U.S. patent application number 11/353427 was filed with the patent office on 2006-08-24 for methods of treating diseases which are mediated by cutaneous lymphocyte antigen positive cells.
Invention is credited to Janine Bilsborough, Jane A. Gross, Donald Y.M. Leung.
Application Number | 20060188499 11/353427 |
Document ID | / |
Family ID | 36685803 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188499 |
Kind Code |
A1 |
Leung; Donald Y.M. ; et
al. |
August 24, 2006 |
Methods of treating diseases which are mediated by cutaneous
lymphocyte antigen positive cells
Abstract
The present invention relates to methods of treating patients
suffering from itching and puritis mediated by cutaneous lymphocyte
antigen positive T cell. In particular, diseases or disorders
including contact dermatitis, drug induced delayed type cutaneous
allergic reactions, toxic epidermal necrolysis, cutaneous T cell
lymphoma, bullous pemphigoid, alopecia aereata, vitiligo, acne
rosacea, prurigo nodularis, and herpes simplex virus, or
combination thereof will benefit from the administration of an
IL-31 antagonist. The invention also includes methods of predicting
a therapeutically responsive patient population.
Inventors: |
Leung; Donald Y.M.;
(Englewood, CO) ; Bilsborough; Janine; (Seattle,
WA) ; Gross; Jane A.; (Seattle, WA) |
Correspondence
Address: |
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
36685803 |
Appl. No.: |
11/353427 |
Filed: |
February 14, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60653114 |
Feb 14, 2005 |
|
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60716762 |
Sep 13, 2005 |
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60749952 |
Dec 13, 2005 |
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Current U.S.
Class: |
424/143.1 |
Current CPC
Class: |
A61P 17/14 20180101;
A61K 2039/545 20130101; C07K 16/2803 20130101; A61K 39/3955
20130101; C07K 2317/76 20130101; G01N 33/505 20130101; A61P 17/00
20180101; G01N 33/6881 20130101; A61P 17/04 20180101; A61K 49/006
20130101; C07K 14/52 20130101; A61P 31/22 20180101; A61K 49/0008
20130101; C07K 16/244 20130101; A61P 17/10 20180101; G01N 33/6869
20130101; A61P 35/00 20180101; A61K 2039/505 20130101; A61P 37/08
20180101; A61P 43/00 20180101; A61P 17/02 20180101; A61P 37/04
20180101 |
Class at
Publication: |
424/143.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating diseased skin comprising administering an
antagonist molecule to a mammal with the diseased skin wherein the
diseased skin is characterized by cutaneous lymphocyte antigen
positive T cells and the antagonist molecule specifically binds to
the polypeptide comprising the amino acid sequence as shown in SEQ
ID NO:2 or SEQ ID NO: 4, and whereby administration of the
antagonist molecule improves, prevents, inhibits or reduces the
diseased skin.
2. The method according to claim 1, wherein the patient has a skin
disorder selected from Contact dermatitis, Drug induced delayed
type cutaneous allergic reactions, Toxic epidermal necrolysis,
Cutaneous T cell Lymphoma, Bullous pemphigoid, Alopecia aereata,
Vitiligo, Acne Rosacea, Prurigo nodularis, and Herpes simplex
virus.
3. The method according to claim 2, wherein the mammal is a
human.
4. The method according to claim 1, wherein the antagonist is an
antibody or antibody fragment.
5. The method according to claim 4, wherin the antagonist molecule
specifically binds to the polypeptide comprising the amino acid
sequence as shown in SEQ ID NO:2.
6. The method according to claim 1, wherein the diseased skin is
pruritic.
7. A method for treating pruritis comprising administering an
antagonist molecule to a mammal with the pruritis wherein the
pruritis is characterized by cutaneous lymphocyte antigen positive
T cells and wherein the antagonist molecule specifically binds to
the polypeptide having the amino acid sequence as shown in SEQ ID
NO:2 or in SEQ ID NO: 4, and whereby administration of the
antagonist molecule improves, prevents, inhibits or reduces the
pruritis.
8. The method according to claim 7, wherein the pruritis is
associated with a skin disorder selected from Contact dermatitis,
Drug induced delayed type cutaneous allergic reactions, Toxic
epidermal necrolysis, Cutaneous T cell Lymphoma, Bullous
pemphigoid, Alopecia aereata, Vitiligo, Acne Rosacea, Prurigo
nodularis, and Herpes simplex virus.
9. The method according to claim 8, wherein the mammal is a
human.
10. The method according to claim 7, wherein the antagonist is an
antibody or antibody fragment.
11. The method according to claim 10, wherin the antagonist
molecule specifically binds to the polypeptide comprising the amino
acid sequence as shown in SEQ ID NO:2.
12. A method for predicting therapeutic response to an IL-31
antagonist in an individual in need of IL-31 antagonist therapy
comprising obtaining a biological sample from the patient,
isolating circulating cutaneous lymphocyte positive T cells from
the biological sample, and detecting IL-31 production from the
isolated cutaneous lymphocyte positive T cells.
13. The method according to claim 12, wherein the IL-31 is detected
by specifically binding to an IL-31 antagonist.
14. The method according to claim 13, wherein the IL-31 antagonist
is an anti-IL-31 antibody or antibody fragment.
15. The method according to claim 12, wherin the antagonist
molecule specifically binds to the polypeptide comprising the amino
acid sequence as shown in SEQ ID NO:2.
16. The method according to claim 12, wherein the individual in
need of IL-31 antagonist therapy has a skin disorder selected from
Contact dermatitis, Drug induced delayed type cutaneous allergic
reactions, Toxic epidermal necrolysis, Cutaneous T cell Lymphoma,
Bullous pemphigoid, Alopecia aereata, Vitiligo, Acne Rosacea,
Prurigo nodularis, and Herpes simplex virus.
17. The method according to claim 10, comprising the additional
step of stimulating or activating the cutaneous lymphocyte antigen
positive T cells.
18. The method according to claim 17, wherein the IL-31 is detected
by specifically binding to an IL-31 antagonist.
19. The method according to claim 17, wherein the IL-31 antagonist
molecule is an anti-IL-31 antibody or antibody fragment.
20. The method according to claim 19, wherin the antagonist
molecule specifically binds to the polypeptide comprising the amino
acid sequence as shown in SEQ ID NO:2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 60/653,114, filed Feb. 14, 2005, U.S.
Provisional Application Ser. No. 60/716,762, filed Sep. 13, 2005,
and U.S. Provisional Application Ser. No. 60/749,952, filed Dec.
13, 2005, all of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The skin plays an important role in the immune system and
consists of layers. Circulating T lymphocytes migrate to the skin
under normal and inflammatory conditions. The cutaneous lymphocyte
antigen (CLA) is considered a homing receptor for T cells with
tropism for the skin. Santamaria-Babi, L., Eur. J. Dermatol.
14:13-18, 2004. CLA is a carbohydrate structure which is expressed
on memory T cells as an epitope of the single cell-surface protein
named P-selectin glycoprotein ligand-1 (PSGL-1) and facilitates
binding of T cells to E-selectin, an inducible adhesion molecule
expressed on vascular endothelium. See Fuhlbrigge R C, et al.,
Nature 1997; 389:978-81.
[0003] Several diseases of the skin are known to express high
levels of CLA+ T cells, including atopic dermatitis, contact
dermatitis, drug-induced allergic reactions, skin-tropic viruses
and viral associated pruritis, vitiligo, cutaneous T cell lymphoma,
alopecia aerata, acne rosacea, acne vulgaris, prurigo nodularis,
and bullous pemphigoid. There is a need to treat such skin T cell
mediated diseases.
[0004] The demonstrated in vivo activities of cytokines illustrate
the enormous clinical potential of, and need for, other cytokines,
cytokine agonists, and cytokine antagonists. The present invention
addresses these needs by providing a method of treating such
diseases by interfering with the actions of IL-31, a newly
identified cytokine. IL-31, when over-expressed in mice, results
initching and dermatitis-like symptoms. Both skin-homing T cells
and epidermal keratinocytes have been implicated in the pathology
of skin diseases in humans.
[0005] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
SUMMARY OF THE INVENTION
[0006] Within one aspect, the invention provides a method of
treating diseased skin comprising administering an antagonist
molecule to a mammal with the diseased skin wherein the diseased
skin is characterized by cutaneous lymphocyte antigen positive T
cells and the antagonist molecule specifically binds to the
polypeptide comprising the amino acid sequence as shown in SEQ ID
NO:2 or SEQ ID NO: 4, and whereby administration of the antagonist
molecule improves, prevents, inhibits or reduces the diseased skin.
Within an embodiment, the patient has a skin disorder selected from
Contact dermatitis, Drug induced delayed type cutaneous allergic
reactions, Toxic epidermal necrolysis, Cutaneous T cell Lymphoma,
Bullous pemphigoid, Alopecia aereata, Vitiligo, Acne Rosacea,
Prurigo nodularis, and Herpes simplex virus. Within a further
embodiment, the mammal is a human. Within another embodiment, the
antagonist is an antibody or antibody fragment. Within a further
emobodiment the antagonist molecule specifically binds to the
polypeptide comprising the amino acid sequence as shown in SEQ ID
NO:2. Within another embodiment, the the diseased skin is
pruritic.
[0007] Within another aspect, the invention provides a method for
treating pruritis comprising administering an antagonist molecule
to a mammal with the pruritis wherein the pruritis is characterized
by cutaneous lymphocyte antigen positive T cells and wherein the
antagonist molecule specifically binds to the polypeptide having
the amino acid sequence as shown in SEQ ID NO:2 or in SEQ ID NO: 4,
and whereby administration of the antagonist molecule improves,
prevents, inhibits or reduces the pruritis. Within an embodiment,
the the pruritis is associated with a skin disorder selected from
Contact dermatitis, Drug induced delayed type cutaneous allergic
reactions, Toxic epidermal necrolysis, Cutaneous T cell Lymphoma,
Bullous pemphigoid, Alopecia aereata, Vitiligo, Acne Rosacea,
Prurigo nodularis, and Herpes simplex virus. Within an further
embodiement, the the mammal is a human. Within a further
embodiment, the antagonist is an antibody or antibody fragment.
Within a further embodiment, the antagonist molecule specifically
binds to the polypeptide comprising the amino acid sequence as
shown in SEQ ID NO:2.
[0008] Within another aspect, the invention provides a method for
predicting therapeutic response to an IL-31 antagonist in an
individual in need of IL-31 antagonist therapy comprising obtaining
a biological sample from the patient, isolating circulating
cutaneous lymphocyte positive T cells from the biological sample,
and detecting IL-31 production from the isolated cutaneous
lymphocyte positive T cells. Within an embodiment, the IL-31 is
detected by specifically binding to an IL-31 antagonist. Within a
further embodiment, the IL-31 antagonist is an anti-IL-31 antibody
or antibody fragment. Within another embodiment, the antagonist
molecule specifically binds to the polypeptide comprising the amino
acid sequence as shown in SEQ ID NO:2. Within another embodiment,
the individual in need of IL-31 antagonist therapy has a skin
disorder selected from Contact dermatitis, Drug induced delayed
type cutaneous allergic reactions, Toxic epidermal necrolysis,
Cutaneous T cell Lymphoma, Bullous pemphigoid, Alopecia aereata,
Vitiligo, Acne Rosacea, Prurigo nodularis, and Herpes simplex
virus. Within another embodiment, the method comprises the
additional step of stimulating or activating the cutaneous
lymphocyte antigen positive T cells. Within a further embodiment,
the IL-31 is detected by specifically binding to an IL-31
antagonist. Within another embodiment, the IL-31 antagonist
molecule is an anti-IL-31 antibody or antibody fragment. Within a
further embodiment, the antagonist molecule specifically binds to
the polypeptide comprising the amino acid sequence as shown in SEQ
ID NO:2.
DETAILED DESCRIPTION OF THE INVENTION
[0009] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0010] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:79524, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0011] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0012] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0013] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0014] The term "complements of a polynucleotide molecule" denotes
a polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to
5'CCCGTGCAT 3'.
[0015] The term "contig" denotes a polynucleotide that has a
contiguous stretch of identical or complementary sequence to
another polynucleotide. Contiguous sequences are said to "overlap"
a given stretch of polynucleotide sequence either in their entirety
or along a partial stretch of the polynucleotide.
[0016] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0017] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0018] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0019] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e., greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0020] The term "neoplastic", when referring to cells, indicates
cells undergoing new and abnormal proliferation, particularly in a
tissue where in the proliferation is uncontrolled and progressive,
resulting in a neoplasm. The neoplastic cells can be either
malignant, i.e., invasive and metastatic, or benign.
[0021] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0022] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0023] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0024] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0025] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0026] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' noncoding regions of genes.
[0027] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0028] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions, include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0029] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0030] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0031] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0032] All references cited herein are incorporated by reference in
their entirety.
[0033] The present invention provides novel methods of using IL-31
polynucleotides, polypeptides, and antagonists in detection,
diagnosis, and treatment of diseases, in particular, diseases that
are mediated by cutaneous lymphocyte antigen (CLA) positive T
cells. The present invention is based in part upon the discovery
that a previously identified cytokine, IL-31 is expressed by
skin-homing T cells, but not gut-homing T cells.
[0034] IL-31 is a recently discovered protein having the structure
of a four-helical-bundle cytokine. This cytokine was previously
identified as IL-31 and is fully described in U.S. patent
application Ser. No. 10/352,554, filed Jan. 21, 2003. See published
U.S. Patent Application No. 2003-0224487, and PCT application WO
03/060090, all herein incorporated by reference. See also, Dillon,
et al., Nature Immunol. 5:752-760, 2004. IL-31 is a ligand with
high specificity for the receptor IL-31RA and at least one
additional subunit comprising OncostatinM receptor beta (OSMRbeta).
The native polynucleotide and polypeptide sequences for human IL-31
are shown in SEQ ID NOs: 1 and 2, respectively. The native
polynucleotide and polypeptide sequences for mouse IL-31 are shown
in. SEQ ID NOs: 3 and 4, respectively. The native polynucleotide
and polypeptide sequences for human. IL-31RA are shown in SEQ ID
NOs: 5 and 6, respectively. The native polynucleotide and
polypeptide sequences for mouse IL-31RA are shown in SEQ ID NOs: 7
and 8, respectively. The native polynucleotide and polypeptide
sequences for human OSMRbeta are shown in SEQ ID NOs: 9 and 10,
respectively.
[0035] The secretory signal sequence of IL-31 is comprised of amino
acid residues 1 (Met) to 23 (Ala), and the mature polypeptide is
comprised of amino acid residues 24 (Ser) to 164 (Thr) as shown in
SEQ ID NO:2. Further N-terminal sequencing analysis of purified
IL-31 from 293T cells showed an N-terminus at residue 27 (Leu) as
shown in SEQ ID NO:2, with the mature polypeptide comprised of
amino acid residues 27 (Leu) to 164 (Thr) as shown in SEQ ID
NO:2.
[0036] As used herein the term, IL-31 means Zcytor17lig, and IL-3
IRA means Zcytor17, as used in U.S. patent publication number
20030224487 (herein incorporated by reference), as shown above. The
heterodimeric receptor for IL-31 was also described in 2003-0096339
(also incorportated herein by reference) as zcytor17 (HUGO name,
IL-31RA) which form a heterodimer with at least one additional
subunit comprising OncostatinM receptor beta (OSMRbeta).
[0037] Both skin-homing T cells and epidermal kerationcytes have
been implicated in the pathology of skin diseases in humans. As
shown herein, IL-31 mRNA and protein expression is restricted to
the skin-homing CLA+ T cell population in both atopic dermatitis
(AD) patients and normal individuals, while analysis of the
receptor for IL-31, IL-31RA, by immunohistochemistry (IHC) suggests
slightly higher levels of IL-31RA expression on skin keratinocytes
in skin biopsies from AD sufferers compared to normal
individuals.
[0038] When over-expressed in mice, IL-31 results in pruritus and
the development of skin dermatitis resembling human atopic
dermatitis (AD). Immunohistochemistry (IHC) studies shown herein
show that IL-31RA protein was expressed by skin keratinocytes and
infiltrating macrophages in skin biopsies from AD patients.
Comparisons between AD patients and normal individuals suggested
that IL-31RA was expressed at higher levels on epidermal
keratinocytes in the AD samples. Skin cell infiltrates, which were
present at greater numbers in skin of AD patients compared to
normal individuals, expressed IL-31 mRNA. Histomorphometric
analysis of these cells suggested a lymphocytic lineage with the
majority of cells staining positive for cutaneous lymphocyte
antigen (CLA) and CD3, demonstrating that skin-homing T cells in
skin express IL-31 mRNA. Upon analysis of peripheral blood T cells
for IL-31, IL-31 mRNA and protein expression is largely restricted
to CD45RO+ CLA+ skin-homing T cells in AD and normal volunteers.
Moreover, circulating CLA+ T cells from AD patients are capable of
producing higher levels of IL-31 compared to CLA+ T cells from
normal individuals, though there is large variability between
patient samples. These results provide strong evidence that IL-31
expression may contribute to the development of AD skin
inflammation and pruritus.
[0039] T As shown herein, IL-31 is produced both locally in the
skin and by skin infiltrating cells. Local production of cytokines
in tissues by T cells is thought to be a key mechanism for disease
pathogenesis in AD and increased numbers of T cells both in
circulation and in skin is thought to correlate with disease.
[0040] Although both AD patients and normal controls have
circulating CLA+ T cells that express IL-31 upon activation, CLA+ T
cells from AD patients are reported to exist in a more activated
state compared to cells from normal individuals. See Akdis M, J
Immunol 159:4611-4619,1997. Consequently, the threshold of
stimulation required for the production of IL-31 by CLA+ T cells
may differ between dermatitis patients and control subjects. As
shown herein, circulating CLA+ T cells from AD patients after 24
hours of stimulation with sub-optimal concentrations of anti-CD3 in
the absence of anti-CD28 have the capacity to produce higher levels
of IL-31 compared to cells from normal individuals. Due to the
variability in IL-31 levels produced by CLA+ T cells from
individual AD patients, there was no significant difference in the
average IL-31 production from circulating CLA+ T cells of AD and
normal individuals. Nevertheless, since more CLA+ T cells are
localized in skin of AD patients, as compared to normal
individuals, there is an increased potential for IL-31 activity in
the AD skin micro-environment.
