human IL-12Rbeta1 amino acid sequence.
[0103] SEQ ID NO: 8 is human IL-12 p40 amino acid sequence.
[0104] SEQ ID NO: 9 is mouse IL-12 p40 amino acid sequence.
[0105] SEQ ID NO: 10 is mouse IL-23 hyperkine SEQ ID NO: 11 is
human IL-23 hyperkine.
[0106] All citations 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.
[0107] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. 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
11 1 570 DNA Homo sapiens CDS (1)..(567) mat_peptide (64)..(567) 1
atg ctg ggg agc aga gct gta atg ctg ctg ttg ctg ctg ccc tgg aca 48
Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr -20
-15 -10 gct cag ggc aga gct gtg cct ggg ggc agc agc cct gcc tgg act
cag 96 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr
Gln -5 -1 1 5 10 tgc cag cag ctt tca cag aag ctc tgc aca ctg gcc
tgg agt gca cat 144 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala
Trp Ser Ala His 15 20 25 cca cta gtg gga cac atg gat cta aga gaa
gag gga gat gaa gag act 192 Pro Leu Val Gly His Met Asp Leu Arg Glu
Glu Gly Asp Glu Glu Thr 30 35 40 aca aat gat gtt ccc cat atc cag
tgt gga gat ggc tgt gac ccc caa 240 Thr Asn Asp Val Pro His Ile Gln
Cys Gly Asp Gly Cys Asp Pro Gln 45 50 55 gga ctc agg gac aac agt
cag ttc tgc ttg caa agg atc cac cag ggt 288 Gly Leu Arg Asp Asn Ser
Gln Phe Cys Leu Gln Arg Ile His Gln Gly 60 65 70 75 ctg att ttt tat
gag aag ctg cta gga tcg gat att ttc aca ggg gag 336 Leu Ile Phe Tyr
Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85 90 cct tct
ctg ctc cct gat agc cct gtg gcg cag ctt cat gcc tcc cta 384 Pro Ser
Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 95 100 105
ctg ggc ctc agc caa ctc ctg cag cct gag ggt cac cac tgg gag act 432
Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr 110
115 120 cag cag att cca agc ctc agt ccc agc cag cca tgg cag cgt ctc
ctt 480 Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu
Leu 125 130 135 ctc cgc ttc aaa atc ctt cgc agc ctc cag gcc ttt gtg
gct gta gcc 528 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val
Ala Val Ala 140 145 150 155 gcc cgg gtc ttt gcc cat gga gca gca acc
ctg agt ccc taa 570 Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser
Pro 160 165 2 189 PRT Homo sapiens 2 Met Leu Gly Ser Arg Ala Val
Met Leu Leu Leu Leu Leu Pro Trp Thr -20 -15 -10 Ala Gln Gly Arg Ala
Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln -5 -1 1 5 10 Cys Gln
Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His 15 20 25
Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr 30
35 40 Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys Asp Pro
Gln 45 50 55 Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile
His Gln Gly 60 65 70 75 Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp
Ile Phe Thr Gly Glu 80 85 90 Pro Ser Leu Leu Pro Asp Ser Pro Val
Ala Gln Leu His Ala Ser Leu 95 100 105 Leu Gly Leu Ser Gln Leu Leu
Gln Pro Glu Gly His His Trp Glu Thr 110 115 120 Gln Gln Ile Pro Ser
Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu 125 130 135 Leu Arg Phe
Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala 140 145 150 155
Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser Pro 160 165 3 1203
DNA Mus musculus CDS (113)..(700) mat_peptide (176)..(700) 3
cgcttagaag tcggactaca gagttagact cagaaccaaa ggaggtggat agggggtcca
60 caggcctggt gcagatcaca gagccagcca gatctgagaa gcagggaaca ag atg
ctg 118 Met Leu -20 gat tgc aga gca gta ata atg cta tgg ctg ttg ccc
tgg gtc act cag 166 Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu Pro