[0041] Example 8 demonstrates that circulating CLA+ T cells from
some AD patients produce higher levels of IL-311 compared to cells
from normal individuals. The detection of IL-31 in patients of such
a subpopulation using the bioassay provided herein, or with any
assay that detects IL-31 produced by circulating T cells in the
blood, may be useful to determine if an IL-31 antagonist will be
useful as treatment for diseases wherein the presence of IL-31
causes inflammation.
[0042] A cell line that is dependent on the OSMRbeta and IL-31RA
linked pathway for survival and growth in the absence of other
growth factors can be used to measure the activity of IL-31. Such
growth factor-dependent cell lines include BaF3, FDC-P1, and MO7e.
For information on the BaF3 cell line, see Palacios and Steinmetz,
(Cell 41: 727-734, 1985) and Mathey-Prevot et al., (Mol. Cell.
Biol. 6: 4133-4135, 1986). For information on the FDC-P1cell line,
see Hapel et al. (Blood 64: 786-790, 1984). For information on the
MO7e cell line, see Kiss et al., (Leukemia 7: 235-240, 1993).
[0043] The amino acid sequence for the OSMR, and IL-31RA receptors
indicated that the encoded receptors belonged to the Class I
cytokine receptor subfamily that includes, but is not limited to,
the receptors for IL-2, IL-4, 1L-7, Lif, IL-12, IL-15, EPO, TPO,
GM-CSF and G-CSF (for a review see, Cosman, "The Hematopoietin
Receptor Superfamily" in Cytokine 5(2): 95-106, 1993). The IL-31RA
receptor is fully described in PCT Patent Application No.
US01/20484 (WIPO publication No. WO 02/00721). Analysis of the
tissue distribution of the mRNA of the IL-31RA receptor revealed
expression in activated CD4+ and CD8+ T-cell subsets, CD14+
monocytes, and weaker expression in CD19+ B-cells. Moreover, the
mRNA was present in both resting or activated monocytic cell lines
THP-1 (ATCC No. TIB-202), U937 (ATCC No. CRL-1593.2) and HL60 (ATCC
No. CCL-240).
[0044] IL-31 is considered a four-alpha helix structure. Referring
to the human IL-31 amino acid sequence shown in SEQ ID NO:2, the
IL-31 helix A is defined by amino acid residues 38-52; helix B by
amino acid residues 83-98; helix C by amino acid residues 104-117;
and helix D by amino acid residues 137-152, and the conserved
cysteine residues within IL-31 correspond to amino acid residues
72, 133, and 147 of SEQ ID NO:2; and 74, 137, and 151 of SEQ ID
NO:8 described herein. Also highly conserved in the IL-31 is the
Glu residue as shown in SEQ ID NO:2 at residue 43.
[0045] The polynucleotide sequence for the mouse ortholog of IL-31
has been identified and is shown in SEQ ID NO:3 and the
corresponding amino acid sequence shown in SEQ ID NO:4. For the
IL-31 mouse cytokine amino acid sequence of SEQ ID NO: 4, helix A
is defined by amino acid residues 38-52; helix B by amino acid
residues 85-98; helix C by amino acid residues 104-118; and helix D
by amino acid residues 141-157. Mature sequence for the mouse IL-31
putatively begins at Met.sub.1, as shown in SEQ ID NO:4, which
corresponds to Met.sub.1, as shown in SEQ ID NO:2, in the human
sequence. Tissue analysis revealed that expression of mouse IL-31
is found in testis, brain, CD90+ cells, prostate cells, salivary
gland and skin. Further N-terminal sequencing analysis of purified
IL-31 from 293T cells showed an N-terminus at residue 31 (Ala) as
shown in SEQ ID NO:4 with the mature polypeptide comprising amino
acid residues 31 (Ala) to 163 (Cys).
[0046] IL-31 is located at the 12q24.31 region of chromosome 12.
Thus, the present invention also provides reagents which will find
use in diagnostic applications. For example, the IL-31 gene, a
probe comprising IL-31 DNA or RNA or a subsequence thereof, can be
used to determine if the IL-31 gene is present on a human
chromosome, such as chromosome 12, or if a gene mutation has
occurred. Detectable chromosomal aberrations at the IL-31 gene
locus include, but are not limited to, aneuploidy, gene copy number
changes, loss of heterozygosity (LOH), translocations, insertions,
deletions, restriction site changes and rearrangements. Such
aberrations can be detected using polynucleotides of the present
invention by employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, and other
genetic linkage analysis techniques known in the art (Sambrook et
al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65,
1995). Detection of chromosomal aberrations may be particularly
important for diseases with a high correlation of cutaneous
lymphocyte antigen. Thus, the present invention includes methods of
detecting changes in the IL-31 gene, including up or down
regulations thereof.
[0047] The proteins of the present invention (or polypeptide
fragments thereof) can be joined to other bioactive molecules,
particularly other cytokines, to provide multi-functional
molecules. For example, one or more helices from IL-31 can be
joined to other cytokines to enhance their biological properties or
efficiency of production.
[0048] The present invention also provides the use of detecting
polypeptide fragments or peptides comprising an epitope-bearing
portion of a IL-31 polypeptide described herein in diseases
mediated by CLA positive T cells. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0049] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies (e.g.,
neutralizing antibodies) that bind with the polypeptides described
herein. Hopp/Woods hydrophilicity profiles can be used to determine
regions that have the most antigenic potential (Hopp et al., 1981,
ibid. and Hopp, 1986, ibid.). For example, in human IL-31,
hydrophilic regions include amino acid residues 54-59 of SEQ ID
NO:2, amino acid residues 129-134 of SEQ ID NO:2, amino acid
residues 53-58 of SEQ ID NO:2, amino acid residues 3540 of SEQ ID
NO:2, and amino acid residues 33-38 of SEQ ID NO:2. For example, in
mouse IL-31, hydrophilic regions include amino acid residues 34-39
of SEQ ID NO:4, amino acid residues 46-51 of SEQ ID NO:4, amino
acid residues 131-136 of SEQ ID NO:4, amino acid residues 158-163
of SEQ ID NO:4, and amino acid residues 157-162 of SEQ ID NO:4.
[0050] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fourteen amino acids, or about fourteen to about thirty amino
acids of SEQ ID NO:2 or SEQ ID NO:4. Such epitope-bearing peptides
and polypeptides can be produced by fragmenting a IL-31
polypeptide, or by chemical peptide synthesis, as described herein.
Moreover, epitopes can be selected by phage display of random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin.
Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.
7:616 (1996)). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an epitope
are described, for example, by Mole, "Epitope Mapping," in Methods
in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992); Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0051] The IL-31 polypeptides of the present invention, including
full-length polypeptides, functional fragments, and fusion
polypeptides, can be produced, purified and refolded by methods
well-known in the art and as described in published U.S. Patent
Application No. 2003-0224487, and PCT application WO 03/060090. It
is preferred to purify the polypeptides of the present invention to
.gtoreq.80% purity, more preferably to .gtoreq.90% purity, even
more preferably .gtoreq.95% purity, and particularly preferred is a
pharmaceutically pure state, that is greater than 99.9% pure with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents. Preferably, a purified polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin.
[0052] The present invention provides methods for using IL-31
antagonists, including anti-IL-31 antibodies for reducing,
inhibiting, or preventing inflammation in cell microenvironments
where one or more cells in the microenvironment is/are T cells that
are positive for the cutaneous lymphocyte antigen. In addition the
present invention provides methods for using IL-31 antagonists,
including anti-IL-31 antibodies for reducing, inhibiting, or
preventing itching and pruritis in cell microenvironments where one
or more cells in the microenvironment is/are T cells that are
positive for the cutaneous lymphocyte antigen.
[0053] Antibodies from an immune response generated by inoculation
of an animal with IL-31 antigens can be isolated and purified are
know in the art and are described herein. Methods for preparing and
isolating polyclonal and monoclonal antibodies are well known in
the art. See, for example, Current Protocols in Immunology,
Cooligan, et al. (eds.), National Institutes of Health, John Wiley
and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989;
and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies:
Techniques and Applications, CRC Press, Inc., Boca Raton, Fla.,
1982.
[0054] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting non-human CDRs onto human framework
and constant regions, or by incorporating the entire non-human
variable domains (optionally "cloaking" them with a human-like
surface by replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized antibodies may
retain non-human residues within the human variable region
framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced. Moreover, human antibodies can
be produced in transgenic, non-human animals that have been
engineered to contain human immunoglobulin genes as disclosed in
WIPO Publication No. WO 98/24893. It is preferred that the
endogenous immunoglobulin genes in these animals be inactivated or
eliminated, such as by homologous recombination.
[0055] Antibodies are considered to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and 2) they do
not significantly cross-react with related polypeptide molecules. A
threshold level of binding is determined if anti-IL-31 antibodies
herein bind to a IL-31 polypeptide, peptide or epitope with an
affinity at least 10-fold greater than the binding affinity to
control (non-IL-31) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (K.sub.a) of 10.sup.6
M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci.
51: 660-672, 1949).
[0056] Antibodies to IL-31 may be used for tagging cells that
express IL-31; for isolating IL-31 by affinity purification; for
diagnostic assays for determining circulating levels of IL-31
polypeptides; for detecting or quantitating soluble IL-31 as a
marker of underlying pathology or disease; in analytical methods
employing FACS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies or as
antagonists to block IL-31 activity in vitro and in vivo. Suitable
direct tags or labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent
markers, magnetic particles and the like; indirect tags or labels
may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to IL-31 or fragments thereof may be used in vitro to
detect denatured IL-31 or fragments thereof in assays, for example,
Western Blots or other assays known in the art.
[0057] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria, toxin, saporin, Pseudomonas exotoxin, ricin,
abrin and the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0058] Binding polypeptides can also act as IL-31 "antagonists" to
block IL-31 binding and signal transduction in vitro and in vivo.
These anti-IL-31 binding polypeptides would be useful for
inhibiting IL-31 activity or protein-binding.
[0059] Both skin-homing T cells and epidermal keratinocytes have
been implicated in the pathology of skin diseases in humans. As
shown in Example 1 herein, of the T cell subsets, IL-31 mRNA and
protein expression is restricted to the skin-homing CLA+ T cell
population in humans. As such, an antagonist to IL-31, including an
antibody or receptor antagonist will be useful in treating skin and
epidermal diseases which are mediated by CLA+ T cells. Such
diseases include, for example, atopic dermatitis, contact
dermatitis, psoriasis, drug-induced allergic reactions, skin-tropic
viruses and viral associated pruritis, vitiligo, cutaneous T cell
lymphoma, alopecia aerata, acne rosacea, acne vulgaris, prurigo
nodularis, and bullous pemphigoid.
[0060] Atopic Dermatitis
[0061] Atopic dermatitis (AD) is a chronically relapsing
inflammatory skin disease with a dramatically increasing incidence
over the last decades. Clinically AD is characterized by highly
pruritic often excoriated plaques and papules that show a chronic
relapsing course. The diagnosis of AD is mostly based on major and
minor clinical findings. See Hanifin J. M., Arch Dermatol: 135,
1551 (1999). Histopathology reveals spongiosis, hyper and focal
parakeratosis in acute lesions, whereas marked epidermal
hyperplasia with hyper and parakeratosis,
acanthosis/hypergranulosis and perivascular infiltration of the
dermis with lymphocytes and abundant mast cells are the hallmarks
of chronic lesions.
[0062] T cells play a central role in the initiation of local
immune responses in tissues and evidence suggests that
skin-infiltrating T cells in particular, may play a key role in the
initiation and maintenance of disregulated immune responses in the
skin. Approximately 90% of infiltrating T cells in cutaneous
inflammatory sites express the cutaneous lymphocyte-associated Ag
(CLA+) which binds E-selectin, an inducible adhesion molecule on
endothelium (reviewed in Santamaria-Babi L. F., et al., Eur J
Dermatol: 14, 13, (2004)). A significant increase in circulating
CLA+ T cells among AD patients compared with control individuals
has been documented (See Teraki Y., et al., Br J Dermatol: 143, 373
(2000)), while others have demonstrated that memory CLA+ T cells
from AD patients preferentially respond to allergen extract
compared to the CLA- population (See Santamaria-Babi, L. F., et
al., J Exp Med: 181, 1935, (1995)). In humans, the pathogenesis of
atopic disorders of the skin have been associated with increases in
CLA+ T cells that express increased levels of Th-2-type cytokines
like IL-5 and IL-13 9, 10. See Akdis M., et al., Eur J Immunol: 30,
3533 (2000); and Hamid Q., et al., J Allergy Clin Immunol: 98, 225
(1996).
[0063] NC/Nga Mice spontaneously develop AD-like lesions that
parallel human AD in many aspects, including clinical course and
signs, histophathology and immunopathology when housed in
non-specified pathogen-free (non-SPF) conditions at around 6-8
weeks of age. In contrast, NC/Nga mice kept under SPF conditions do
not develop skin lesions. However, onset of spontaneous skin
lesions and scratching behaviour can be synchronized in NC/Nga mice
housed in a SPF facility by weekly intradermal injection of crude
dust mite antigen. See Matsuoka H., et al., Allergy: 58, 139
(2003). Therefore, the development of AD in NC/Nga is a useful
model for the evaluation of novel therapeutics for the treatment of
AD.
[0064] In addition to the NC/Nga model of spontaneous AD,
epicutaneous sensitization of mice using OVA can also be used as a
model to induce antigen-dependent epidermal and dermal thickening
with a mononuclear infiltrate in skin of sensitized mice. This
usually coincides with elevated serum levels of total and specific
IgE, however no skin barrier dysfunction or pruritus normally
occurs in this model. See Spergel J. M., et al., J Clin Invest,
101: 1614, (1998). This protocol can be modified in order to induce
skin barrier disregulation and pruritis by sensitizing DO11.10 OVA
TCR transgenic mice with OVA. Increasing the number of
antigen-specific T cells that could recognize the sensitizing
antigen may increase the level of inflammation in the skin to
induce visible scratching behaviour and lichenification/scaling of
the skin.
[0065] Both the NC/Nga spontaneous AD model and the OVA
epicutaneous DO11.10 model are used to investigate expression of
IL-31 and IL-31RA in AD. See Example 3.
[0066] An IL-31 neutralizing antagonist could be effective in
inhibiting, reducing, minimizing or preventing atopic dermatitis
reactions.
[0067] Contact Dermatitis
[0068] Allergic contact dermatitis is defined as a T cell mediated
immune reaction to an antigen that comes into contact with the
skin. The CLA+ T cell population is considered to be involved in
the initiation of dermatitis since allergen dependent T cell
responses are largely confined to the CLA+ population of cells (See
Santamaria-Babi, L. F., et al., J Exp Med:181, 1935, (1995)).
Recent data has found that only memory (CD45RO+) CD4+ CLA+ and not
CD8+ T cells proliferate and produce both type-I (IFN-.gamma.) and
type-2 (IL-5) cytokines in response to nickel, a common contact
hypersensitivity allergen. Furthermore, cells expressing CLA in
combination with CD4, CD45RO (memory) or CD69 are increased after
nickel-specific stimulation and express the chemokine receptors
CXCR3, CCR4, CCR10 but not CCR6. See Moed H., et al., Br J
Dermatol:51, 32, (2004).
[0069] In animal models, it has been demonstrated that allergic
contact dermatitis is T-cell dependent and that the
allergic-responsive T cells migrate to the site of allergen
application. See generally: Engeman T. M., et al., J Immunol: 164,
5207, (2000); Ferguson T. A. & Kupper T. S. J Immunol: 150,
1172, (1993); and Gorbachev, A. V. & Fairchild R. L. Crit Rev
Immunol: 21, 451(2001). Since CLA+ T cells produce IL-31 and IL-31
stimulation of skin keratinocytes can induce pro-inflammatory
chemokines, IL-31 may be involved in the pathophysiology of contact
dermatitis. See Example 2 for an in vivo model of contact
dermatitis.
[0070] An IL-31 neutralizing antagonist could be effective in
inhibiting, reducing, minimizing or preventing contact dermatitis
reactions.
[0071] Drug-Induced Delayed Type Cutaneous Allergic Reactions
[0072] Drug-induced delayed type cutaneous allergic reactions are
very heterogeneous and may mirror many distinct pathophysiological
events. See Brockow K., et al., Allergy: 57, 45 (2002).
Immunological mechanisms involved in these reactions have been
shown as either antibody or cell mediated. In immediate drug
allergy an IgE-mediated antibody reaction can be demonstrated by a
positive skin prick and/or intradermal test after 20 min, whereas
non-immediate reactions to drugs can occur more than one hour after
last drug intake and are often T-cell mediated. Non-immediate
T-cell mediated delayed type reactions can occur in patients with
adverse drug reactions to penicillins for example. Proliferative T
cell responses to penicillins have been shown to be restricted to
the memory (CD45RO+) CLA+ subpopulation of T cells from penicillin
allergic patients whereas the CD45RO+CLA-subset shows no
proliferative response. See Blanca M., Leyva L., et al., Blood
Cells Mol Dis:31, 75 (2003). Delayed-type hypersensitivity (DTH)
reactions can be artificially reproduced in mice, allowing
assessment of factors that may be involved in the initiation and
perpetuation of the DTH response. An IL-31 neutralizing antagonist
could be effective in inhibiting, reducing, minimizing or
preventing delayed type hypersensitivity reactions. See Example 4
for an in vivo model of DTH.
[0073] Toxic epidermal necrolysis (TEN) is a very rare but
extremely severe drug reaction characterized by widespread
apoptosis of epidermis with extensive blisters. Studies have shown
that lymphocytes infiltrating the blister are CLA+ T cells and can
exhibit cytotoxicity towards epidermal keratinocytes. See Leyva L.,
et al.,. J Allergy Clin Immunol: 105, 157 (2000); and Nassif A.,
Bensussan A., et al., J Allergy Clin Immunol: 114, 1209 2004). A
transgenic mouse system, whereby OVA is expressed under the control
of the keratin-5 (K5) promoter in the epidermal and hair follicular
keratinocytes of mice, has been generated to establish an animal
model for TEN. OVA specific CD8+ T cells, when adoptively
transferred into K5-OVA mice, undergo activation and proliferation
in the skin-draining lymph nodes and target the skin of K5-OVA
mice, resulting in development of skin lesions that are reminiscent
of TEN. See Azukizawa H., et al., Eur J Immunol: 33, 1879 (2003).
An IL-31 neutralizing antagonist could be effective in inhibiting,
reducing, minimizing or preventing TEN reactions.
[0074] Bullous Pemphigoid
[0075] Bullous pemphigoid is a subepidermal disorder which
manifests as subepidermal blisters with a dermal infiltrate of
neutrophils and eosinophils. Diagnosis is characterized by the
presence of antigen-specific antibodies against specific adhesion
proteins of the epidermis and dermal-epidermal junction. See Jordon
R. E., et al., JAMA: 200, 751 (1967). Studies analyzing the role of
T cells in the pathogenesis of bullous pemphigoid by analysis of
PBL and skin blister T cells have found a predominance of CLA+ T
cells expressing increased levels of Th2-cytokines like IL-4 and
IL-13. See Teraki Y., et al., J Invest Dermatol: 117, 1097 (2001).
In bullous pemphigoid patients following therapy with systemic
corticosteroids, the frequency of CLA+, but not CLA-,
interleukin-13-producing cells is significantly decreased.
Decreases in CLA+ cells following corticosteroid treatment is
associated with clinical improvement. See Teraki, ibid.
Neutralization of IL-31 may improve clinical outcome of bullous
pemohigoid. An IL-31 neutralizing antagonist could be effective in
inhibiting, reducing, minimizing or preventing bullous
pemphigoid.
[0076] Alopecia Greata
[0077] Alopecia greata (AA) is regarded as a tissue-restricted
autoimmune disease of hair follicles in which follicular activity
is arrested because of the continued activity of lymphocytic
infiltrates. AA results in patches of complete hair loss anywhere
on the body, though actual loss of hair follicles does not occur,
even in hairless lesions. Although clinical signs of inflammation
are absent, skin biopsies from sites of active disease show
perifollicular lymphocytic inflammation of primarily CD4+ cells,
along with a CD8+ intrafollicular infiltrate. See Kalish R. S.
& Gilhar A. J Investig Dernatol Symp Proc: 8, 164 (2003).
[0078] Studies have shown that scalp skin infiltrating CD4+ or CD8+
lymphocytes express CLA and, in peripheral blood of individuals
with AA, the percent of CLA+CD4+ or CD8+ lymphocytes is
significantly higher than that of normal controls. Furthermore,
patients with severe or progressive AA show a much higher
CLA-positivity compared to patients recovering from the disease and
a decrease in percent CLA+ cells parallels a good clinical course.
See Yano S., et al., Acta Derm Venereol: 82, 82 (2002). These
studies therefore suggest that CLA+ lymphocytes may play an
important role in AA. Xenograft models have demonstrated that
activated T cells are likely to play a role in the pathogenesis of
AA. Lesional scalp from AA patients grafted onto nude mice regrows
hair coincident with a loss of infiltrating lymphocytes from the
graft and, transfer of activated lesional T cells to SCID mice can
transfer hair loss to human scalp explants on SCID mice. See Kalish
R. S. & Gilhar A. J Investig Dermatol Symp Proc: 8, 164
(2003).
[0079] A variety of immunomodulating therapies are part of the
usual treatment for this disorder however none of these treatments
have been consistent in their efficacy. See Tang L., et al., J
Invest Dermatol: 120, 400 (2003); Tang L., et al., (2004); and Tang
L., et al., J Am Acad Dermatol: 49, 1013 (2003). Neutralizing
anti-IL-31 antibody may be effective to limit, reduce, inhbit, or
prevent the effects of the development of AA.
[0080] Acne Vulgaris/Acne Rosacea
[0081] Acne vulgaris, a disorder of the pilosebaceous apparatus, is
the most common skin problem of adolescence. Abnormalities in
follicular keratinization are thought to produce the acne lesion.
Acne rosacea is differentiated from acne vulagaris by the presence
of red papules, pustules, cysts and extensive telangiectasias, but
the absence of comedones (white heads). Increased sebum excretion
from sebaceous glands is a major factor in the pathophysiology of
acne vulgaris. Other sebaceous gland functions are also associated
with the development of acne, including sebaceous proinflammatory
lipids; different cytokines produced locally; periglandular
peptides and neuropeptides, such as corticotrophin-releasing
hormone, which is produced by sebocytes; and substance P, which is
expressed in the nerve endings at the vicinity of healthy-looking
glands of acne patients. See Zouboulis C. C. Clin Dermatol: 22, 360
(2004).
[0082] Although the pathophysiology of acne vulgaris and acne
rosacea remains unknown, clinical observations and histopathologic
studies suggest that inflammation of the pilosebaceous follicle may
be central to the pathogenesis of rosacea and acne vulgaris. Early
studies on analysis of T cell subsets infiltrating rosacea legions
indicated that the majority of T cells expressed CD4. See Rufli T.
& Buchner S. A. Dermatologica: 169, 1 (1984).
[0083] CD4+ T cells produce IL-31 and IHC analysis of skin for
IL-31 expression suggests that IL-31 is expressed in sebaceous and
sweat glands. L-31 stimulation of epidermal keratinocytes induces
expression of chemokines which likely results in cellular
infiltration suggesting that IL-31 may contribute to the
pro-inflammatory response in skin. IL-31 may therefore contribute
to the pathophysiology of acne rosacea and acne vulgaris.
Neutralization of IL-31 may improve clinical outcome of acne
vulgaris and acne rosacea. An IL-31 neutralizing antagonist could
be effective in inhibiting, reducing, minimizing or preventing acne
vulgaris and acne rosacea.
[0084] Prurigo Nodularis
[0085] Prurigo nodularis is an eruption of lichenified or
excoriated nodules caused by intractable pruritus that is difficult
to treat. While chronic rubbing results in lichenification, and
scratching in linear excoriations, individuals who pick and gouge
at their itchy, irritated skin tend to produce markedly thickened
papules known as prurigo nodules. Although prurigo nodularis is not
specific to atopic dermatitis, many patients with these nodules
also have an atopic reaction, which manifests as allergic rhinitis,
asthma, or food allergy. T cells represent the majority of
infiltrating cells in prurigo lesions and these lesions often
represents the most pruritic skin lesion in atopy patients.
[0086] Topical treatment of prurigo nodularis with capsaicin, an
anti-pruritic alkaloid that interferes with the perception of
pruritis and pain by depletion of neuropeptides like substance P in
small sensory cutaneous nerves, has proven to be an effective and
safe regimen resulting in clearing of the skin lesions. See Stander
S., et al., J Am Acad Dermatol: 44, 471 (2001). Studies of the itch
response in NC/Nga mice using capsaicin treatment showed that the
spontaneous development of dermatitis lesions was almost completely
prevented. Furthermore, the elevation of serum IgE levels was
significantly suppressed and infiltrating eosinophils and mast cell
numbers in lesional skin of capsaicin treated mice were reduced.
See Mihara K., et al., Br J Dermatol: 151, 335 (2004). The
observations from this group suggest that scratching behaviour
might contribute to the development of dermatitis by enhancing
various immunological responses, therefore implying that prevention
of the itch sensation and/or itch-associated scratching behaviour
might be an effective treatment for AD. See Mihara K., et al., Br J
Dermatol: 151, 335 (2004).
[0087] Chronic delivery of IL-31 induces pruritis and alopecia in
mice followed by the development of skin lesions resembling
dermatitis suggesting that IL-31 induces itching. See See Dillon S.
R., et al., Nat Immunol: 5, 752 (2004). Neutralization of IL-31 in
IL-31 treated mice to prevent pruritis and alopecia was tested in
Example 10. Neutralization of IL-31 may improve clinical outcome of
prurigo nodularis. An IL-31 neutralizing antagonist could be
effective in inhibiting, reducing, minimizing or preventing prurigo
nodularis.
[0088] Skin-Tropic Viruses and Viral Associated Pruritis
[0089] Herpes Simplex Virus (HSV)-specific CD8+ T cells in the
peripheral blood and HSV-specific CD8+ T cells recovered from
herpes lesions express high levels of CLA whereas non-skin-tropic
herpes virus-specific CD8+ T cells lack CLA expression. See Koelle
D. M., et al., J Clin Invest: 110, 537 (2002). HSV-2 reactive CD4+
T lymphocytes also express CLA, but at levels lower than those
previously observed for CD8+ T lymphocytes. See Gonzalez J. C., et
al., J Infect Dis: 191, 243 (2005). Pruritis has also been
associated with herpes viral infections (See Hung K. Y., et al.,
Blood Purif. 16, 147 (1998), though other viral diseases, like HIV,
have also been associated with pruritic skin lesions. Severe,
intractable pruritus, often associated with erythematopapular skin
lesions and hypereosinophilia, is a condition observed in some
nonatopic, HIV-infected patients 36. See Singh F. & Rudikoff D,
Am J Clin Dermatol; 4, 177 (2003); and Milazzo F., Piconi S., et
al., Allergy: 54, 266 (1999).
[0090] The association of skin-tropic viruses with pruritis and
CLA+ T cells suggests that IL-31 producing T cells may be involved
in the pathophysiology of viral infections. Thus, an IL-31
neutralizing antagonist could be effective in inhibiting, reducing,
minimizing or preventing viarl associated pruritis, and
neutralization of IL-31 may improve clinical outcome of viral
associated pruritis.
[0091] IL-31 has been shown to induce several chemokine and
cytokine genes in normal human epidermal ketatinocytes (NHEKs),
including genes encoding GRO.alpha., (CXCL1), TARC (CCl17),
MIP3.beta., (CCL19), MDC (CCL22), MIP-3 (CCL23), MIP-1 .beta.
(CCL4), and I-309. See Dillon S. R., et al., Nat Immunol: 5, 752
(2004). TARC and MDC bind CCR4, a chemokine receptor associated
with Th2-type T cells and predominantly expressed by CLA+ T cells
in peripheral blood. Both chemokines have been implicated in the
recruitment of T cells into the skin of AD patients suggesting that
these chemokines contribute to the inflammatory process associated
with the pathogenesis of AD. See Example 9 for a model to measure
the reduction in TARC and MDC levels in CLA+ T cell mediated
disease by administering an IL-31 antagonist.
[0092] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. IL-31RA
polypeptides, soluble heterodimeric and multimeric receptor
polypeptides, or anti-IL-31 antibodies or binding partners of the
present invention, and the like, could serve as a valuable
therapeutic to reduce inflammation and pathological effects in
psoriasis, other inflammatory skin diseases, skin and mucosal
allergies, and related diseases.
[0093] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0094] Using methods known in the art, and disclosed herein, one of
skill could readily detect IL-31 in diseases that have a high
correlation of CLA+ T cells. Such methods involve taking a
biological sample from a patient, such as blood, saliva, or biopsy,
and comparing it to a normal control sample. Histological,
cytological, flow cytometric, biochemical and other methods can be
used to determine the relative levels or localization of IL-31, or
cells expressing IL-31, i.e., monocytes, in the patient sample
compared to the normal control. A change in the level (increase or
decrease) of IL-31 expression, or a change in number or
localization of monocytes (e.g., increase or infiltration of
monocytic cells in tissues where they are not normally present)
compared to a control would be indicative of disease. Such
diagnostic methods can also include measuring TARC and MDC, for
example. Such methods are well known in the art and disclosed
herein.
[0095] L-31 polypeptides that bind IL-31RA receptor polypeptides,
and antibodies thereto are useful to antagonize or block signaling
via IL-31RA-comprising receptors in the treatment of atopic
dermatitis, contact dermatitis, drug induced delayed type cutaneous
allergic reactions, toxic epidermal necrolysis, cutaneous T cell
lymphoma, bullous pemphigoid, alopecia greata, vitiligo, acne
rosacea, prurigo nodularis, and Herpes simplex virus.
[0096] L-31 may also be used within diagnostic systems for the
detection of circulating levels of ligand, and in the detection of
diseases that are mediated by CLA+ T cells. IL-31 may also be used
within diagnostic systems for the detection of circulating levels
of ligand, and in the detection of diseases that have a high
correlation of CLA+ T cells. Within a related embodiment,
antibodies or other agents that specifically bind to IL-31 can be
used to detect circulating IL-31 polypeptides; conversely, IL-31
itself can be used to detect circulating or locally-acting receptor
polypeptides. Elevated or depressed levels of ligand or receptor
polypeptides may be indicative of pathological conditions,
including inflammation and pruritis.
[0097] Generally, the dosage of administered IL-31 antibody will
vary depending upon such factors as the patient's age, weight,
height, sex, general medical condition and previous medical
history. One skilled in the art can readily determine such dosages,
and adjustments thereto, using methods known in the art.
[0098] Administration of an anti-IL-31 antibody to a subject can be
topical, intradermal, as an inhalant, intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering therapeutic proteins by
injection, the administration may be by continuous infusion or by
single or multiple boluses.
[0099] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising IL-31 can be prepared and inhaled with the aid of
dry-powder dispersers, liquid aerosol generators, or nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al.,
Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated
by the AERX diabetes management system, which is a hand-held
electronic inhaler that delivers aerosolized insulin into the
lungs. Studies have shown that proteins as large as 48,000 kDa have
been delivered across skin at therapeutic concentrations with the
aid of low-frequency ultrasound, which illustrates the feasibility
of trascutaneous administration (Mitragotri et al., Science 269:850
(1995)). Transdermal delivery using electroporation provides
another means to administer a molecule having IL-31 binding
activity (Potts et al., Pharm. Biotechnol. 10:213 (1997)).
[0100] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having IL-31 binding activity can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing
Company 1995).
[0101] For purposes of therapy, molecules having IL-31 binding
activity and a pharmaceutically acceptable carrier are administered
to a patient in a therapeutically effective amount. A combination
of a protein, polypeptide, or peptide having IL-31 binding activity
and a pharmaceutically acceptable carrier is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. An agent is
physiologically significant if its presence results in a detectable
change in the physiology of a recipient patient. For example, an
agent used to treat inflammation is physiologically significant if
its presence alleviates at least a portion of the inflammatory
response. Similalry, an agent used to treat itching and pruritis
associated with a disease mediated CLA+ T cells, or a disease with
a high correlation of CLA+ Tcells, is physiologically significant
if its presence alleviates at least a portion of the pruritic or
itch response.
[0102] A pharmaceutical composition comprising an IL-31 antiobdy
can be furnished in liquid form, in an aerosol, or in solid form.
Liquid forms, are illustrated by injectable solutions, aerosols,
droplets, topological solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms. The latter form is illustrated by miniosmotic pumps and
implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade,
"Implants in Drug Delivery," in Drug Delivery Systems, Ranade and
Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,
"Protein Delivery with Infusion Pumps," in Protein Delivery:
Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum
Press 1997); Yewey et al., "Delivery of Proteins from a Controlled
Release Injectable Implant," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
Other solid forms include creams, pastes, other topological
applications, and the like.
[0103] Polypeptides having IL-31 binding activity can be
encapsulated within liposomes using standard techniques of protein
microencapsulation (see, for example, Anderson et al., Infect.
Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et
al. "Preparation and Use of Liposomes in Immunological Studies," in
Liposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page
317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124
(1987)). As noted above, therapeutically useful liposomes may
contain a variety of components. For example, liposomes may
comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,
Biochim. Biophys. Acta 1150:9 (1993)).
[0104] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0105] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0106] Determination of Human Primary T Cell Types that Express
IL-31 Upon Stimulation
[0107] A. Selection of Study Subjects and Biopsies
[0108] Twelve patients with AD (moderate to severe disease; median
age was 32 years old with skin involvement of 5-45%), 6 patients
with psoriasis (median age was 56 years old with skin involvement
of 10-65%) and 12 healthy individuals (median age 34 years) were
included in A study after informed consent. None of the patients
had received any systemic corticosteroids previously. All patients
were off topical corticosteroids for one week before their skin
biopsy or blood drawing. Two mm punch biopsies were taken from 1)
acute erythematous AD lesions of less than three days' onset, 2)
chronic, lichenified AD lesions of greater than two weeks'
duration, 3) chronic psoriasis lesions, and 4) normal skin. The
skin samples were immediately frozen at -70.degree. C. for
immunohistochemistry or Western and immuno-dot blotting.
[0109] B. Isolation and Activation of Primary Human T Cell
Subsets:
[0110] To isolate various T cell subsets, human PBMCs from the
donors were isolated using standard Ficoll gradient centrifugation.
Total T cells were then isolated using the T Cell Isolation Kit II
(Miltenyi Biotec) according to the manufacturer's instructions.
Separation efficiency was assessed using standard flow cytometry
and determined to be >95% T cells. To separate CD45RA+ "naive" T
cells from the CD45RO+"memory" T cells, the total T cell population
was incubated with anti-CD45RO microbeads (Miltenyi Biotec) for 15
minutes at +4.degree. C. and magnetically separated according to
the manufacturers instructions. The naive and memory T cell
populations were determined to be >90% pure by flow
cytometry.
[0111] CD45RO+ memory T cells are often tissue specific and
cutaneous lymphocyte antigen (CLA) is used to differentiate
skin-homing T cells from gut-homing T cells expressing
.alpha.4/.beta.7 on their surface. To determine which of these cell
types produce IL-31, CLA+ T cells were isolated from total T cells,
activated and conditioned media was collected for the IL-31
bioassay. To do this, total T cells were isolated and then
incubated on ice for 20 minutes in 1 mL of a 1:50 dilution of
anti-CLA-FITC antibody (PharMingen). Cells were then washed,
resuspended in MACS buffer and incubated with anti-FITC microbeads
(Miltenyi Biotec) for 15 minutes at +4.degree. C. The cells were
then washed, resuspended and magnetically separated over an LS
column according to the manufacturers instructions. The labeled T
cells were later determined to be >80% pure while the
CLA-depleted T cells were >98% CLA-. Both CLA+ and CLA- T cells
were collected and cultured concurrently.