Trp Val Thr Gln -15 -10 -5 ggc ctg gct gtg cct agg agt agc agt cct
gac tgg gct cag tgc cag 214 Gly Leu Ala Val Pro Arg Ser Ser Ser Pro
Asp Trp Ala Gln Cys Gln -1 1 5 10 cag ctc tct cgg aat ctc tgc atg
cta gcc tgg aac gca cat gca cca 262 Gln Leu Ser Arg Asn Leu Cys Met
Leu Ala Trp Asn Ala His Ala Pro 15 20 25 gcg gga cat atg aat cta
cta aga gaa gaa gag gat gaa gag act aaa 310 Ala Gly His Met Asn Leu
Leu Arg Glu Glu Glu Asp Glu Glu Thr Lys 30 35 40 45 aat aat gtg ccc
cgt atc cag tgt gaa gat ggt tgt gac cca caa gga 358 Asn Asn Val Pro
Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro Gln Gly 50 55 60 ctc aag
gac aac agc cag ttc tgc ttg caa agg atc cgc caa ggt ctg 406 Leu Lys
Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly Leu 65 70 75
gct ttt tat aag cac ctg ctt gac tct gac atc ttc aaa ggg gag cct 454
Ala Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu Pro 80
85 90 gct cta ctc cct gat agc ccc atg gag caa ctt cac acc tcc cta
cta 502 Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser Leu
Leu 95 100 105 gga ctc agc caa ctc ctc cag cca gag gat cac ccc cgg
gag acc caa 550 Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg
Glu Thr Gln 110 115 120 125 cag atg ccc agc ctg agt tct agt cag cag
tgg cag cgc ccc ctt ctc 598 Gln Met Pro Ser Leu Ser Ser Ser Gln Gln
Trp Gln Arg Pro Leu Leu 130 135 140 cgt tcc aag atc ctt cga agc ctc
cag gcc ttt ttg gcc ata gct gcc 646 Arg Ser Lys Ile Leu Arg Ser Leu
Gln Ala Phe Leu Ala Ile Ala Ala 145 150 155 cgg gtc ttt gcc cac gga
gca gca act ctg act gag ccc tta gtg cca 694 Arg Val Phe Ala His Gly
Ala Ala Thr Leu Thr Glu Pro Leu Val Pro 160 165 170 aca gct
taaggatgcc caggttccca tggctaccat gataagacta atctatcagc 750 Thr Ala
175 ccagacatct accagttaat taacccatta ggacttgtgc tgttcttgtt
tcgtttgttt 810 tgcgtgaagg gcaaggacac cattattaaa gagaaaagaa
acaaacccca gagcaggcag 870 ctggctagag aaaggagctg gagaagaaga
ataaagtctc gagcccttgg ccttggaagc 930 gggcaagcag ctgcgtggcc
tgaggggaag ggggcggtgg catcgagaaa ctgtgagaaa 990 acccagagca
tcagaaaaag tgagcccagg ctttggccat tatctgtaag aaaaacaaga 1050
aaaggggaac attatacttt cctgggtggc tcagggaaat gtgcagatgc acagtactcc
1110 agacagcagc tctgtacctg cctgctctgt ccctcagttc taacagaatc
tagtcactaa 1170 gaactaacag gactaccaat acgaactgac aaa 1203 4 196 PRT
Mus musculus 4 Met Leu Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu
Pro Trp Val -20 -15 -10 Thr Gln Gly Leu Ala Val Pro Arg Ser Ser Ser
Pro Asp Trp Ala Gln -5 -1 1 5 10 Cys Gln Gln Leu Ser Arg Asn Leu
Cys Met Leu Ala Trp Asn Ala His 15 20 25 Ala Pro Ala Gly His Met
Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu 30 35 40 Thr Lys Asn Asn
Val Pro Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro 45 50 55 Gln Gly
Leu Lys Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln 60 65 70 75
Gly Leu Ala Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly 80
85 90 Glu Pro Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr
Ser 95 100 105 Leu Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His
Pro Arg Glu 110 115 120 Thr Gln Gln Met Pro Ser Leu Ser Ser Ser Gln
Gln Trp Gln Arg Pro 125 130 135 Leu Leu Arg Ser Lys Ile Leu Arg Ser
Leu Gln Ala Phe Leu Ala Ile 140 145 150 155 Ala Ala Arg Val Phe Ala
His Gly Ala Ala Thr Leu Thr Glu Pro Leu 160 165 170 Val Pro Thr Ala
175 5 2859 DNA Homo sapiens CDS (119)..(2005) mat_peptide
(188)..(2005) 5 gtggtacggg aattccattg tgttgggcag ccaacaaggg
tggcagcctg gctctgaagt 60 ggaattatgt gcttcaaaca ggttgaaaga