[0112] To activate the CD45RA+ and CD45RO+ T cell subsets, cells
were cultured overnight in 24-well tissue culture plates pretreated
with 2.0 .mu.g/mL anti-CD3 antibody (Southern Biotechnology). The
cells were plated at a concentration of 2.5.times.10.sup.6 cells/mL
in tissue culture media (RPMI, 5% fetal bovine serum, L-Glutamine
and Sodium Pyruvate (all Gibco)) supplemented with 2.0 .mu.g/mL
anti-CD28 (Southern Biotechnology) and placed in a +37.degree. C.
incubator. After four hours, half of the wells were harvested,
cells pelleted and conditioned media frozen at -20.degree. C. until
time of IL-31 bioassay.
[0113] The CLA+ and CLA- T cell subsets were activated similarly in
48-well tissue culture plates that were pretreated with 2.0
.mu.g/mL anti-CD3 antibody (Southern Biotechnology). The cells were
activated for 16 hours or 24 hours in a +37.degree. C. incubator at
a concentration of 6.25.times.10.sup.5 cells/mL. Samples were
harvested, cells pelleted and conditioned media frozen at
-20.degree. C. until time of IL-31 bioassay. For suboptimal
activation, CLA+ T cells were cultured in plates pre-treated with
0.5 ug/ml of anti-CD3 antibody.
[0114] C. Human IL-31 Bioassay Protocol:
[0115] BAF3 cells transfected with hIL-31RA, hOSMRB, and KZ134 (a
signal transducer and activator of transcription-activated
luciferase reporter) were grown to 5.times.10.sup.5 and
1.times.10.sup.6 cells/mL. Cells were washed with assay media (RPMI
1640, 10% FBS, L-Glutamine, Sodium Pyruvate, and Pen/Strep (all
Gibco)) and resuspended at 3.times.10.sup.5 cell/mL in assay
medium. In a 96-well opaque plate, hIL-31 standards were titered in
duplicate from 600 pg/mL to 9.38 pg/mL in assay medium via a 100
.mu.L/well, 1:2 serial dilution. Quality control standards were
added in duplicate to the plate at 350 .mu.g/mL and 35 .mu.g/mL in
100 .mu.L. Test samples were often diluted 1:2 or 1:4 and added in
duplicate to the sample wells. 100 .mu.L of the washed BAF3 cells
were then added to each well for a final concentration of
3.times.10.sup.4 cells/well. The plate was then incubated for 16-24
hours at +37.degree. C. in a 5% CO.sub.2 incubator. The plate was
then centrifuged at 1200 RPM for 5 minutes, media flicked off and
25 .mu.L/well of lysis buffer (Promega) added to each well. After
10 minutes the plate was read on a luminometer (Berthold). The
luminometer added 40 .mu.L/well of luciferase substrate mix
(Promega) and integrated the luminescence for a period of 4
seconds. Luminescence values were exported to a spreadsheet where
they were analyzed and converted into picograms of IL-31 per
10.sup.6 cells per mL of volume. The data is summarized in Table
1.
[0116] D. Results of IL-31 Bioassay:
[0117] The results from the CD45RA+ and the CD45RO+ T cell samples
revealed that IL-31 was primarily produced by activated CD45RO+
memory T cells. The CD45RA+ and CD45RO+ T cells from both donors
produced no detectable IL-31 when unstimulated. However, the
CD45RO+ samples from both donors #3 and #4 generated significant
levels of IL-31 following a 24 hour activation with plate-bound
anti-CD3 and soluble anti-CD28 (110.4 pg/10.sup.6 cells/mL and
145.6 pg/10.sup.6 cells/mL respectively). Conversely, when the
CD45RA+ T cells from donors #3 and #4 were activated with anti-CD3
and anti-CD28, they produced very low amounts of IL-31 (13.1
pg/10.sup.6 cells/mL and 12.7 pg/10.sup.6 cells/mL
respectively).
[0118] The CLA+ and CLA- T cell samples revealed that IL-31 seems
to be made almost entirely by activated CLA+ T cells. The
CLA-population of T cells (which includes naive T cells,
.alpha.4/.beta.7 gut-homing memory T cells, and tissue uncommitted
T cells) from both donors generated no detectable levels of IL-31
regardless of time point or activation condition. The CLA+ T cells
on the other hand, generated very high levels of IL-31 when
stimulated with 2.0 .mu.g/mL plate-bound anti-CD3 antibody. Donor
#5 generated 1385.7 pg/10.sup.6 cells/mL IL-31 by 16 hours and
>1920 pg/10.sup.6 cells/mL by 24 hours. Donor #6 generated 121.3
pg/10.sup.6 cells/mL IL-31 at 16 hours and 328.9 pg/10.sup.6
cells/mL IL-31 at 24 hours. These results clearly demonstrate that
of the T cell subsets, IL-31 seems to be made specifically by
cutaneous (CLA+) T cells under standard activation conditions.
TABLE-US-00001 TABLE 1 IL-31 (pg/10.sup.6 IL-31 Donor# Cell Type
Activation cells/mL) (pg/10.sup.6 cells/mL) 6 hr 24 hr 3 CD45RA+
.alpha.CD3 + .alpha.CD28 Below 13.1 Detection 3 CD45RO+ .alpha.CD3
+ .alpha.CD28 8.6 110.4 4 CD45RA+ .alpha.CD3 + .alpha.CD28 6.7 12.7
4 CD45RO+ .alpha.CD3 + .alpha.CD28 11.9 145.6 16 hr 24 hr 5 CLA+ T
Unstimulated Below Below Cells Detection Detection 5 CLA+ T
.alpha.CD3 1385.7 >1920 Cells 5 CLA- T Unstimulated Below Below
Cells Detection Detection 5 CLA- T .alpha.CD3 Below Below Cells
Detection Detection 6 CLA+ T Unstimulated Below Below Cells
Detection Detection 6 CLA+ T .alpha.CD3 121.3 328.9 Cells 6 CLA- T
Unstimulated Below Below Cells Detection Detection 6 CLA- T
.alpha.CD3 Below Below Cells Detection Detection
Example 2
[0119] IL-31 Involvement in Initiation and Perpetuation of Contact
Hyper-Sensitivity
[0120] A. Method I
[0121] BALB/c mice are painted on shaved mid-back with 25 ul of
0.5% DNFB dissolved (2,4,dinitro-fluoro-benzene, Sigma, St. Louis
Mo.) in acetone:olive oil (4:1) solution using a pipettor. A
vehicle control group receives 25 ul of acetone:olive oil only.
After 5 days, mice are anaesthetized with isofluorane in an
inhalation chamber and both ear pinnae of experimental and control
animals are measured with an engineer's micrometer (Mitutoyo) to
obtain a baseline measurement. Mice are then challenged by applying
10 ul of 0.25% DNFB in acetone:olive oil (4:1) to both sides of
each ear of all mice. Contact hyper-sensitivity is measured at 24 h
and 48 h later as the difference between the right ear (challenged)
and the left ear (unchallenged). All measurements are done with an
engineer's micrometer. Background values are determined by the
difference in ear swelling between the challenged and unchallenged
ears of naive mice.
[0122] Whole blood and serum for FACS and/or ELISA analysis are
collected prior to sacrifice and ears are collected for
histology.
[0123] Method II (Induces Th2 responses)
[0124] BALB/c mice are painted on shaved mid-back with 100 ul of
0.5% FITC (fluorescein isothiocyanate) in a 1:1 solution of
acetone/dibutyl phthalate (MSDS available using pipettor on days 1,
2 and 8. On day 13, mice are anaesthetized with isofluorane in an
inhalation chamber and both ear pinnae of experimental and control
animals are measured with an engineer's micrometer (Mitutoyo) to
obtain a baseline measurement. Mice are challenged by applying 25
ul of 0.5% FITC (in 1:1 acetone/dibutyl phthalate) to the dorsal
surface of each ear. Contact hyper-sensitivity is measured at 24 h
and 48 h later as the difference between the right ear (challenged)
and the left ear (unchallenged). All measurements are done with an
engineer's micrometer. Background values are determined by the
difference in ear swelling between the challenged and unchallenged
ears of naive mice. Whole blood and serum for FACS and/or ELISA
analysis are collected prior to sacrifice and ears are collected
for histology.
[0125] Method III (Induces Th1 Responses)
[0126] BALB/c mice are painted on shaved mid-back with 25 ul of 2%
oxazalone (in 4:lacetone/olive oil) using pipettor. On day 7, mice
are anaesthetized with isofluorane in an inhalation chamber and
both ear pinnae of experimental and control animals are measured
with an engineer's micrometer (Mitutoyo) to obtain a baseline
measurement. Mice are challenged by applying 8 ul of oxazalone to
the dorsal surface of each ear. Contact hyper-sensitivity is
measured at 24 h and 48 h later as the difference between the right
ear (challenged) and the left ear (unchallenged). All measurements
are done with an engineer's micrometer. Background values are
determined by the difference in ear swelling between the challenged
and unchallenged ears of naive mice. Whole blood and serum for FACS
and/or ELISA analysis are collected prior to sacrifice and ears are
collected for histology.
[0127] Involvement of IL-31 in the initiation and perpetuation of
contact hyper-sensitivity is tested using a neutralizing antibody
against IL-31 both at the sensitization and challenge phases of the
experiment.
Example 3
[0128] IL-31 Involvement in Atopic Dermatitis
[0129] A. Methods I (Sensitization of NC/Nga Mice)
[0130] Male NC/Nga mice were purchased from Charles River
Laboratories, Japan. The mice were 4 weeks old on arrival and
housed in SPF quarantine conditions for 4 weeks to acclimate. The
mice were approximately 10-11 weeks old at the start of the antigen
sensitization. Mice were anaesthetized with isofluorane and backs
were shaved with electric clippers. Approximately 10 ug of
Dermatophagoides pteronyssinus (Dp) (Indoor Biotechnologies,
Charlottesville, Virginia, special order) extract was injected
intradermally at the nape of the neck 3 times per week for 5 to 6
weeks until mice developed skin lesions. Control animals received
10 ul PBS intradermal injections 3 times per week. The Dp extract
was prepared according to method by Matsuoka and colleagues.
Matsuoka H., et al., Allergy: 58, 139 (2003). Briefly, 595 mg Dp
lyophilized spent culture extract was dissolved in 12 mL sterile
PBS (Gibco). Dp was mixed in a 50 mL Falcon tube on a shaking
rocker for 30 minutes. The extract was spun for 10 minutes at 2000
rpm and the supernatant was collected and aliquoted into 1 mL
cryovial tubes and stored at -20.degree. C.
[0131] B. Method II (Sensitization of DO11.10 mice)
[0132] DO11.10 transgenic mice were bred from an in-house colony
and were between 9.5 and 14 weeks old at start of antigen
sensitization. 24 hours prior to epicutaneous sensitization mice
were anaesthetized with isofluorane and the entire trunk (back and
abdomen) of mice were shaved with electric clippers. The mice were
then tape stripped with Elastin surgical tape (Johnson and Johnson)
on the back. 1 cm2 sterile gauze patches were wetted with either
500 ug oyalbumin (Calbiochem 32467) or sterile PBS (Gibco) and
adhered to left backside of mice with DuoDerm Extra Thin Dressing
(ConvaTec 187932). The patch and dressing were then covered in a
body wrap of the Elastin surgical tape so mice could not remove or
destroy the patches. Patches were worn for 7 days and removed. The
mice were rested for two weeks before having another round of
epicutaneous sensitization. Mice received a total of three one-week
sensitizations.
[0133] Results:
[0134] Immunohistochemical analysis of IL-31RA expression in
lesional and non-lesional skin from dust mite sensitized NC/Nga and
OVA sensitized DO11.10 animals showed that IL-3 IRA is expressed by
epidermal keratinocytes in mice, however no significant difference
in levels of expression was found between antigen sensitized versus
PBS sensitized animals in this study.
Example 4
[0135] IL-31 Involvement Delayed Type Hypersenstitvity
[0136] A. Methods
[0137] To generate a DTH response, mice were sensitized to antigen
on day 0 by subcutaneous immunization at the base of the tail with
100 ug ovalbumin (OVA) in complete Freund's adjuvant (CFA, 50-100
ul total volume). One week later mice were anesthetized with
isofluorane in an inhalation chamber and both ear pinnae of
experimental and control animals were measured with an engineer's
micrometer (Mitutoyo) to obtain a baseline measurement. Mice were
challenged intradermally with 10 ug OVA in PBS in a total volume of
10 ul into the left ear pinnae, just below the skin without hitting
any veins. As a control, mice also received an injection of 10 ul
PBS in the right ear pinnae. In some cases, a separate control
group given an i.d. injection of OVA in the ear may also be treated
with topical corticosteroids as a positive control to inhibit the
reaction. At 24 and 48 hr after challenge, mice were anesthetized
and ear thickness was measured. Results were expressed as: Specific
ear swelling=(24 hr measurement-0 hr measurement) for experimental
ear-(24 hr measurement-0 hr measurement) for negative control ear.
Induration, the hallmark of DTH, is detectable by 18 hours after
injection of sensitized antigen and is maximal by 2448 hours. The
lag in the onset of palpable induration is the reason for naming
the response "delayed type."
[0138] B. Results
[0139] L-31 transgenic mice were tested for DTH, however, due to an
increase in ear thickness in unchallenged IL-31 transgenic animals,
no statistically significant difference in DTH could be determined
between IL-31 Tg animals compared to wildtype controls in this
study. IL-31 receptor knockout animals were also tested in a DTH
response and no significant difference in the DTH response could be
observed between receptor knockout and wildtype animals.
Example 5
[0140] Immunohistochemical (IHC) Staining of IL-31 in Skin Lesions
from Uninvolved Psoriatic, and Atopic Dermatitis
[0141] Uninvolved psoriatic, atopic dermatitis and normal skin were
tested for the IL-31 ligand by IHC. Positive control cells
consisted of BHK cells transfected with IL-31. Negative controls
performed included: (1) un-transfected BHK cells, (2) staining
representative tissues and cells with protein A purified Normal
Rabbit serum and detecting antibody binding as usual. Antibody
reagent was E5758 (Rabbit anti-huIL-31 CEE, Aff. Purified at 1.0
mg/ml). Control cells included C02-6020: BHK cells expressing
zcytor17 Lig hu-CEE/21, and a BHK wild type. Tissues tested
included acute atopic dermatitis skin samples, chronic atopic
dermatitis skin samples, unaffected area skin samples, and normal
control skin samples and other in-house control samples.
[0142] The cells and tissues described above were fixed overnight
in 10% NBF and embedded in paraffin using standard techniques.
[0143] 5 .mu.M sections were baked at 61.degree. C. for 30 min for
tissue adhesion. Slides were subsequently dewaxed in 3.times.5' in
xylene and rehydrated through graded alcohols as follows:
2.times.2' in 100% EtOH, 2.times.2' in X95% EtOH, 1.times.2' in 70%
EtOH. Slides were rinsed in dH20, and then heat induced epitope
retrieval (HIER) was performed for 20 minutes under steam followed
by 20 minutes cooling to RT in 10 mM Tris, 1 mM EDTA, pH 9.0
[0144] Slides were loaded onto a DakoCytomation Autostainer. Slides
were rinsed with TBS/Tween buffer (TBST), prepared as recommend by
manufacturer. Endogenous biotin was blocked with a 10 minute
incubation in avidin solution, washed in TBST followed by a 10
minute incubation in biotin solution. Slides were washed in TBST. A
protein block (PBSB) (0.5% Blocking Powder in PBS, Perkin Elmer
NEL700001KT.) was applied for 30 minutes and rinsed off slides. The
primary antibody was diluted to 500 ng/ml and was applied for 60
minutes in ChemMate Antibody Dilution Buffer (part# ADB250, Ventana
Medical systems).
[0145] Tissues washed twice in TBST, and then incubated 45 minutes
in biotinylated Goat anti-Rabbit Ab, 750 ng/ml in PBSB (catalog #
BA-1000, Vector Labs). Slides washed twice in TBST. Vectastain
Elite ABC Reagent (catalog# PK-7100, Vector Labs) was incubated for
45 minutes. Slides washed twice in TBST. Signals were developed
with DAB+ (catalog# K-3468, DakoCytomation) for 10 minutes at room
temperature. Tissue slides were then counterstained in hematoxylin
(catalog# H-3401 Vector Labs), dehydrated and coverslipped in
VectorMount (catalog# H-5000, Vector Labs).
[0146] Results:
[0147] 1) Cell Controls:
[0148] BHK cells transfected with IL-31 was positively stained with
IL-31 antibody E5758 while un-transfected cells was negative for
this antibody. The same transfected and un-transfected cells were
negative with anti-rabbit sera.
[0149] 2) Atopic Dermatitis Skin Analysis:
[0150] The staining pattern for IL31 in the AD skin samples is
identical to that of psoriasis skins reported previously:
keratinocyte and CD3 positive T-cells stained negative for IL31. A
weak but rather uniform staining of the epithelial cells in the
secretory portion of the sweat glands was present, but a strong
signal was observed in the inner layer of epithelium in the duct
portion. Sebaceous gland was positive for IL31. There was no
difference in the IL31 staining between AD and normal skin.
[0151] Immunohistochemical (IHC) staining of uninvolved psoriatic,
atopic dermatitis and normal skin showed strong staining of IL-31
in the holocrine secretion of the sebaceous glands. Considering the
phenotype of IL 31 transgenic mice, it is interesting to note that
the sebaceous glands originate as an epithelial bud from the outer
root sheath of hair follicles. In addition to sebaceous glands weak
but rather uniform staining of IL-31 was observed in the epithelial
cells in the secretory portion of the sweat glands and a strong
signal in the inner layer of epithelium was observed in the duct
portion of sweat glands.
Example 6
Immunohistochemical (IHC) Staining of IL-3 IRA in Uninvolved
Psoriatic, and Atopic Dermatitis
[0152] Uninvolved psoriatic, atopic dermatitis and normal skin were
tested for the IL-3 IRA by IHC. Positive control cells consisted of
BHK cells dual transfected with IL-31RA and OSMR. Negative controls
performed included: (1) un-transfected BHK cells, (2) staining
representative tissues and cells with protein A purified Normal
Rabbit serum and detecting antibody binding as usual. Antibody
reagent was E6292 (Rabbit anti-huIL-31RAs-CEE v.4 at 1.33 mg/ml).
Control cells included C02-5117 BHK cells expressing human IL-31RA
and human OSMR (Total cells in the pellet: 3.9.times.10.sup.6,
vitality was >90%) and CO.sub.4-1587: BHK wild type (Total cells
in the pellet: 5.times.10.sup.6). Other tissues examined included:
5 Acute atopic dermatitis skin samples, 10 Chronic atopic
dermatitis skin samples, 10 Unaffected area skin samples, Normal
control skin samples, and other in-house skin samples.
[0153] The cells and tissues described above were fixed overnight
in 10% NBF and embedded in paraffin using standard techniques.
[0154] 5 .mu.M sections were baked at 61.degree. C. for 30 min for
tissue adhesion. Slides were subsequently dewaxed in 3.times.5' in
xylene and rehydrated through graded alcohols as follows:
2.times.2' in 100% EtOH, 2.times.2' in X95% EtOH, 1.times.2' in 70%
EtOH. Slides were rinsed in dH20, and then heat induced epitope
retrieval (HIER) was performed for 20 minutes under steam followed
by 20 minutes cooling to RT in 10 mM Tris, 1 mM EDTA, pH 9.0
[0155] Slides were loaded onto a DakoCytomation Autostainer. Slides
were rinsed with TBS/Tween buffer (TBST), prepared as recommend by
manufacturer. Endogenous biotin was blocked with a 10-minute
incubation in avidin solution, washed in TBST followed by a
10-minute incubation in biotin solution. Slides were washed in
TBST. A protein block (PBSB) (0.5% Blocking Powder in PBS, Perkin
Elmer NEL700001KT.) was applied for 30 minutes and rinsed off
slides. Primary antibodies diluted from 665 ng/ml to 1330 ng/ml for
IL31RA were applied for 60 minutes in ChemMate Antibody Dilution
Buffer (part# ADB250, Ventana Medical systems).
[0156] Tissues were washed twice in TBST, and then incubated 45
minutes in biotinylated Goat anti-Rabbit Ab, 750 ng/ml in PBSB
(catalog # BA-1000, Vector Labs). Slides were washed twice in TBST.
Vectastain Elite ABC Reagent (catalog# PK-7100, Vector Labs) was
incubated for 45 minutes. Slides were washed twice in TBST. Signals
were developed with DAB+(catalog# K-3468, DakoCytomation) for 10
minutes at room temperature. Tissue slides were then counterstained
in hematoxylin (catalog# H-3401 Vector Labs), dehydrated and
coverslipped in VectorMount (catalog# H-5000, Vector Labs).
[0157] Results are shown in Table 2. TABLE-US-00002 TABLE 2 Results
of IHC for IL-31RA in skin biopsy specimens from patients with
involved and uninvolved AD compared to healthy volunteers IL-31RA
IHC CASE ID SCORE* CD3 IHC SCORE* AD-1 2-3 0-1 AD-2 2-3 2 AD-3 2-3
1-2 AD-4 3 1 AD-5 2 2 UAD-1 1-2 1 UAD-2 1 0-1 UAD-5 1-2 0-1 UAD-6
2-3 ND UAD-7 2 1 UAD-8 1 1 UAD-9 1-2 1 UAD-10 2 ND Normal-1 1 0-1
Normal-2 0-1 0-1 Normal-3 1 0-1 Abbreviations: AD: atopic
dermatitis; UAD: uninvolved AD; ND: Not Done *IHC signal was scored
from 0 (no signal) to 4 (intense signal)
[0158] There was a slight up regulation of IL31RA in the epidermis
of AD skin samples. Possibly a small percentage of CD3 positive
T-cells were positive for IL31RA in the AD skins. There were CLA
positive cells in all skin samples tested. AD skins may have more
CLA positive cells than that of the normal or UAD samples.
[0159] The receptor for IL-31, IL-31RA was also expressed in the
epithelial cells of eccrine sweat glands with the cuboidal
epithelial cells in the secretory portion of the eccrine glands
demonstrating slightly higher level of IL-31RA protein compared to
the duct portion.
[0160] Collectively, these data demonstrate that IL-31RA is
expressed by epidermal keratinocytes from both control volunteers
and AD patients. However, the levels of IL-31RA expressed on
keratinocytes from AD skin biopsies were higher than the levels
observed in skin biopsies from normal controls, indicating a
potential for increased responsiveness to IL-31 in the context of
AD.
[0161] L-31RA was also found expressed on a subset of perivascular
infiltrating cells present in skin biopsies from AD patients but
was not present in control skin biopsies. These IL-31RA+ cells were
recognized by an antibody specific for the tissue macrophage marker
CD68, indicating these cells were skin-infiltrating tissue
macrophages.
Example 7
[0162] Isolation of Skin Infiltrating cells by Laser Capture
Microscopy and Analysis of IL-31mRNA by RT-PCR
[0163] The presence of skin infiltrating T cells is a
distinguishing feature in skin biopsies from AD patients compared
to normal individuals. Since IL-31 is a T cell associated cytokine,
the expression of IL-31 in skin-infiltrating T cells in tissue
biopsies from AD patients was examined. First, the presence of
increased numbers of CD3+ T cells in skin tissue biopsies from AD
patients compared to normal individuals was confirmed by IHC. See
Table 2. Next, laser capture microscopy was used to specifically
isolate skin infiltrating cells for analysis of IL-31 mRNA by
RT-PCR. IL-31 mRNA was expressed by skin infiltrating cells from
acute AD patients. In normal tissues, infiltrating cells are not
normally found and therefore could not be tested. However, the
epidermal keratinocyte layer, which is present in both AD and
normal skin, was analyzed for IL-31 mRNA expression and lower
levels of IL-31 mRNA were found in normal samples compared to the
epidermal keratinocyte layer of AD samples. Semi-quantitative
analysis of IL-31 mRNA expression compared to an internal control
gene (HPRT) showed that although IL-31 mRNA levels were not
significantly different between AD and normal samples, there was a
trend towards higher IL-31 expression in skin from AD patients.
Example 8
[0164] IL-31 is Produced by Memory T Cells with a Skin-Homing
Phenotype
[0165] Analysis of skin biopsies confirmed that the infiltrating
CD3+ T cells in the skin, which express IL-31 mRNA, express the
skin-homing marker cutaneous lymphocyte antigen (CLA). Of the total
T cell population in normal human peripheral blood, IL-31
expression was found to be largely restricted to CD45RO+
memory/effector cells as opposed to the CD45RA+ naive T cell
population.
[0166] In order to determine if IL-31 production was associated
with CLA+ skin-homing T cells, CLA+ and CLA- T cells were isolated
from peripheral blood of patients diagnosed with AD and control
volunteers and compared IL-31 mRNA and protein levels following
anti-CD3 plus anti-CD28 stimulation. Our results indicate that
IL-31 mRNA was significantly elevated in CLA+ T cells from both AD
and normal individuals at both 4 h (p0.0087 and p0.0022 CLA+
compared to CLA- for AD and normal, respectively) and 24 h (p0.0022
CLA+ compared to CLA- for both AD and normal samples) post
stimulation. Analysis of IL-31 protein levels in culture
supernatants confirmed that IL-31 was produced predominantly by
CLA+ T cells as there was no detectible IL-31 in culture
supernatants from CLA- T cells from both AD and control
individuals. There were no significant differences in IL-31 levels
between AD and normal patients. We also analysed the production of
IL-31 by peripheral blood T cells that express other
tissue-specific homing markers, such as the gut-specific homing
marker .alpha.4.beta.7, from normal volunteers. Comparison of the
IL-31 levels produced by CLA+ T cells and .alpha.4.beta.7+ cells
demonstrated CLA+ T cells preferentially produce IL-31 compared to
the .alpha.4.beta.7+ cells (average of 34.5 pg/ml and 14.42 pg/ml
IL-31, respectively).
[0167] Although both AD patients and normal controls have
circulating CLA+ T cells that express IL-31 upon activation, CLA+ T
cells from AD patients are reported to exist in a more activated
state compared to cells from normal individuals. Consequently, the
threshold of stimulation required for the production of IL-31 by
CLA+ T cells may differ between dermatitis patients and control
subjects. To test this hypothesis, we stimulated CLA+ T cells from
AD patients and control individuals with sub-optimal concentrations
of anti-CD3 in the absence of anti-CD28 and analyzed the production
of IL-31 in culture supernatants at 24 h after stimulation. Our
results demonstrate that circulating CLA+ T cells from some AD
patients produce higher levels of IL-31 compared to cells from
normal individuals in this study with maximum levels reaching 1200
pg/mL, whereas maximal detected levels in normal CLA+ supernatants
was only 400 pg/ml and maximal detected levels for psorasis
patients was 73 pg/ml at suboptimal concentrations of anti-CD3
stimulation. Five of eleven AD patients showed IL-31 levels below
the limit of detection of our assay suggesting there might be a
subset of AD patients where IL-31 is produced at low levels. This
may reflect variations in the stage of disease of our study
population. Nevertheless, more than half of the AD patients showed
a trend towards higher IL-31 levels compared to psoriasis patients
and normal individuals following suboptimal stimulation with
anti-CD3. Since more CLA+ T cells are localized in skin of AD
patients as compared to normal individuals, our studies suggest
that there is an increased potential for IL-31 activity in the AD
skin micro-environment. Thus, this study may suggest a
subpopulation of AD patients, which have more activated CLA+ T
cells producing IL-31.
Example 9
[0168] Reduction of TARC and MDC in Response to Anti-II-31 Antibody
in AD Mouse Models
[0169] Method I
[0170] Six-week old male NC/Nga mice (CRL Japan) were sensitized
intradermally with 50 .mu.g dust mite extract (D. pteronyssinus,
Indoor Biotechnologies) three times a week on the back and scored
for AD-like lesions. After 5 weeks of sensitization the mice were
euthanized and the right ears were excised and placed into a single
well of a 48-well culture dish (Corning) supplemented with RPMI+2%
FBS (GIBCO Invitrogen). Plates were placed in 5% CO.sub.2 humidity
controlled incubators. Supernatants were collected after 24 hours
and frozen at -20.degree. C. until further analysis.
[0171] Method I
[0172] Twelve-week old female NC/Nga mice (CRL Japan) were
sensitized intradermally with 10 .mu.g SEB (Toxin Technology) in
the ear and on the back three times per week. The mice were scored
for AD-like lesions. After 5 weeks of sensitization the mice were
euthanized and 6 mm biopsy punches were taken from the injected ear
of each mouse and placed into a single well of a 48-well culture
dish supplemented with RPMI+2% FBS. Plates were placed in 5%
CO.sub.2 humidity controlled incubators. Supernatants were
collected after 24 hours and frozen at -20.degree. C. until further
analysis.
[0173] Groups of mice in both studies were treated with either a
rat anti-mouse IL-31 monoclonal antibody at 10 mg/kg or vehicle,
intraperitoneally two times each week starting after 1 to 2 weeks
of sensitization.
[0174] TARC and MDC concentrations in the 24-hour supernatant
samples were measured by conventional ELISA (R&D Systems).
[0175] TARC and MDC concentrations were lower in ear supernatants
from anti-IL-31 treated mice compared to control mice in both
studies, however, these results were not statistically significant
when analyzed by ANOVA, probably due to small sample size. When the
data from both experiments is combined and analyzed there is a
statistically significant difference between treated groups.
Example 10
[0176] Administration of IL-31 neutralizing antibody
[0177] Normal female BALB/c mice (CRL) approximately 8 to 12 weeks
old were implanted subcutaneously with 14-day osmotic pumps (Alzet,
#2002) delivering 1 ug/day mIL-31. Groups of mice received
intraperitoneal (i.p.) injections of rat anti-mouse IL-31
monoclonal antibody 10 mg/kg (200 ug/mouse) twice weekly starting 1
week prior to IL-31 delivery. Control groups of mice received i.p.
injections of vehicle (PBS/0.1% BSA) with the identical dosing
schedules. Mice were scored daily for alopecia and pruritis using
the following criteria: 0=no scratching, animal appears normal,
1=thinning of coat in small areas, scratching noted, 2=minor hair
loss (small patches), scratching, 3=moderate hair loss, scratching,
and 4=severe hair loss, excessive scratching.
[0178] In all experiments, mice treated with rat anti-mL-31 mAb had
a delay in onset of symptoms of approximately 5 to 7 days and a
lower overall score for alopecia and pruritis. All groups of mAb
treated mice (regardless of dose frequency or concentration)
developed alopecia and pruritis similar to control mice by 13 day
of the study. These data suggest that neutralization of IL-31 can
delay the onset of the scratch/hairloss response induced by
IL-31.