gggaaacagt cttttcctgc ttccagac 118 atg aat cak gtc act att caa tgg
gat gca gta ata gcc ctt tac ata 166 Met Asn Xaa Val Thr Ile Gln Trp
Asp Ala Val Ile Ala Leu Tyr Ile -20 -15 -10 ctc ttc agc tgg tgt cat
gga gga att aca aat ata aac tgc tct ggc 214 Leu Phe Ser Trp Cys His
Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly -5 -1 1 5 cac atc tgg gta
gaa cca gcc aca att ttt aag atg ggt atg aat atc 262 His Ile Trp Val
Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 10 15 20 25 tct ata
tat tgc caa gca gca att aag aac tgc caa cca agg aaa ctt 310 Ser Ile
Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 30 35 40
cat ttt tat aaa aat ggc atc aaa gaa aga ttt caa atc aca agg att 358
His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile 45
50 55 aat aaa aca aca gct cgg ctt tgg tat aaa aac ttt ctg gaa cca
cat 406 Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro
His 60 65 70 gct tct atg tac tgc act gct gaa tgt ccc aaa cat ttt
caa gag aca 454 Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe
Gln Glu Thr 75 80 85 ctg ata tgt gga aaa gac att tct tct gga tat
ccg cca gat att cct 502 Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr
Pro Pro Asp Ile Pro 90 95 100 105 gat gaa gta acc tgt gtc att tat
gaa tat tca ggc aac atg act tgc 550 Asp Glu Val Thr Cys Val Ile Tyr
Glu Tyr Ser Gly Asn Met Thr Cys 110 115 120 acc tgg aat gct rgg aag
ctc acc tac ata gac aca aaa tac gtg gta 598 Thr Trp Asn Ala Xaa Lys
Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val 125 130 135 cat gtg aag agt
tta gag aca gaa gaa gag caa cag tat ctc acc tca 646 His Val Lys Ser
Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 140 145 150 agc tat
att aac atc tcc act gat tca tta caa ggt ggc aag aag tac 694 Ser Tyr
Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 155 160 165
ttg gtt tgg gtc caa gca gca aac gca cta ggc atg gaa gag tca aaa 742
Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 170
175 180 185 caa ctg caa att cac ctg gat gat ata gtg ata cct tct gca
gcc gtc 790 Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala
Ala Val 190 195 200 att tcc agg gct gag act ata aat gct aca gtg ccc
aag acc ata att 838 Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro
Lys Thr Ile Ile 205 210 215 tat tgg gat agt caa aca aca att gaa aag
gtt tcc tgt gaa atg aga 886 Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys
Val Ser Cys Glu Met Arg 220 225 230 tac aag gct aca aca aac caa act
tgg aat gtt aaa gaa ttt gac acc 934 Tyr Lys Ala Thr Thr Asn Gln Thr
Trp Asn Val Lys Glu Phe Asp Thr 235 240 245 aat ttt aca tat gtg caa
cag tca gaa ttc tac ttg gag cca aac att 982 Asn Phe Thr Tyr Val Gln
Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 250 255 260 265 aag tac gta
ttt caa gtg aga tgt caa gaa aca ggc aaa agg tac tgg 1030 Lys Tyr
Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 270 275 280
cag cct tgg agt tca ccg ttt ttt cat aaa aca cct gaa aca gtt ccc
1078 Gln Pro Trp Ser Ser Pro Phe Phe His Lys Thr Pro Glu Thr Val
Pro 285 290 295 cag gtc aca tca aaa gca ttc caa cat gac aca tgg aat
tct ggg cta 1126 Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp
Asn Ser Gly Leu 300 305 310 aca gtt gct tcc atc tct aca ggg cac ctt
act tct gac aac aga gga 1174 Thr Val Ala Ser Ile Ser Thr Gly His
Leu Thr Ser Asp Asn Arg Gly 315 320 325 gac att gga ctt tta ttg gga
atg atc gtc ttt gct gtt atg ttg tca 1222 Asp Ile Gly Leu Leu Leu
Gly Met Ile Val Phe Ala Val Met Leu Ser 330 335 340 345 att ctt tct
ttg att ggg ata ttt aac aga tca ttc cga act ggg att 1270 Ile Leu
Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile 350 355 360
aaa aga agg atc tta ttg tta ata cca aag tgg ctt tat gaa gat att
1318 Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp
Ile 365 370 375 cct aat atg aaa aac agc aat gtt gtg aaa atg cta cag
gaa aat agt 1366 Pro Asn Met Lys Asn Ser Asn Val Val Lys Met Leu
Gln Glu Asn Ser 380 385 390 gaa ctt atg aat aat aat tcc agt gag cag
gtc cta tat gtt gat ccc 1414 Glu Leu Met Asn Asn Asn Ser Ser Glu
Gln Val Leu Tyr Val Asp Pro 395 400 405 atg att aca gag ata aaa gaa
atc ttc atc cca gaa cac aag cct aca 1462 Met Ile Thr Glu Ile Lys
Glu Ile Phe Ile Pro Glu His Lys Pro Thr 410 415 420 425 gac tac aag
aag gag aat aca gga ccc ctg gag aca aga gac tac ccg 1510 Asp Tyr
Lys Lys Glu Asn Thr Gly Pro Leu Glu Thr Arg Asp Tyr Pro 430 435 440
caa aac tcg cta ttc gac aat act aca gtt gta tat att cct gat ctc
1558 Gln Asn Ser Leu Phe Asp Asn Thr Thr Val Val Tyr Ile Pro Asp
Leu 445 450 455 aac act gga tat aaa ccc caa att tca aat ttt ctg cct
gag gga agc 1606 Asn Thr Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu
Pro Glu Gly Ser 460 465 470 cat ctc agc aat aat aat gaa att act tcc
tta aca ctt aaa cca cca 1654 His Leu Ser Asn Asn Asn Glu Ile Thr
Ser Leu Thr Leu Lys Pro Pro 475 480 485 gtt gat tcc tta gac tca gga
aat aat ccc agg tta caa aag cat cct 1702 Val Asp Ser Leu Asp Ser
Gly Asn Asn Pro Arg Leu Gln Lys His Pro 490 495 500 505 aat ttt gct
ttt tct gtt tca agt gtg aat tca cta agc aac aca ata 1750 Asn Phe
Ala Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile 510 515 520
ttt ctt gga gaa tta agc ctc ata tta aat caa gga gaa tgc agt tct
1798 Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser
Ser 525 530 535 cct gac ata caa aac tca gta gag gag gaa acc acc atg
ctt ttg gaa 1846 Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr
Met Leu Leu Glu 540 545 550 aat gat tca ccc agt gaa act att cca gaa
cag acc ctg ctt cct gat 1894 Asn Asp Ser Pro Ser Glu Thr Ile Pro
Glu Gln Thr Leu Leu Pro Asp 555 560 565 gaa ttt gtc tcc tgt ttg ggg
atc gtg aat gag gag ttg cca tct att 1942 Glu Phe Val Ser Cys Leu
Gly Ile Val Asn Glu Glu Leu Pro Ser Ile 570 575 580 585 aat act tat
ttt cca caa aat att ttg gaa agc cac ttc aat agg att 1990 Asn Thr
Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn Arg Ile 590 595 600
tca ctc ttg gaa aag tagagctgtg tggtcaaaat caatatgaga aagctgcctt
2045 Ser Leu Leu Glu Lys 605 gcaatctgaa cttgggtttt ccctgcaata
gaaattgaat tctgcctctt tttgaaaaaa 2105 atgtattcac atacaaatct
tcacatggac acatgttttc atttcccttg gataaatacc 2165 taggtagggg
attgctgggc catatgataa gcatatgttt cagttctacc aatcttgttt 2225
ccagagtagt gacatttctg tgctcctacc atcaccatgt aagaattccc gggagctcca
2285 tgccttttta attttagcca ttcttctgcc tmatttctta aaattagaga
attaaggtcc 2345 cgaaggtgga acatgcttca tggtcacaca tacaggcaca
aaaacagcat tatgtggacg 2405 cctcatgtat tttttataga gtcaactatt
tcctctttat tttccctcat tgaaagatgc 2465 aaaacagctc tctattgtgt
acagaaaggg taaataatgc aaaatacctg gtagtaaaat 2525 aaatgctgaa
aattttcctt taaaatagaa tcattaggcc aggcgtggtg gctcatgctt 2585
gtaatcccag cactttggta ggctgaggtr ggtggatcac ctgaggtcag gagttcgagt
2645 ccagcctggc caatatgctg aaaccctgtc tctactaaaa ttacaaaaat
tagccggcca 2705 tggtggcagg tgcttgtaat cccagctact tgggaggctg
aggcaggaga atcacttgaa 2765 ccaggaaggc agaggttgca ctgagctgag
attgtgccac tgcactccag cctgggcaac 2825 aagagcaaaa ctctgtctgg
aaaaaaaaaa aaaa 2859 6 629 PRT Homo sapiens misc_feature
(-21)..(-21) The 'Xaa' at location -21 stands for Gln, or His.