[0179] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
10 1 904 DNA Homo sapiens CDS (28)...(519) 1 ctgaagctgg ccttgctctc
tctcgcc atg gcc tct cac tca ggc ccc tcg acg 54 Met Ala Ser His Ser
Gly Pro Ser Thr 1 5 tct gtg ctc ttt ctg ttc tgc tgc ctg gga ggc tgg
ctg gcc tcc cac 102 Ser Val Leu Phe Leu Phe Cys Cys Leu Gly Gly Trp
Leu Ala Ser His 10 15 20 25 acg ttg ccc gtc cgt tta cta cga cca agt
gat gat gta cag aaa ata 150 Thr Leu Pro Val Arg Leu Leu Arg Pro Ser
Asp Asp Val Gln Lys Ile 30 35 40 gtc gag gaa tta cag tcc ctc tcg
aag atg ctt ttg aaa gat gtg gag 198 Val Glu Glu Leu Gln Ser Leu Ser
Lys Met Leu Leu Lys Asp Val Glu 45 50 55 gaa gag aag ggc gtg ctc
gtg tcc cag aat tac acg ctg ccg tgt ctc 246 Glu Glu Lys Gly Val Leu
Val Ser Gln Asn Tyr Thr Leu Pro Cys Leu 60 65 70 agc cct gac gcc
cag ccg cca aac aac atc cac agc cca gcc atc cgg 294 Ser Pro Asp Ala
Gln Pro Pro Asn Asn Ile His Ser Pro Ala Ile Arg 75 80 85 gca tat
ctc aag aca atc aga cag cta gac aac aaa tct gtt att gat 342 Ala Tyr
Leu Lys Thr Ile Arg Gln Leu Asp Asn Lys Ser Val Ile Asp 90 95 100
105 gag atc ata gag cac ctc gac aaa ctc ata ttt caa gat gca cca gaa
390 Glu Ile Ile Glu His Leu Asp Lys Leu Ile Phe Gln Asp Ala Pro Glu
110 115 120 aca aac att tct gtg cca aca gac acc cat gaa tgt aaa cgc
ttc atc 438 Thr Asn Ile Ser Val Pro Thr Asp Thr His Glu Cys Lys Arg
Phe Ile 125 130 135 ctg act att tct caa cag ttt tca gag tgc atg gac
ctc gca cta aaa 486 Leu Thr Ile Ser Gln Gln Phe Ser Glu Cys Met Asp
Leu Ala Leu Lys 140 145 150 tca ttg acc tct gga gcc caa cag gcc acc
act taaggccatc tcttcctttc 539 Ser Leu Thr Ser Gly Ala Gln Gln Ala
Thr Thr 155 160 ggattggcag gaacttaagg agccttaaaa agatgaccga
cagctaagtg tgggaactct 599 gccgtgattc cttaagtaca tttttccaat
gaataatctc agggacccct catatgggct 659 agtcccggga gggctgagat
gtgaatttgt gaattacctt gaaaaacatt aggttattgt 719 tattagtctt
ggtatttatg gaatgctttt cttctgcagg cttaagtctt acttattata 779
ccctcgtgag ggtgggaggt ggcagctatg ttaatttatt gatatttatt gtactaagag
839 ttgtcaatgc tccctggggg agccctcgga atctatttaa taaattatat
tgaatttttc 899 tcata 904 2 164 PRT Homo sapiens 2 Met Ala Ser His
Ser Gly Pro Ser Thr Ser Val Leu Phe Leu Phe Cys 1 5 10 15 Cys Leu
Gly Gly Trp Leu Ala Ser His Thr Leu Pro Val Arg Leu Leu 20 25 30
Arg Pro Ser Asp Asp Val Gln Lys Ile Val Glu Glu Leu Gln Ser Leu 35
40 45 Ser Lys Met Leu Leu Lys Asp Val Glu Glu Glu Lys Gly Val Leu
Val 50 55 60 Ser Gln Asn Tyr Thr Leu Pro Cys Leu Ser Pro Asp Ala
Gln Pro Pro 65 70 75 80 Asn Asn Ile His Ser Pro Ala Ile Arg Ala Tyr
Leu Lys Thr Ile Arg 85 90 95 Gln Leu Asp Asn Lys Ser Val Ile Asp
Glu Ile Ile Glu His Leu Asp 100 105 110 Lys Leu Ile Phe Gln Asp Ala
Pro Glu Thr Asn Ile Ser Val Pro Thr 115 120 125 Asp Thr His Glu Cys
Lys Arg Phe Ile Leu Thr Ile Ser Gln Gln Phe 130 135 140 Ser Glu Cys
Met Asp Leu Ala Leu Lys Ser Leu Thr Ser Gly Ala Gln 145 150 155 160
Gln Ala Thr Thr 3 755 DNA Mus musculus CDS (1)...(489) 3 atg atc
ttc cac aca gga aca acg aag cct acc ctg gtg ctg ctt tgc 48 Met Ile
Phe His Thr Gly Thr Thr Lys Pro Thr Leu Val Leu Leu Cys 1 5 10 15
tgt ata gga acc tgg ctg gcc acc tgc agc ttg tcc ttc ggt gcc cca 96
Cys Ile Gly Thr Trp Leu Ala Thr Cys Ser Leu Ser Phe Gly Ala Pro 20
25 30 ata tcg aag gaa gac tta aga act aca att gac ctc ttg aaa caa
gag 144 Ile Ser Lys Glu Asp Leu Arg Thr Thr Ile Asp Leu Leu Lys Gln
Glu 35 40 45 tct cag gat ctt tat aac aac tat agc ata aag cag gca
tct ggg atg 192 Ser Gln Asp Leu Tyr Asn Asn Tyr Ser Ile Lys Gln Ala
Ser Gly Met 50 55 60 tca gca gac gaa tca ata cag ctg ccg tgt ttc
agc ctg gac cgg gaa 240 Ser Ala Asp Glu Ser Ile Gln Leu Pro Cys Phe
Ser Leu Asp Arg Glu 65 70 75 80 gca tta acc aac atc tcg gtc atc ata
gca cat ctg gag aaa gtc aaa 288 Ala Leu Thr Asn Ile Ser Val Ile Ile
Ala His Leu Glu Lys Val Lys 85 90 95 gtg ttg agc gag aac aca gta
gat act tct tgg gtg ata aga tgg cta 336 Val Leu Ser Glu Asn Thr Val
Asp Thr Ser Trp Val Ile Arg Trp Leu 100 105 110 aca aac atc agc tgt
ttc aac cca ctg aat tta aac att tct gtg cct 384 Thr Asn Ile Ser Cys
Phe Asn Pro Leu Asn Leu Asn Ile Ser Val Pro 115 120 125 gga aat act
gat gaa tcc tat gat tgt aaa gtg ttc gtg ctt acg gtt 432 Gly Asn Thr
Asp Glu Ser Tyr Asp Cys Lys Val Phe Val Leu Thr Val 130 135 140 tta
aag cag ttc tca aac tgc atg gca gaa ctg cag gct aag gac aat 480 Leu
Lys Gln Phe Ser Asn Cys Met Ala Glu Leu Gln Ala Lys Asp Asn 145 150
155 160 act aca tgc tgagtgatgg gggggggggg ggtgcagtgt cctcagcagt 529
Thr Thr Cys gcctgtcctt cgagggctga gcttgcaacc caggacttaa ctccaaaggg
actgtgcggt 589 cattactagt catgttattt atgtttttat tttgtccact
gaaatcttgt tctgctaccc 649 tgtagggact ggaagtggca gctatattta
tttatttatg tactgagttt gttaacgctc 709 catggaggag ccttcagagt
ctatttaata aattatattg acatga 755 4 163 PRT Mus musculus 4 Met Ile
Phe His Thr Gly Thr Thr Lys Pro Thr Leu Val Leu Leu Cys 1 5 10 15
Cys Ile Gly Thr Trp Leu Ala Thr Cys Ser Leu Ser Phe Gly Ala Pro 20
25 30 Ile Ser Lys Glu Asp Leu Arg Thr Thr Ile Asp Leu Leu Lys Gln
Glu 35 40 45 Ser Gln Asp Leu Tyr Asn Asn Tyr Ser Ile Lys Gln Ala
Ser Gly Met 50 55 60 Ser Ala Asp Glu Ser Ile Gln Leu Pro Cys Phe
Ser Leu Asp Arg Glu 65 70 75 80 Ala Leu Thr Asn Ile Ser Val Ile Ile
Ala His Leu Glu Lys Val Lys 85 90 95 Val Leu Ser Glu Asn Thr Val
Asp Thr Ser Trp Val Ile Arg Trp Leu 100 105 110 Thr Asn Ile Ser Cys
Phe Asn Pro Leu Asn Leu Asn Ile Ser Val Pro 115 120 125 Gly Asn Thr
Asp Glu Ser Tyr Asp Cys Lys Val Phe Val Leu Thr Val 130 135 140 Leu
Lys Gln Phe Ser Asn Cys Met Ala Glu Leu Gln Ala Lys Asp Asn 145 150
155 160 Thr Thr Cys 5 1557 DNA Homo sapiens CDS (1)...(1557) 5 atg
atg tgg acc tgg gca ctg tgg atg ctc ccc tca ctc tgc aaa ttc 48 Met
Met Trp Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys Lys Phe 1 5 10
15 agc ctg gca gct ctg cca gct aag cct gag aac att tcc tgt gtc tac
96 Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu Asn Ile Ser Cys Val Tyr
20 25 30 tac tat agg aaa aat tta acc tgc act tgg agt cca gga aag
gaa acc 144 Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp Ser Pro Gly Lys
Glu Thr 35 40 45 agt tat acc cag tac aca gtt aag aga act tac gct
ttt gga gaa aaa 192 Ser Tyr Thr Gln Tyr Thr Val Lys Arg Thr Tyr Ala
Phe Gly Glu Lys 50 55 60 cat gat aat tgt aca acc aat agt tct aca
agt gaa aat cgt gct tcg 240 His Asp Asn Cys Thr Thr Asn Ser Ser Thr
Ser Glu Asn Arg Ala Ser 65 70 75 80 tgc tct ttt ttc ctt cca aga ata
acg atc cca gat aat tat acc att 288 Cys Ser Phe Phe Leu Pro Arg Ile
Thr Ile Pro Asp Asn Tyr Thr Ile 85 90 95 gag gtg gaa gct gaa aat
gga gat ggt gta att aaa tct cat atg aca 336 Glu Val Glu Ala Glu Asn
Gly Asp Gly Val Ile Lys Ser His Met Thr 100 105 110 tac tgg aga tta
gag aac ata gcg aaa act gaa cca cct aag att ttc 384 Tyr Trp Arg Leu
Glu Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe 115 120 125 cgt gtg
aaa cca gtt ttg ggc atc aaa cga atg att caa att gaa tgg 432 Arg Val
Lys Pro Val Leu Gly Ile Lys Arg Met Ile Gln Ile Glu Trp 130 135 140
ata aag cct gag ttg gcg cct gtt tca tct gat tta aaa tac aca ctt 480
Ile Lys Pro Glu Leu Ala Pro Val Ser Ser Asp Leu Lys Tyr Thr Leu 145
150 155 160 cga ttc agg aca gtc aac agt acc agc tgg atg gaa gtc aac
ttc gct 528 Arg Phe Arg Thr Val Asn Ser Thr Ser Trp Met Glu Val Asn
Phe Ala 165 170 175 aag aac cgt aag gat aaa aac caa acg tac aac ctc
acg ggg ctg cag 576 Lys Asn Arg Lys Asp Lys Asn Gln Thr Tyr Asn Leu
Thr Gly Leu Gln 180 185 190 cct ttt aca gaa tat gtc ata gct ctg cga
tgt gcg gtc aag gag tca 624 Pro Phe Thr Glu Tyr Val Ile Ala Leu Arg
Cys Ala Val Lys Glu Ser 195 200 205 aag ttc tgg agt gac tgg agc caa
gaa aaa atg gga atg act gag gaa 672 Lys Phe Trp Ser Asp Trp Ser Gln
Glu Lys Met Gly Met Thr Glu Glu 210 215 220 gaa gct cca tgt ggc ctg
gaa ctg tgg aga gtc ctg aaa cca gct gag 720 Glu Ala Pro Cys Gly Leu
Glu Leu Trp Arg Val Leu Lys Pro Ala Glu 225 230 235 240 gcg gat gga
aga agg cca gtg cgg ttg tta tgg aag aag gca aga gga 768 Ala Asp Gly
Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly 245 250 255 gcc
cca gtc cta gag aaa aca ctt ggc tac aac ata tgg tac tat cca 816 Ala
Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr Tyr Pro 260 265
270 gaa agc aac act aac ctc aca gaa aca atg aac act act aac cag cag
864 Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn Thr Thr Asn Gln Gln
275 280 285 ctt gaa ctg cat ctg gga ggc gag agc ttt tgg gtg tct atg
att tct 912 Leu Glu Leu His Leu Gly Gly Glu Ser Phe Trp Val Ser Met
Ile Ser 290 295 300 tat aat tct ctt ggg aag tct cca gtg gcc acc ctg
agg att cca gct 960 Tyr Asn Ser Leu Gly Lys Ser Pro Val Ala Thr Leu
Arg Ile Pro Ala 305 310 315 320 att caa gaa aaa tca ttt cag tgc att
gag gtc atg cag gcc tgc gtt 1008 Ile Gln Glu Lys Ser Phe Gln Cys
Ile Glu Val Met Gln Ala Cys Val 325 330 335 gct gag gac cag cta gtg
gtg aag tgg caa agc tct gct cta gac gtg 1056 Ala Glu Asp Gln Leu
Val Val Lys Trp Gln Ser Ser Ala Leu Asp Val 340 345 350 aac act tgg
atg att gaa tgg ttt ccg gat gtg gac tca gag ccc acc 1104 Asn Thr
Trp Met Ile Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr 355 360 365
acc ctt tcc tgg gaa tct gtg tct cag gcc acg aac tgg acg atc cag
1152 Thr Leu Ser Trp Glu Ser Val Ser Gln Ala Thr Asn Trp Thr Ile
Gln 370 375 380 caa gat aaa tta aaa cct ttc tgg tgc tat aac atc tct
gtg tat cca 1200 Gln Asp Lys Leu Lys Pro Phe Trp Cys Tyr Asn Ile
Ser Val Tyr Pro 385 390 395 400 atg ttg cat gac aaa gtt ggc gag cca
tat tcc atc cag gct tat gcc 1248 Met Leu His Asp Lys Val Gly Glu
Pro Tyr Ser Ile Gln Ala Tyr Ala 405 410 415 aaa gaa ggc gtt cca tca
gaa ggt cct gag acc aag gtg gag aac att 1296 Lys Glu Gly Val Pro
Ser Glu Gly Pro Glu Thr Lys Val Glu Asn Ile 420 425 430 ggc gtg aag
acg gtc acg atc aca tgg aaa gag att ccc aag agt gag 1344 Gly Val
Lys Thr Val Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu 435 440 445
aga aag ggt atc atc tgc aac tac acc atc ttt tac caa gct gaa ggt
1392 Arg Lys Gly Ile Ile Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu
Gly 450 455 460 gga aaa gga ttc tcc aag aca gtc aat tcc agc atc ttg
cag tac ggc 1440 Gly Lys Gly Phe Ser Lys Thr Val Asn Ser Ser Ile
Leu Gln Tyr Gly 465 470 475 480 ctg gag tcc ctg aaa cga aag acc tct
tac att gtt cag gtc atg gcc 1488 Leu Glu Ser Leu Lys Arg Lys Thr
Ser Tyr Ile Val Gln Val Met Ala 485 490 495 agc acc agt gct ggg gga
acc aac ggg acc agc ata aat ttc aag aca 1536 Ser Thr Ser Ala Gly
Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys Thr 500 505 510 ttg tca ttc
agt gtc ttt gag 1557 Leu Ser Phe Ser Val Phe Glu 515 6 519 PRT Homo
sapiens 6 Met Met Trp Thr Trp Ala Leu Trp Met Leu Pro Ser Leu Cys
Lys Phe 1 5 10 15 Ser Leu Ala Ala Leu Pro Ala Lys Pro Glu Asn Ile
Ser Cys Val Tyr 20 25 30 Tyr Tyr Arg Lys Asn Leu Thr Cys Thr Trp
Ser Pro Gly Lys Glu Thr 35 40 45 Ser Tyr Thr Gln Tyr Thr Val Lys
Arg Thr Tyr Ala Phe Gly Glu Lys 50 55 60 His Asp Asn Cys Thr Thr
Asn Ser Ser Thr Ser Glu Asn Arg Ala Ser 65 70 75 80 Cys Ser Phe Phe
Leu Pro Arg Ile Thr Ile Pro Asp Asn Tyr Thr Ile 85 90 95 Glu Val
Glu Ala Glu Asn Gly Asp Gly Val Ile Lys Ser His Met Thr 100 105 110
Tyr Trp Arg Leu Glu Asn Ile Ala Lys Thr Glu Pro Pro Lys Ile Phe 115
120 125 Arg Val Lys Pro Val Leu Gly Ile Lys Arg Met Ile Gln Ile Glu
Trp 130 135 140 Ile Lys Pro Glu Leu Ala Pro Val Ser Ser Asp Leu Lys
Tyr Thr Leu 145 150 155 160 Arg Phe Arg Thr Val Asn Ser Thr Ser Trp
Met Glu Val Asn Phe Ala 165 170 175 Lys Asn Arg Lys Asp Lys Asn Gln
Thr Tyr Asn Leu Thr Gly Leu Gln 180 185 190 Pro Phe Thr Glu Tyr Val
Ile Ala Leu Arg Cys Ala Val Lys Glu Ser 195 200 205 Lys Phe Trp Ser
Asp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu 210 215 220 Glu Ala
Pro Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro Ala Glu 225 230 235
240 Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp Lys Lys Ala Arg Gly
245 250 255 Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr Asn Ile Trp Tyr
Tyr Pro 260 265 270 Glu Ser Asn Thr Asn Leu Thr Glu Thr Met Asn Thr
Thr Asn Gln Gln 275 280 285 Leu Glu Leu His Leu Gly Gly Glu Ser Phe
Trp Val Ser Met Ile Ser 290 295 300 Tyr Asn Ser Leu Gly Lys Ser Pro
Val Ala Thr Leu Arg Ile Pro Ala 305 310 315 320 Ile Gln Glu Lys Ser
Phe Gln Cys Ile Glu Val Met Gln Ala Cys Val 325 330 335 Ala Glu Asp
Gln Leu Val Val Lys Trp Gln Ser Ser Ala Leu Asp Val 340 345 350 Asn
Thr Trp Met Ile Glu Trp Phe Pro Asp Val Asp Ser Glu Pro Thr 355 360
365 Thr Leu Ser Trp Glu Ser Val Ser Gln Ala Thr Asn Trp Thr Ile Gln
370 375 380 Gln Asp Lys Leu Lys Pro Phe Trp Cys Tyr Asn Ile Ser Val
Tyr Pro 385 390 395 400 Met Leu His Asp Lys Val Gly Glu Pro Tyr Ser
Ile Gln Ala Tyr Ala 405 410 415 Lys Glu Gly Val Pro Ser Glu Gly Pro
Glu Thr Lys Val Glu Asn Ile 420 425 430 Gly Val Lys Thr Val Thr Ile
Thr Trp Lys Glu Ile Pro Lys Ser Glu 435 440 445 Arg Lys Gly Ile Ile
Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly 450 455 460 Gly Lys Gly
Phe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr Gly 465 470 475 480
Leu Glu Ser Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln Val Met Ala 485
490 495 Ser Thr Ser Ala Gly Gly Thr Asn Gly Thr Ser Ile Asn Phe Lys
Thr 500 505 510 Leu Ser Phe Ser Val Phe Glu 515 7 2748 DNA Mus
musculus CDS (237)...(2222) 7 gatggggccc tgaatgttga tctgacagaa
ttccagacca acctggtggt tattgtcctt 60 ttcatctggt catgctgaat
atactctcaa gatgtgctgg agaaggtgct gctgtccggg 120 ctctcagaga
aggcagtgct ggaggcgttc ctggcccggg tctcctccta ctgttcctgg 180
tagcccagcc ttctcggggt ggaaggagaa gctggccagg tgagctctga ggaagc atg
239 Met 1 ctg agc agc cag aag gga tcc tgc agc cag gaa cca ggg gca
gcc cac 287 Leu Ser Ser Gln Lys Gly Ser Cys Ser Gln Glu Pro Gly Ala
Ala His 5 10 15
gtc cag cct ctg ggt gtg aac gct gga ata atg tgg acc ttg gca ctg 335
Val Gln Pro Leu Gly Val Asn Ala Gly Ile Met Trp Thr Leu Ala Leu 20
25 30 tgg gca ttc tct ttc ctc tgc aaa ttc agc ctg gca gtc ctg ccg
act 383 Trp Ala Phe Ser Phe Leu Cys Lys Phe Ser Leu Ala Val Leu Pro
Thr 35 40 45 aag cca gag aac att tcc tgc gtc ttt tac ttc gac aga
aat ctg act 431 Lys Pro Glu Asn Ile Ser Cys Val Phe Tyr Phe Asp Arg
Asn Leu Thr 50 55 60 65 tgc act tgg aga cca gag aag gaa acc aat gat
acc agc tac att gtg 479 Cys Thr Trp Arg Pro Glu Lys Glu Thr Asn Asp
Thr Ser Tyr Ile Val 70 75 80 act ttg act tac tcc tat gga aaa agc
aat tat agt gac aat gct aca 527 Thr Leu Thr Tyr Ser Tyr Gly Lys Ser
Asn Tyr Ser Asp Asn Ala Thr 85 90 95 gag gct tca tat tct ttt ccc
cgt tcc tgt gca atg ccc cca gac atc 575 Glu Ala Ser Tyr Ser Phe Pro
Arg Ser Cys Ala Met Pro Pro Asp Ile 100 105 110 tgc agt gtt gaa gta
caa gct caa aat gga gat ggt aaa gtt aaa tct 623 Cys Ser Val Glu Val
Gln Ala Gln Asn Gly Asp Gly Lys Val Lys Ser 115 120 125 gac atc aca
tat tgg cat tta atc tcc ata gca aaa acc gaa cca cct 671 Asp Ile Thr
Tyr Trp His Leu Ile Ser Ile Ala Lys Thr Glu Pro Pro 130 135 140 145
ata att tta agt gtg aat cca att tgt aat aga atg ttc cag ata caa 719
Ile Ile Leu Ser Val Asn Pro Ile Cys Asn Arg Met Phe Gln Ile Gln 150
155 160 tgg aaa ccg cgt gaa aag act cgt ggg ttt cct tta gta tgc atg
ctt 767 Trp Lys Pro Arg Glu Lys Thr Arg Gly Phe Pro Leu Val Cys Met
Leu 165 170 175 cgg ttc aga act gtc aac agt agc cgc tgg acg gaa gtc
aat ttt gaa 815 Arg Phe Arg Thr Val Asn Ser Ser Arg Trp Thr Glu Val
Asn Phe Glu 180 185 190 aac tgt aaa cag gtc tgc aac ctc aca gga ctt
cag gct ttc aca gaa 863 Asn Cys Lys Gln Val Cys Asn Leu Thr Gly Leu
Gln Ala Phe Thr Glu 195 200 205 tat gtc ctg gct cta cga ttc agg ttc
aat gac tca aga tat tgg agc 911 Tyr Val Leu Ala Leu Arg Phe Arg Phe
Asn Asp Ser Arg Tyr Trp Ser 210 215 220 225 aag tgg agc aaa gaa gaa
acc aga gtg act atg gag gaa gtt cca cat 959 Lys Trp Ser Lys Glu Glu
Thr Arg Val Thr Met Glu Glu Val Pro His 230 235 240 gtc ctg gac ctg
tgg aga att ctg gaa cca gca gac atg aac gga gac 1007 Val Leu Asp
Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn Gly Asp 245 250 255 agg
aag gtg cga ttg ctg tgg aag aag gca aga gga gcc ccc gtc ttg 1055
Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala Pro Val Leu 260
265 270 gag aaa aca ttt ggc tac cac ata cag tac ttt gca gag aac agc
act 1103 Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe Ala Glu Asn
Ser Thr 275 280 285 aac ctc aca gag ata aac aac atc acc acc cag cag
tat gaa ctg ctt 1151 Asn Leu Thr Glu Ile Asn Asn Ile Thr Thr Gln
Gln Tyr Glu Leu Leu 290 295 300 305 ctg atg agc cag gca cac tct gtg
tcc gtg act tct ttt aat tct ctt 1199 Leu Met Ser Gln Ala His Ser
Val Ser Val Thr Ser Phe Asn Ser Leu 310 315 320 ggc aag tcc caa gag
acc atc ctg agg atc cca gat gtc cat gag aag 1247 Gly Lys Ser Gln
Glu Thr Ile Leu Arg Ile Pro Asp Val His Glu Lys 325 330 335 acc ttc
cag tac att aag agc atg cag gcc tac ata gcc gag ccc ctg 1295 Thr
Phe Gln Tyr Ile Lys Ser Met Gln Ala Tyr Ile Ala Glu Pro Leu 340 345
350 ttg gtg gtg aac tgg caa agc tcc att cct gcg gtg gac act tgg ata
1343 Leu Val Val Asn Trp Gln Ser Ser Ile Pro Ala Val Asp Thr Trp
Ile 355 360 365 gtg gag tgg ctc cca gaa gct gcc atg tcg aag ttc cct
gcc ctt tcc 1391 Val Glu Trp Leu Pro Glu Ala Ala Met Ser Lys Phe
Pro Ala Leu Ser 370 375 380 385 tgg gaa tct gtg tct cag gtc acg aac
tgg acc atc gag caa gat aaa 1439 Trp Glu Ser Val Ser Gln Val Thr
Asn Trp Thr Ile Glu Gln Asp Lys 390 395 400 cta aaa cct ttc aca tgc
tat aat ata tca gtg tat cca gtg ttg gga 1487 Leu Lys Pro Phe Thr
Cys Tyr Asn Ile Ser Val Tyr Pro Val Leu Gly 405 410 415 cac cga gtt
gga gag ccg tat tca atc caa gct tat gcc aaa gaa gga 1535 His Arg
Val Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu Gly 420 425 430
act cca tta aaa ggt cct gag acc agg gtg gag aac atc ggt ctg agg
1583 Thr Pro Leu Lys Gly Pro Glu Thr Arg Val Glu Asn Ile Gly Leu
Arg 435 440 445 aca gcc acg atc aca tgg aag gag att cct aag agt gct
agg aat gga 1631 Thr Ala Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser
Ala Arg Asn Gly 450 455 460 465 ttt atc aac aat tac act gta ttt tac
caa gct gaa ggt gga aaa gaa 1679 Phe Ile Asn Asn Tyr Thr Val Phe
Tyr Gln Ala Glu Gly Gly Lys Glu 470 475 480 ctc tcc aag act gtt aac
tct cat gcc ctg cag tgt gac ctg gag tct 1727 Leu Ser Lys Thr Val
Asn Ser His Ala Leu Gln Cys Asp Leu Glu Ser 485 490 495 ctg aca cga
agg acc tct tat act gtt tgg gtc atg gcc agc acc aga 1775 Leu Thr
Arg Arg Thr Ser Tyr Thr Val Trp Val Met Ala Ser Thr Arg 500 505 510
gct gga ggt acc aac ggg gtg aga ata aac ttc aag aca ttg tca atc
1823 Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe Lys Thr Leu Ser
Ile 515 520 525 agt gtg ttt gaa att gtc ctt cta aca tct cta gtt gga
gga ggc ctt 1871 Ser Val Phe Glu Ile Val Leu Leu Thr Ser Leu Val
Gly Gly Gly Leu 530 535 540 545 ctt cta ctt agc atc aaa aca gtg act
ttt ggc ctc aga aag cca aac 1919 Leu Leu Leu Ser Ile Lys Thr Val
Thr Phe Gly Leu Arg Lys Pro Asn 550 555 560 cgg ttg act ccc ctg tgt
tgt cct gat gtt ccc aac cct gct gaa agt 1967 Arg Leu Thr Pro Leu
Cys Cys Pro Asp Val Pro Asn Pro Ala Glu Ser 565 570 575 agt tta gcc
aca tgg ctc gga gat ggt ttc aag aag tca aat atg aag 2015 Ser Leu
Ala Thr Trp Leu Gly Asp Gly Phe Lys Lys Ser Asn Met Lys 580 585 590
gag act gga aac tct ggg aac aca gaa gac gtg gtc cta aaa cca tgt
2063 Glu Thr Gly Asn Ser Gly Asn Thr Glu Asp Val Val Leu Lys Pro
Cys 595 600 605 ccc gtc ccc gcg gat ctc att gac aag ctg gta gtg aac
ttt gag aat 2111 Pro Val Pro Ala Asp Leu Ile Asp Lys Leu Val Val
Asn Phe Glu Asn 610 615 620 625 ttt ctg gaa gta gtt ttg aca gag gaa
gct gga aag ggt cag gcg agc 2159 Phe Leu Glu Val Val Leu Thr Glu
Glu Ala Gly Lys Gly Gln Ala Ser 630 635 640 att ttg gga gga gaa gcg
aat gag tat atc tta tcc cag gaa cca agc 2207 Ile Leu Gly Gly Glu
Ala Asn Glu Tyr Ile Leu Ser Gln Glu Pro Ser 645 650 655 tgt cct ggc
cat tgc tgaagctacc ctcagggtcc aggacagctg tcttgttggc 2262 Cys Pro
Gly His Cys 660 acttgactct ggcaggaacc tgatctctac ttttcttctc
cctgtctccg gacactttct 2322 ctccttcatg cagagaccag gactagagcg
gattcctcat ggtttgccag gctcctcagt 2382 ccttgctcgg gctcaggatc
ttcaacaatg ccctttctgg gacactccat catccactta 2442 tatttatttt
ttgcaacatt gtggattgaa cccagggact tgtttatgcg cgcaacttca 2502
gtaactgtgg cagagactta ggaatggaga tctgaccctt tgcagaaggt ttctggacat
2562 ccgtccctgt gtgagcctca gacagcattg tctttacttt gaatcagctt
ccaagttaat 2622 aaaagaaaaa cagagaggtg gcataacagc tcctgcttcc
tgacctgctt gagttccagt 2682 tctgacttcc tttggtgatg aacagcaatg
tgggaagtgt aagctgaata aaccctttcc 2742 tcccca 2748 8 662 PRT Mus
musculus 8 Met Leu Ser Ser Gln Lys Gly Ser Cys Ser Gln Glu Pro Gly
Ala Ala 1 5 10 15 His Val Gln Pro Leu Gly Val Asn Ala Gly Ile Met
Trp Thr Leu Ala 20 25 30 Leu Trp Ala Phe Ser Phe Leu Cys Lys Phe
Ser Leu Ala Val Leu Pro 35 40 45 Thr Lys Pro Glu Asn Ile Ser Cys
Val Phe Tyr Phe Asp Arg Asn Leu 50 55 60 Thr Cys Thr Trp Arg Pro
Glu Lys Glu Thr Asn Asp Thr Ser Tyr Ile 65 70 75 80 Val Thr Leu Thr
Tyr Ser Tyr Gly Lys Ser Asn Tyr Ser Asp Asn Ala 85 90 95 Thr Glu
Ala Ser Tyr Ser Phe Pro Arg Ser Cys Ala Met Pro Pro Asp 100 105 110
Ile Cys Ser Val Glu Val Gln Ala Gln Asn Gly Asp Gly Lys Val Lys 115
120 125 Ser Asp Ile Thr Tyr Trp His Leu Ile Ser Ile Ala Lys Thr Glu
Pro 130 135 140 Pro Ile Ile Leu Ser Val Asn Pro Ile Cys Asn Arg Met
Phe Gln Ile 145 150 155 160 Gln Trp Lys Pro Arg Glu Lys Thr Arg Gly
Phe Pro Leu Val Cys Met 165 170 175 Leu Arg Phe Arg Thr Val Asn Ser
Ser Arg Trp Thr Glu Val Asn Phe 180 185 190 Glu Asn Cys Lys Gln Val
Cys Asn Leu Thr Gly Leu Gln Ala Phe Thr 195 200 205 Glu Tyr Val Leu
Ala Leu Arg Phe Arg Phe Asn Asp Ser Arg Tyr Trp 210 215 220 Ser Lys
Trp Ser Lys Glu Glu Thr Arg Val Thr Met Glu Glu Val Pro 225 230 235
240 His Val Leu Asp Leu Trp Arg Ile Leu Glu Pro Ala Asp Met Asn Gly
245 250 255 Asp Arg Lys Val Arg Leu Leu Trp Lys Lys Ala Arg Gly Ala
Pro Val 260 265 270 Leu Glu Lys Thr Phe Gly Tyr His Ile Gln Tyr Phe
Ala Glu Asn Ser 275 280 285 Thr Asn Leu Thr Glu Ile Asn Asn Ile Thr
Thr Gln Gln Tyr Glu Leu 290 295 300 Leu Leu Met Ser Gln Ala His Ser
Val Ser Val Thr Ser Phe Asn Ser 305 310 315 320 Leu Gly Lys Ser Gln
Glu Thr Ile Leu Arg Ile Pro Asp Val His Glu 325 330 335 Lys Thr Phe
Gln Tyr Ile Lys Ser Met Gln Ala Tyr Ile Ala Glu Pro 340 345 350 Leu
Leu Val Val Asn Trp Gln Ser Ser Ile Pro Ala Val Asp Thr Trp 355 360
365 Ile Val Glu Trp Leu Pro Glu Ala Ala Met Ser Lys Phe Pro Ala Leu
370 375 380 Ser Trp Glu Ser Val Ser Gln Val Thr Asn Trp Thr Ile Glu
Gln Asp 385 390 395 400 Lys Leu Lys Pro Phe Thr Cys Tyr Asn Ile Ser
Val Tyr Pro Val Leu 405 410 415 Gly His Arg Val Gly Glu Pro Tyr Ser
Ile Gln Ala Tyr Ala Lys Glu 420 425 430 Gly Thr Pro Leu Lys Gly Pro
Glu Thr Arg Val Glu Asn Ile Gly Leu 435 440 445 Arg Thr Ala Thr Ile
Thr Trp Lys Glu Ile Pro Lys Ser Ala Arg Asn 450 455 460 Gly Phe Ile
Asn Asn Tyr Thr Val Phe Tyr Gln Ala Glu Gly Gly Lys 465 470 475 480
Glu Leu Ser Lys Thr Val Asn Ser His Ala Leu Gln Cys Asp Leu Glu 485
490 495 Ser Leu Thr Arg Arg Thr Ser Tyr Thr Val Trp Val Met Ala Ser
Thr 500 505 510 Arg Ala Gly Gly Thr Asn Gly Val Arg Ile Asn Phe Lys
Thr Leu Ser 515 520 525 Ile Ser Val Phe Glu Ile Val Leu Leu Thr Ser
Leu Val Gly Gly Gly 530 535 540 Leu Leu Leu Leu Ser Ile Lys Thr Val
Thr Phe Gly Leu Arg Lys Pro 545 550 555 560 Asn Arg Leu Thr Pro Leu
Cys Cys Pro Asp Val Pro Asn Pro Ala Glu 565 570 575 Ser Ser Leu Ala
Thr Trp Leu Gly Asp Gly Phe Lys Lys Ser Asn Met 580 585 590 Lys Glu
Thr Gly Asn Ser Gly Asn Thr Glu Asp Val Val Leu Lys Pro 595 600 605
Cys Pro Val Pro Ala Asp Leu Ile Asp Lys Leu Val Val Asn Phe Glu 610
615 620 Asn Phe Leu Glu Val Val Leu Thr Glu Glu Ala Gly Lys Gly Gln
Ala 625 630 635 640 Ser Ile Leu Gly Gly Glu Ala Asn Glu Tyr Ile Leu
Ser Gln Glu Pro 645 650 655 Ser Cys Pro Gly His Cys 660 9 2964 DNA
Homo sapiens CDS (13)...(2949) 9 gaattcgcca cc atg gct cta ttt gca
gtc ttt cag aca aca ttc ttc tta 51 Met Ala Leu Phe Ala Val Phe Gln
Thr Thr Phe Phe Leu 1 5 10 aca ttg ctg tcc ttg agg act tac cag agt
gaa gtc ttg gct gaa cgt 99 Thr Leu Leu Ser Leu Arg Thr Tyr Gln Ser
Glu Val Leu Ala Glu Arg 15 20 25 tta cca ttg act cct gta tca ctt
aaa gtt tcc acc aat tct acg cgt 147 Leu Pro Leu Thr Pro Val Ser Leu
Lys Val Ser Thr Asn Ser Thr Arg 30 35 40 45 cag agt ttg cac tta caa
tgg act gtc cac aac ctt cct tat cat cag 195 Gln Ser Leu His Leu Gln
Trp Thr Val His Asn Leu Pro Tyr His Gln 50 55 60 gaa ttg aaa atg
gta ttt cag atc cag atc agt agg att gaa aca tcc 243 Glu Leu Lys Met
Val Phe Gln Ile Gln Ile Ser Arg Ile Glu Thr Ser 65 70 75 aat gtc
atc tgg gtg ggg aat tac agc acc act gtg aag tgg aac cag 291 Asn Val
Ile Trp Val Gly Asn Tyr Ser Thr Thr Val Lys Trp Asn Gln 80 85 90
gtt ctg cat tgg agc tgg gaa tct gag ctc cct ttg gaa tgt gcc aca 339
Val Leu His Trp Ser Trp Glu Ser Glu Leu Pro Leu Glu Cys Ala Thr 95
100 105 cac ttt gta aga ata aag agt ttg gtg gac gat gcc aag ttc cct
gag 387 His Phe Val Arg Ile Lys Ser Leu Val Asp Asp Ala Lys Phe Pro
Glu 110 115 120 125 cca aat ttc tgg agc aac tgg agt tcc tgg gag gaa
gtc agt gta caa 435 Pro Asn Phe Trp Ser Asn Trp Ser Ser Trp Glu Glu
Val Ser Val Gln 130 135 140 gat tct act gga cag gat ata ttg ttc gtt
ttc cct aaa gat aag ctg 483 Asp Ser Thr Gly Gln Asp Ile Leu Phe Val
Phe Pro Lys Asp Lys Leu 145 150 155 gtg gaa gaa ggc acc aat gtt acc