misc_feature (126)..(126) The 'Xaa' at location 126 stands for Gly,
or Arg. 6 Met Asn Xaa Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu
Tyr Ile -20 -15 -10 Leu Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile
Asn Cys Ser Gly -5 -1 1 5 His Ile Trp Val Glu Pro Ala Thr Ile Phe
Lys Met Gly Met Asn Ile 10 15 20 25 Ser Ile Tyr Cys Gln Ala Ala Ile
Lys Asn Cys Gln Pro Arg Lys Leu 30 35 40 His Phe Tyr Lys Asn Gly
Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile 45 50 55 Asn Lys Thr Thr
Ala Arg Leu Trp Tyr Lys Asn Phe
Leu Glu Pro His 60 65 70 Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro
Lys His Phe Gln Glu Thr 75 80 85 Leu Ile Cys Gly Lys Asp Ile Ser
Ser Gly Tyr Pro Pro Asp Ile Pro 90 95 100 105 Asp Glu Val Thr Cys
Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 110 115 120 Thr Trp Asn
Ala Xaa Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val 125 130 135 His
Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 140 145
150 Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr
155 160 165 Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu
Ser Lys 170 175 180 185 Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile
Pro Ser Ala Ala Val 190 195 200 Ile Ser Arg Ala Glu Thr Ile Asn Ala
Thr Val Pro Lys Thr Ile Ile 205 210 215 Tyr Trp Asp Ser Gln Thr Thr
Ile Glu Lys Val Ser Cys Glu Met Arg 220 225 230 Tyr Lys Ala Thr Thr
Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 235 240 245 Asn Phe Thr
Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 250 255 260 265
Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 270
275 280 Gln Pro Trp Ser Ser Pro Phe Phe His Lys Thr Pro Glu Thr Val
Pro 285 290 295 Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp Asn
Ser Gly Leu 300 305 310 Thr Val Ala Ser Ile Ser Thr Gly His Leu Thr
Ser Asp Asn Arg Gly 315 320 325 Asp Ile Gly Leu Leu Leu Gly Met Ile
Val Phe Ala Val Met Leu Ser 330 335 340 345 Ile Leu Ser Leu Ile Gly
Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile 350 355 360 Lys Arg Arg Ile
Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile 365 370 375 Pro Asn
Met Lys Asn Ser Asn Val Val Lys Met Leu Gln Glu Asn Ser 380 385 390
Glu Leu Met Asn Asn Asn Ser Ser Glu Gln Val Leu Tyr Val Asp Pro 395
400 405 Met Ile Thr Glu Ile Lys Glu Ile Phe Ile Pro Glu His Lys Pro
Thr 410 415 420 425 Asp Tyr Lys Lys Glu Asn Thr Gly Pro Leu Glu Thr
Arg Asp Tyr Pro 430 435 440 Gln Asn Ser Leu Phe Asp Asn Thr Thr Val
Val Tyr Ile Pro Asp Leu 445 450 455 Asn Thr Gly Tyr Lys Pro Gln Ile
Ser Asn Phe Leu Pro Glu Gly Ser 460 465 470 His Leu Ser Asn Asn Asn
Glu Ile Thr Ser Leu Thr Leu Lys Pro Pro 475 480 485 Val Asp Ser Leu
Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys His Pro 490 495 500 505 Asn
Phe Ala Phe Ser Val Ser Ser Val Asn Ser Leu Ser Asn Thr Ile 510 515
520 Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn Gln Gly Glu Cys Ser Ser
525 530 535 Pro Asp Ile Gln Asn Ser Val Glu Glu Glu Thr Thr Met Leu
Leu Glu 540 545 550 Asn Asp Ser Pro Ser Glu Thr Ile Pro Glu Gln Thr
Leu Leu Pro Asp 555 560 565 Glu Phe Val Ser Cys Leu Gly Ile Val Asn
Glu Glu Leu Pro Ser Ile 570 575 580 585 Asn Thr Tyr Phe Pro Gln Asn
Ile Leu Glu Ser His Phe Asn Arg Ile 590 595 600 Ser Leu Leu Glu Lys
605 7 862 PRT Homo sapiens 7 Met Ala His Thr Phe Arg Gly Cys Ser
Leu Ala Phe Met Phe Ile Ile 1 5 10 15 Thr Trp Leu Leu Ile Lys Ala
Lys Ile Asp Ala Cys Lys Arg Gly Asp 20 25 30 Val Thr Val Lys Pro
Ser His Val Ile Leu Leu Gly Ser Thr Val Asn 35 40 45 Ile Thr Cys
Ser Leu Lys Pro Arg Gln Gly Cys Phe His Tyr Ser Arg 50 55 60 Arg
Asn Lys Leu Ile Leu Tyr Lys Phe Asp Arg Arg Ile Asn Phe His 65 70
75 80 His Gly His Ser Leu Asn Ser Gln Val Thr Gly Leu Pro Leu Gly
Thr 85 90 95 Thr Leu Phe Val Cys Lys Leu Ala Cys Ile Asn Ser Asp
Glu Ile Gln 100 105 110 Ile Cys Gly Ala Glu Ile Phe Val Gly Val Ala
Pro Glu Gln Pro Gln 115 120 125 Asn Leu Ser Cys Ile Gln Lys Gly Glu
Gln Gly Thr Val Ala Cys Thr 130 135 140 Trp Glu Arg Gly Arg Asp Thr
His Leu Tyr Thr Glu Tyr Thr Leu Gln 145 150 155 160 Leu Ser Gly Pro
Lys Asn Leu Thr Trp Gln Lys Gln Cys Lys Asp Ile 165 170 175 Tyr Cys
Asp Tyr Leu Asp Phe Gly Ile Asn Leu Thr Pro Glu Ser Pro 180 185 190
Glu Ser Asn Phe Thr Ala Lys Val Thr Ala Val Asn Ser Leu Gly Ser 195
200 205 Ser Ser Ser Leu Pro Ser Thr Phe Thr Phe Leu Asp Ile Val Arg
Pro 210 215 220 Leu Pro Pro Trp Asp Ile Arg Ile Lys Phe Gln Lys Ala
Ser Val Ser 225 230 235 240 Arg Cys Thr Leu Tyr Trp Arg Asp Glu Gly
Leu Val Leu Leu Asn Arg 245 250 255 Leu Arg Tyr Arg Pro Ser Asn Ser
Arg Leu Trp Asn Met Val Asn Val 260 265 270 Thr Lys Ala Lys Gly Arg
His Asp Leu Leu Asp Leu Lys Pro Phe Thr 275 280 285 Glu Tyr Glu Phe
Gln Ile Ser Ser Lys Leu His Leu Tyr Lys Gly Ser 290 295 300 Trp Ser
Asp Trp Ser Glu Ser Leu Arg Ala Gln Thr Pro Glu Glu Glu 305 310 315
320 Pro Thr Gly Met Leu Asp Val Trp Tyr Met Lys Arg His Ile Asp Tyr
325 330 335 Ser Arg Gln Gln Ile Ser Leu Phe Trp Lys Asn Leu Ser Val
Ser Glu 340 345 350 Ala Arg Gly Lys Ile Leu His Tyr Gln Val Thr Leu
Gln Glu Leu Thr 355 360 365 Gly Gly Lys Ala Met Thr Gln Asn Ile Thr
Gly His Thr Ser Trp Thr 370 375 380 Thr Val Ile Pro Arg Thr Gly Asn
Trp Ala Val Ala Val Ser Ala Ala 385 390 395 400 Asn Ser Lys Gly Ser
Ser Leu Pro Thr Arg Ile Asn Ile Met Asn Leu 405 410 415 Cys Glu Ala
Gly Leu Leu Ala Pro Arg Gln Val Ser Ala Asn Ser Glu 420 425 430 Gly
Met Asp Asn Ile Leu Val Thr Trp Gln Pro Pro Arg Lys Asp Pro 435 440
445 Ser Ala Val Gln Glu Tyr Val Val Glu Trp Arg Glu Leu His Pro Gly
450 455 460 Gly Asp Thr Gln Val Pro Leu Asn Trp Leu Arg Ser Arg Pro
Tyr Asn 465 470 475 480 Val Ser Ala Leu Ile Ser Glu Asn Ile Lys Ser
Tyr Ile Cys Tyr Glu 485 490 495 Ile Arg Val Tyr Ala Leu Ser Gly Asp
Gln Gly Gly Cys Ser Ser Ile 500 505 510 Leu Gly Asn Ser Lys His Lys
Ala Pro Leu Ser Gly Pro His Ile Asn 515 520 525 Ala Ile Thr Glu Glu
Lys Gly Ser Ile Leu Ile Ser Trp Asn Ser Ile 530 535 540 Pro Val Gln
Glu Gln Met Gly Cys Leu Leu His Tyr Arg Ile Tyr Trp 545 550 555 560
Lys Glu Arg Asp Ser Asn Ser Gln Pro Gln Leu Cys Glu Ile Pro Tyr 565
570 575 Arg Val Ser Gln Asn Ser His Pro Ile Asn Ser Leu Gln Pro Arg
Val 580 585 590 Thr Tyr Val Leu Trp Met Thr Ala Leu Thr Ala Ala Gly
Glu Ser Ser 595 600 605 His Gly Asn Glu Arg Glu Phe Cys Leu Gln Gly
Lys Ala Asn Trp Met 610 615 620 Ala Phe Val Ala Pro Ser Ile Cys Ile
Ala Ile Ile Met Val Gly Ile 625 630 635 640 Phe Ser Thr His Tyr Phe
Gln Gln Lys Val Phe Val Leu Leu Ala Ala 645 650 655 Leu Arg Pro Gln
Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser 660 665 670 Thr Cys
Ala Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro 675 680 685
Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro 690
695 700 Leu Val Ile Ser Glu Val Leu His Gln Val Thr Pro Val Phe Arg
His 705 710 715 720 Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu Lys Gly
Ile Gln Gly His 725 730 735 Gln Ala Ser Glu Lys Asp Met Met His Ser
Ala Ser Ser Pro Pro Pro 740 745 750 Pro Arg Ala Leu Gln Ala Glu Ser
Arg Gln Leu Val Asp Leu Tyr Lys 755 760 765 Val Leu Glu Ser Arg Gly
Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys 770 775 780 Pro Trp Thr Val
Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly Tyr 785 790 795 800 Leu
Pro Ser Asn Ile Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala 805 810
815 Asp Ser Leu Glu Glu Leu Glu Pro Gln His Ile Ser Leu Ser Val Phe
820 825 830 Pro Ser Ser Ser Leu His Pro Leu Thr Phe Ser Cys Gly Asp
Lys Leu 835 840 845 Thr Leu Asp Gln Leu Lys Met Arg Cys Asp Ser Leu
Met Leu 850 855 860 8 328 PRT Homo sapiens 8 Met Cys His Gln Gln
Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro
Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val
Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40
45 Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
50 55 60 Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln
Val Lys 65 70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys
Gly Gly Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys
Lys Glu Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln
Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile
Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly
Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170
175 Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu
Pro Ile 195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr
Glu Asn Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
Pro Asp Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys
Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val
Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val
Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295
300 Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
305 310 315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 9 335 PRT Mus
musculus 9 Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val
Leu Leu 1 5 10 15 Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys
Asp Val Tyr Val 20 25 30 Val Glu Val Asp Trp Thr Pro Asp Ala Pro
Gly Glu Thr Val Asn Leu 35 40 45 Thr Cys Asp Thr Pro Glu Glu Asp
Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60 Arg His Gly Val Ile Gly
Ser Gly Lys Thr Leu Thr Ile Thr Val Lys 65 70 75 80 Glu Phe Leu Asp
Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr 85 90 95 Leu Ser
His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110
Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115
120 125 Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val
Gln 130 135 140 Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser
Ser Ser Pro 145 150 155 160 Asp Ser Arg Ala Val Thr Cys Gly Met Ala
Ser Leu Ser Ala Glu Lys 165 170 175 Val Thr Leu Asp Gln Arg Asp Tyr
Glu Lys Tyr Ser Val Ser Cys Gln 180 185 190 Glu Asp Val Thr Cys Pro
Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205 Ala Leu Glu Ala
Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220 Phe Phe
Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln 225 230 235
240 Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro
245 250 255 Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe
Phe Val 260 265 270 Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr
Glu Glu Gly Cys 275 280 285 Asn Gln Lys Gly Ala Phe Leu Val Glu Lys
Thr Ser Thr Glu Val Gln 290 295 300 Cys Lys Gly Gly Asn Val Cys Val
Gln Ala Gln Asp Arg Tyr Tyr Asn 305 310 315 320 Ser Ser Cys Ser Lys
Trp Ala Cys Val Pro Cys Arg Val Arg Ser 325 330 335 10 531 PRT Mus
musculus 10 Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala Ala Val
Ala Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Leu Met Trp Glu Leu
Glu Lys Asp Val 20 25 30 Tyr Val Val Glu Val Asp Trp Thr Pro Asp
Ala Pro Gly Glu Thr Val 35 40 45 Asn Leu Thr Cys Asp Thr Pro Glu
Glu Asp Asp Ile Thr Trp Thr Ser 50 55 60 Asp Gln Arg His Gly Val
Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr 65 70 75 80 Val Lys Glu Phe
Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly 85 90 95 Glu Thr
Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly 100 105 110
Ile Trp Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu 115
120 125 Lys Cys Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp
Leu 130 135 140 Val Gln Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser
Ser Ser Ser 145 150 155 160 Ser Pro Asp Ser Arg Ala Val Thr Cys Gly
Met Ala Ser Leu Ser Ala 165 170 175 Glu Lys Val Thr Leu Asp Gln Arg
Asp Tyr Glu Lys Tyr Ser Val Ser 180 185 190 Cys Gln Glu Asp Val Thr
Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile 195 200 205 Glu Leu Ala Leu
Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser 210 215 220 Thr Ser
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn 225 230 235
240 Leu Gln Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu
245 250 255 Tyr Pro Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu
Lys Phe 260 265 270 Phe Val Arg Ile Gln Arg Lys Lys Glu Lys Met Lys
Glu Thr Glu Glu 275 280 285 Gly Cys Asn Gln Lys Gly Ala Phe Leu Val