att tgt tac gtt tct agg aac att 531 Val Glu Glu Gly Thr Asn Val Thr
Ile Cys Tyr Val Ser Arg Asn Ile 160 165 170 caa aat aat gta tcc tgt
tat ttg gaa ggg aaa cag att cat gga gaa 579 Gln Asn Asn Val Ser Cys
Tyr Leu Glu Gly Lys Gln Ile His Gly Glu 175 180 185 caa ctt gat cca
cat gta act gca ttc aac ttg aat agt gtg cct ttc 627 Gln Leu Asp Pro
His Val Thr Ala Phe Asn Leu Asn Ser Val Pro Phe 190 195 200 205 att
agg aat aaa ggg aca aat atc tat tgt gag gca agt caa gga aat 675 Ile
Arg Asn Lys Gly Thr Asn Ile Tyr Cys Glu Ala Ser Gln Gly Asn 210 215
220 gtc agt gaa ggc atg aaa ggc atc gtt ctt ttt gtc tca aaa gta ctt
723 Val Ser Glu Gly Met Lys Gly Ile Val Leu Phe Val Ser Lys Val Leu
225 230 235 gag gag ccc aag gac ttt tct tgt gaa acc gag gac ttc aag
act ttg 771 Glu Glu Pro Lys Asp Phe Ser Cys Glu Thr Glu Asp Phe Lys
Thr Leu 240 245 250 cac tgt act tgg gat cct ggg acg gac act gcc ttg
ggg tgg tct aaa 819 His Cys Thr Trp Asp Pro Gly Thr Asp Thr Ala Leu
Gly Trp Ser Lys 255 260 265 caa cct tcc caa agc tac act tta ttt gaa
tca ttt tct ggg gaa aag 867 Gln Pro Ser Gln Ser Tyr Thr Leu Phe Glu
Ser Phe Ser Gly Glu Lys 270 275 280 285 aaa ctt tgt aca cac aaa aac
tgg tgt aat tgg caa ata act caa gac 915 Lys Leu Cys Thr His Lys Asn
Trp Cys Asn Trp Gln Ile Thr Gln Asp 290 295 300 tca caa gaa acc tat
aac ttc aca ctc ata gct gaa aat tac tta agg 963 Ser Gln Glu Thr Tyr
Asn Phe Thr Leu Ile Ala Glu Asn Tyr Leu Arg 305 310 315 aag aga agt
gtc aat atc ctt ttt aac ctg act cat cga gtt tat tta 1011 Lys Arg
Ser Val Asn Ile Leu Phe Asn Leu Thr His Arg Val Tyr Leu 320 325 330
atg aat cct ttt agt gtc aac ttt gaa aat gta aat gcc aca aat gcc
1059 Met Asn Pro Phe Ser Val Asn Phe Glu Asn Val Asn Ala Thr Asn
Ala 335 340 345 atc atg acc tgg aag gtg cac tcc ata agg aat aat ttc
aca tat ttg 1107 Ile Met Thr Trp Lys Val His Ser Ile Arg Asn Asn
Phe Thr Tyr Leu 350 355 360 365 tgt cag att gaa ctc cat ggt gaa gga
aaa atg atg caa tac aat gtt 1155 Cys Gln Ile Glu Leu His Gly Glu
Gly Lys Met Met Gln Tyr Asn Val 370 375 380 tcc atc aag gtg aac ggt
gag tac ttc tta agt gaa ctg gaa cct gcc 1203 Ser Ile Lys Val Asn
Gly Glu Tyr Phe Leu Ser Glu Leu Glu Pro Ala 385 390
395 aca gag tac atg gcg cga gta cgg tgt gct gat gcc agc cac ttc tgg
1251 Thr Glu Tyr Met Ala Arg Val Arg Cys Ala Asp Ala Ser His Phe
Trp 400 405 410 aaa tgg agt gaa tgg agt ggt cag aac ttc acc aca ctt
gaa gct gct 1299 Lys Trp Ser Glu Trp Ser Gly Gln Asn Phe Thr Thr
Leu Glu Ala Ala 415 420 425 ccc tca gag gcc cct gat gtc tgg aga att
gtg agc ttg gag cca gga 1347 Pro Ser Glu Ala Pro Asp Val Trp Arg
Ile Val Ser Leu Glu Pro Gly 430 435 440 445 aat cat act gtg acc tta
ttc tgg aag cca tta tca aaa ctg cat gcc 1395 Asn His Thr Val Thr
Leu Phe Trp Lys Pro Leu Ser Lys Leu His Ala 450 455 460 aat gga aag
atc ctg ttc tat aat gta gtt gta gaa aac cta gac aaa 1443 Asn Gly
Lys Ile Leu Phe Tyr Asn Val Val Val Glu Asn Leu Asp Lys 465 470 475
cca tcc agt tca gag ctc cat tcc att cca gca cca gcc aac agc aca
1491 Pro Ser Ser Ser Glu Leu His Ser Ile Pro Ala Pro Ala Asn Ser
Thr 480 485 490 aaa cta atc ctt gac agg tgt tcc tac caa atc tgc gtc
ata gcc aac 1539 Lys Leu Ile Leu Asp Arg Cys Ser Tyr Gln Ile Cys
Val Ile Ala Asn 495 500 505 aac agt gtg ggt gct tct cct gct tct gta
ata gtc atc tct gca gac 1587 Asn Ser Val Gly Ala Ser Pro Ala Ser
Val Ile Val Ile Ser Ala Asp 510 515 520 525 ccc gaa aac aaa gag gtt
gag gaa gaa aga att gca ggc aca gag ggt 1635 Pro Glu Asn Lys Glu
Val Glu Glu Glu Arg Ile Ala Gly Thr Glu Gly 530 535 540 gga ttc tct
ctg tct tgg aaa ccc caa cct gga gat gtt ata ggc tat 1683 Gly Phe
Ser Leu Ser Trp Lys Pro Gln Pro Gly Asp Val Ile Gly Tyr 545 550 555
gtt gtg gac tgg tgt gac cat acc cag gat gtg ctc ggt gat ttc cag
1731 Val Val Asp Trp Cys Asp His Thr Gln Asp Val Leu Gly Asp Phe
Gln 560 565 570 tgg aag aat gta ggt ccc aat acc aca agc aca gtc att
agc aca gat 1779 Trp Lys Asn Val Gly Pro Asn Thr Thr Ser Thr Val
Ile Ser Thr Asp 575 580 585 gct ttt agg cca gga gtt cga tat gac ttc
aga att tat ggg tta tct 1827 Ala Phe Arg Pro Gly Val Arg Tyr Asp
Phe Arg Ile Tyr Gly Leu Ser 590 595 600 605 aca aaa agg att gct tgt
tta tta gag aaa aaa aca gga tac tct cag 1875 Thr Lys Arg Ile Ala
Cys Leu Leu Glu Lys Lys Thr Gly Tyr Ser Gln 610 615 620 gaa ctt gct
cct tca gac aac cct cac gtg ctg gtg gat aca ttg aca 1923 Glu Leu
Ala Pro Ser Asp Asn Pro His Val Leu Val Asp Thr Leu Thr 625 630 635
tcc cac tcc ttc act ctg agt tgg aaa gat tac tct act gaa tct caa
1971 Ser His Ser Phe Thr Leu Ser Trp Lys Asp Tyr Ser Thr Glu Ser
Gln 640 645 650 cct ggt ttt ata caa ggg tac cat gtc tat ctg aaa tcc
aag gcg agg 2019 Pro Gly Phe Ile Gln Gly Tyr His Val Tyr Leu Lys
Ser Lys Ala Arg 655 660 665 cag tgc cac cca cga ttt gaa aag gca gtt
ctt tca gat ggt tca gaa 2067 Gln Cys His Pro Arg Phe Glu Lys Ala
Val Leu Ser Asp Gly Ser Glu 670 675 680 685 tgt tgc aaa tac aaa att
gac aac ccg gaa gaa aag gca ttg att gtg 2115 Cys Cys Lys Tyr Lys
Ile Asp Asn Pro Glu Glu Lys Ala Leu Ile Val 690 695 700 gac aac cta
aag cca gaa tcc ttc tat gag ttt ttc atc act cca ttc 2163 Asp Asn
Leu Lys Pro Glu Ser Phe Tyr Glu Phe Phe Ile Thr Pro Phe 705 710 715
act agt gct ggt gaa ggc ccc agt gct acg ttc acg aag gtc acg act
2211 Thr Ser Ala Gly Glu Gly Pro Ser Ala Thr Phe Thr Lys Val Thr
Thr 720 725 730 ccg gat gaa cac tcc tcg atg ctg att cat atc cta ctg
ccc atg gtt 2259 Pro Asp Glu His Ser Ser Met Leu Ile His Ile Leu
Leu Pro Met Val 735 740 745 ttc tgc gtc ttg ctc atc atg gtc atg tgc
tac ttg aaa agt cag tgg 2307 Phe Cys Val Leu Leu Ile Met Val Met
Cys Tyr Leu Lys Ser Gln Trp 750 755 760 765 atc aag gag acc tgt tat
cct gac atc cct gac cct tac aag agc agc 2355 Ile Lys Glu Thr Cys
Tyr Pro Asp Ile Pro Asp Pro Tyr Lys Ser Ser 770 775 780 atc ctg tca
tta ata aaa ttc aag gag aac cct cac cta ata ata atg 2403 Ile Leu
Ser Leu Ile Lys Phe Lys Glu Asn Pro His Leu Ile Ile Met 785 790 795
aat gtc agt gac tgt atc cca gat gct att gaa gtt gta agc aag cca
2451 Asn Val Ser Asp Cys Ile Pro Asp Ala Ile Glu Val Val Ser Lys
Pro 800 805 810 gaa ggg aca aag ata cag ttc cta ggc act agg aag tca
ctc aca gaa 2499 Glu Gly Thr Lys Ile Gln Phe Leu Gly Thr Arg Lys
Ser Leu Thr Glu 815 820 825 acc gag ttg act aag cct aac tac ctt tat
ctc ctt cca aca gaa aag 2547 Thr Glu Leu Thr Lys Pro Asn Tyr Leu
Tyr Leu Leu Pro Thr Glu Lys 830 835 840 845 aat cac tct ggc cct ggc
ccc tgc atc tgt ttt gag aac ttg acc tat 2595 Asn His Ser Gly Pro
Gly Pro Cys Ile Cys Phe Glu Asn Leu Thr Tyr 850 855 860 aac cag gca
gct tct gac tct ggc tct tgt ggc cat gtt cca gta tcc 2643 Asn Gln
Ala Ala Ser Asp Ser Gly Ser Cys Gly His Val Pro Val Ser 865 870 875
cca aaa gcc cca agt atg ctg gga cta atg acc tca cct gaa aat gta
2691 Pro Lys Ala Pro Ser Met Leu Gly Leu Met Thr Ser Pro Glu Asn
Val 880 885 890 cta aag gca cta gaa aaa aac tac atg aac tcc ctg gga
gaa atc cca 2739 Leu Lys Ala Leu Glu Lys Asn Tyr Met Asn Ser Leu
Gly Glu Ile Pro 895 900 905 gct gga gaa aca agt ttg aat tat gtg tcc
cag ttg gct tca ccc atg 2787 Ala Gly Glu Thr Ser Leu Asn Tyr Val
Ser Gln Leu Ala Ser Pro Met 910 915 920 925 ttt gga gac aag gac agt
ctc cca aca aac cca gta gag gca cca cac 2835 Phe Gly Asp Lys Asp
Ser Leu Pro Thr Asn Pro Val Glu Ala Pro His 930 935 940 tgt tca gag
tat aaa atg caa atg gca gtc tcc ctg cgt ctt gcc ttg 2883 Cys Ser
Glu Tyr Lys Met Gln Met Ala Val Ser Leu Arg Leu Ala Leu 945 950 955
cct ccc ccg acc gag aat agc agc ctc tcc tca att acc ctt tta gat
2931 Pro Pro Pro Thr Glu Asn Ser Ser Leu Ser Ser Ile Thr Leu Leu
Asp 960 965 970 cca ggt gaa cac tac tgc taaccagcac tcgag 2964 Pro
Gly Glu His Tyr Cys 975 10 979 PRT Homo sapiens 10 Met Ala Leu Phe
Ala Val Phe Gln Thr Thr Phe Phe Leu Thr Leu Leu 1 5 10 15 Ser Leu
Arg Thr Tyr Gln Ser Glu Val Leu Ala Glu Arg Leu Pro Leu 20 25 30
Thr Pro Val Ser Leu Lys Val Ser Thr Asn Ser Thr Arg Gln Ser Leu 35
40 45 His Leu Gln Trp Thr Val His Asn Leu Pro Tyr His Gln Glu Leu
Lys 50 55 60 Met Val Phe Gln Ile Gln Ile Ser Arg Ile Glu Thr Ser
Asn Val Ile 65 70 75 80 Trp Val Gly Asn Tyr Ser Thr Thr Val Lys Trp
Asn Gln Val Leu His 85 90 95 Trp Ser Trp Glu Ser Glu Leu Pro Leu
Glu Cys Ala Thr His Phe Val 100 105 110 Arg Ile Lys Ser Leu Val Asp
Asp Ala Lys Phe Pro Glu Pro Asn Phe 115 120 125 Trp Ser Asn Trp Ser
Ser Trp Glu Glu Val Ser Val Gln Asp Ser Thr 130 135 140 Gly Gln Asp
Ile Leu Phe Val Phe Pro Lys Asp Lys Leu Val Glu Glu 145 150 155 160
Gly Thr Asn Val Thr Ile Cys Tyr Val Ser Arg Asn Ile Gln Asn Asn 165
170 175 Val Ser Cys Tyr Leu Glu Gly Lys Gln Ile His Gly Glu Gln Leu
Asp 180 185 190 Pro His Val Thr Ala Phe Asn Leu Asn Ser Val Pro Phe
Ile Arg Asn 195 200 205 Lys Gly Thr Asn Ile Tyr Cys Glu Ala Ser Gln
Gly Asn Val Ser Glu 210 215 220 Gly Met Lys Gly Ile Val Leu Phe Val
Ser Lys Val Leu Glu Glu Pro 225 230 235 240 Lys Asp Phe Ser Cys Glu
Thr Glu Asp Phe Lys Thr Leu His Cys Thr 245 250 255 Trp Asp Pro Gly
Thr Asp Thr Ala Leu Gly Trp Ser Lys Gln Pro Ser 260 265 270 Gln Ser
Tyr Thr Leu Phe Glu Ser Phe Ser Gly Glu Lys Lys Leu Cys 275 280 285
Thr His Lys Asn Trp Cys Asn Trp Gln Ile Thr Gln Asp Ser Gln Glu 290
295 300 Thr Tyr Asn Phe Thr Leu Ile Ala Glu Asn Tyr Leu Arg Lys Arg
Ser 305 310 315 320 Val Asn Ile Leu Phe Asn Leu Thr His Arg Val Tyr
Leu Met Asn Pro 325 330 335 Phe Ser Val Asn Phe Glu Asn Val Asn Ala
Thr Asn Ala Ile Met Thr 340 345 350 Trp Lys Val His Ser Ile Arg Asn
Asn Phe Thr Tyr Leu Cys Gln Ile 355 360 365 Glu Leu His Gly Glu Gly
Lys Met Met Gln Tyr Asn Val Ser Ile Lys 370 375 380 Val Asn Gly Glu
Tyr Phe Leu Ser Glu Leu Glu Pro Ala Thr Glu Tyr 385 390 395 400 Met
Ala Arg Val Arg Cys Ala Asp Ala Ser His Phe Trp Lys Trp Ser 405 410
415 Glu Trp Ser Gly Gln Asn Phe Thr Thr Leu Glu Ala Ala Pro Ser Glu
420 425 430 Ala Pro Asp Val Trp Arg Ile Val Ser Leu Glu Pro Gly Asn
His Thr 435 440 445 Val Thr Leu Phe Trp Lys Pro Leu Ser Lys Leu His
Ala Asn Gly Lys 450 455 460 Ile Leu Phe Tyr Asn Val Val Val Glu Asn
Leu Asp Lys Pro Ser Ser 465 470 475 480 Ser Glu Leu His Ser Ile Pro
Ala Pro Ala Asn Ser Thr Lys Leu Ile 485 490 495 Leu Asp Arg Cys Ser
Tyr Gln Ile Cys Val Ile Ala Asn Asn Ser Val 500 505 510 Gly Ala Ser
Pro Ala Ser Val Ile Val Ile Ser Ala Asp Pro Glu Asn 515 520 525 Lys
Glu Val Glu Glu Glu Arg Ile Ala Gly Thr Glu Gly Gly Phe Ser 530 535
540 Leu Ser Trp Lys Pro Gln Pro Gly Asp Val Ile Gly Tyr Val Val Asp
545 550 555 560 Trp Cys Asp His Thr Gln Asp Val Leu Gly Asp Phe Gln
Trp Lys Asn 565 570 575 Val Gly Pro Asn Thr Thr Ser Thr Val Ile Ser
Thr Asp Ala Phe Arg 580 585 590 Pro Gly Val Arg Tyr Asp Phe Arg Ile
Tyr Gly Leu Ser Thr Lys Arg 595 600 605 Ile Ala Cys Leu Leu Glu Lys
Lys Thr Gly Tyr Ser Gln Glu Leu Ala 610 615 620 Pro Ser Asp Asn Pro
His Val Leu Val Asp Thr Leu Thr Ser His Ser 625 630 635 640 Phe Thr
Leu Ser Trp Lys Asp Tyr Ser Thr Glu Ser Gln Pro Gly Phe 645 650 655
Ile Gln Gly Tyr His Val Tyr Leu Lys Ser Lys Ala Arg Gln Cys His 660
665 670 Pro Arg Phe Glu Lys Ala Val Leu Ser Asp Gly Ser Glu Cys Cys
Lys 675 680 685 Tyr Lys Ile Asp Asn Pro Glu Glu Lys Ala Leu Ile Val
Asp Asn Leu 690 695 700 Lys Pro Glu Ser Phe Tyr Glu Phe Phe Ile Thr
Pro Phe Thr Ser Ala 705 710 715 720 Gly Glu Gly Pro Ser Ala Thr Phe
Thr Lys Val Thr Thr Pro Asp Glu 725 730 735 His Ser Ser Met Leu Ile
His Ile Leu Leu Pro Met Val Phe Cys Val 740 745 750 Leu Leu Ile Met
Val Met Cys Tyr Leu Lys Ser Gln Trp Ile Lys Glu 755 760 765 Thr Cys
Tyr Pro Asp Ile Pro Asp Pro Tyr Lys Ser Ser Ile Leu Ser 770 775 780
Leu Ile Lys Phe Lys Glu Asn Pro His Leu Ile Ile Met Asn Val Ser 785
790 795 800 Asp Cys Ile Pro Asp Ala Ile Glu Val Val Ser Lys Pro Glu
Gly Thr 805 810 815 Lys Ile Gln Phe Leu Gly Thr Arg Lys Ser Leu Thr
Glu Thr Glu Leu 820 825 830 Thr Lys Pro Asn Tyr Leu Tyr Leu Leu Pro
Thr Glu Lys Asn His Ser 835 840 845 Gly Pro Gly Pro Cys Ile Cys Phe
Glu Asn Leu Thr Tyr Asn Gln Ala 850 855 860 Ala Ser Asp Ser Gly Ser
Cys Gly His Val Pro Val Ser Pro Lys Ala 865 870 875 880 Pro Ser Met
Leu Gly Leu Met Thr Ser Pro Glu Asn Val Leu Lys Ala 885 890 895 Leu
Glu Lys Asn Tyr Met Asn Ser Leu Gly Glu Ile Pro Ala Gly Glu 900 905
910 Thr Ser Leu Asn Tyr Val Ser Gln Leu Ala Ser Pro Met Phe Gly Asp
915 920 925 Lys Asp Ser Leu Pro Thr Asn Pro Val Glu Ala Pro His Cys
Ser Glu 930 935 940 Tyr Lys Met Gln Met Ala Val Ser Leu Arg Leu Ala
Leu Pro Pro Pro 945 950 955 960 Thr Glu Asn Ser Ser Leu Ser Ser Ile
Thr Leu Leu Asp Pro Gly Glu 965 970 975 His Tyr Cys
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