Glu Lys Thr Ser Thr Glu 290 295 300 Val Gln Cys Lys Gly Gly Asn Val
Cys Val Gln Ala Gln Asp Arg Tyr 305 310 315 320 Tyr Asn Ser Ser Cys
Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg 325 330 335 Ser Ser Arg
Gly Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser 340 345 350 Lys
Leu Leu Ala Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln Cys 355 360
365 Gln Gln Leu Ser Arg Asn Leu Cys Met Leu Ala
Trp Asn Ala His Ala 370 375 380 Pro Ala Gly His Met Asn Leu Leu Arg
Glu Glu Glu Asp Glu Glu Thr 385 390 395 400 Lys Asn Asn Val Pro Arg
Ile Gln Cys Glu Asp Gly Cys Asp Pro Gln 405 410 415 Gly Leu Lys Asp
Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly 420 425 430 Leu Val
Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu 435 440 445
Pro Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser Leu 450
455 460 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu
Thr 465 470 475 480 Gln Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp
Gln Arg Pro Leu 485 490 495 Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln
Ala Phe Leu Ala Ile Ala 500 505 510 Ala Arg Val Phe Ala His Gly Ala
Ala Thr Leu Thr Glu Pro Leu Val 515 520 525 Pro Thr Ala 530 11 521
PRT Homo sapiens 11 Met Ser Ala Leu Leu Ile Leu Ala Leu Val Gly Ala
Ala Val Ala Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Leu Ile Trp
Glu Leu Lys Lys Asp Val 20 25 30 Tyr Val Val Glu Leu Asp Trp Tyr
Pro Asp Ala Pro Gly Glu Met Val 35 40 45 Val Leu Thr Cys Asp Thr
Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu 50 55 60 Asp Gln Ser Ser
Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln 65 70 75 80 Val Lys
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly 85 90 95
Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly 100
105 110 Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn
Lys 115 120 125 Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg
Phe Thr Cys 130 135 140 Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr
Phe Ser Val Lys Ser 145 150 155 160 Ser Arg Gly Ser Ser Asp Pro Gln
Gly Val Thr Cys Gly Ala Ala Thr 165 170 175 Leu Ser Ala Glu Arg Val
Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser 180 185 190 Val Glu Cys Gln
Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu 195 200 205 Pro Ile
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn 210 215 220
Tyr Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro 225
230 235 240 Asn Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val
Glu Val 245 250 255 Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His
Ser Tyr Phe Ser 260 265 270 Leu Thr Phe Cys Val Gln Val Gln Gly Lys
Ser Lys Arg Glu Lys Lys 275 280 285 Asp Arg Val Phe Thr Asp Lys Thr
Ser Ala Thr Val Ile Cys Arg Lys 290 295 300 Asn Ala Ser Ile Ser Val
Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser 305 310 315 320 Trp Ser Glu
Trp Ala Ser Val Pro Cys Ser Gly Ser Gly Ser Ser Arg 325 330 335 Gly
Gly Ser Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Lys Leu Arg 340 345
350 Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln Cys Gln Gln Leu
355 360 365 Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser Ala His Pro Leu
Val Gly 370 375 380 His Met Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr
Thr Asn Asp Val 385 390 395 400 Pro His Ile Gln Cys Gly Asp Gly Cys
Asp Pro Gln Gly Leu Arg Asp 405 410 415 Asn Ser Gln Phe Cys Leu Gln
Arg Ile His Gln Gly Leu Ile Phe Tyr 420 425 430 Glu Lys Leu Leu Gly
Ser Asp Ile Phe Thr Gly Glu Pro Ser Leu Leu 435 440 445 Pro Asp Ser
Pro Val Ala Gln Leu His Ala Ser Leu Leu Gly Leu Ser 450 455 460 Gln
Leu Leu Gln Pro Glu Gly His His Trp Glu Thr Gln Gln Ile Pro 465 470
475 480 Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu Leu Arg Phe
Lys 485 490 495 Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val Ala Ala
Arg Val Phe 500 505 510 Ala His Gly Ala Ala Thr Leu Ser Pro 515
520
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