U.S. patent application number 13/509072 was filed with the patent office on 2012-12-27 for methods and compositions for modulating the activity of the interleukin-35 receptor complex.
This patent application is currently assigned to St. Jude Children's Research Hospital. Invention is credited to Lauren W. Collison, Dario Aa Vignali.
Application Number | 20120328637 13/509072 |
Document ID | / |
Family ID | 44060356 |
Filed Date | 2012-12-27 |
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United States Patent
Application |
20120328637 |
Kind Code |
A1 |
Vignali; Dario Aa ; et
al. |
December 27, 2012 |
METHODS AND COMPOSITIONS FOR MODULATING THE ACTIVITY OF THE
INTERLEUKIN-35 RECEPTOR COMPLEX
Abstract
The receptor for Interleukin 35 (IL-35) is provided. The
Interleukin 35 Receptor (IL-35R) comprises a heterodimeric complex
of the Interluekin12R.beta.2 receptor and the gp130 receptor.
Various compositions comprising the IL-35R complex, along with
polynucleotides encoding the same and kits and methods for the
detection of the same the same are provided. Methods of modulating
the activity of IL-35R or modulating effector T cell functions are
also provided. Such methods employ various IL-35R antagonists and
agonists that modulate the activity of the IL-35R complex and, in
some embodiments, modulate effector T cell function. Further
provided are methods for screening for IL-35R binding agents and
for IL-35R modulating agents. Various methods of treatment are
further provided.
Inventors: |
Vignali; Dario Aa;
(Germantown, TN) ; Collison; Lauren W.; (Memphis,
TN) |
Assignee: |
St. Jude Children's Research
Hospital
Memphis
TN
|
Family ID: |
44060356 |
Appl. No.: |
13/509072 |
Filed: |
November 19, 2010 |
PCT Filed: |
November 19, 2010 |
PCT NO: |
PCT/US10/57369 |
371 Date: |
July 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61263058 |
Nov 20, 2009 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
435/325; 435/6.1; 435/6.11; 435/6.12; 435/7.9; 435/7.92; 436/501;
514/1.1; 514/19.3; 530/350; 530/387.3; 530/387.9; 536/23.4;
536/23.5; 800/13 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/244 20130101; C07K 14/7155 20130101; A01K 2217/05 20130101;
A01K 2267/0387 20130101; C12Q 1/6886 20130101; C07K 16/2866
20130101; A61K 2039/505 20130101; C07K 2317/76 20130101; C12Q
2600/158 20130101; C12Q 1/6883 20130101; A61P 29/00 20180101; G01N
33/505 20130101 |
Class at
Publication: |
424/185.1 ;
435/6.1; 435/6.11; 435/6.12; 435/7.9; 435/7.92; 435/325; 436/501;
514/1.1; 514/19.3; 530/350; 530/387.3; 530/387.9; 536/23.4;
536/23.5; 800/13 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53; C12N 5/10 20060101 C12N005/10; A61P 29/00 20060101
A61P029/00; A61P 35/00 20060101 A61P035/00; C07K 14/715 20060101
C07K014/715; C07K 16/18 20060101 C07K016/18; C07H 21/04 20060101
C07H021/04; A01K 67/027 20060101 A01K067/027; A61K 39/00 20060101
A61K039/00; G01N 33/566 20060101 G01N033/566 |
Claims
1. A soluble Interleukin 35 receptor (IL-35R) complex comprising:
a) a first polypeptide comprising the extracellular domain of a
gp130 polypeptide or a biologically active variant or fragment
thereof; and, b) a second polypeptide comprising the extracellular
domain of an IL12R.beta.2 polypeptide or a biologically active
fragment or variant thereof; wherein said soluble complex bind
IL-35.
2. The soluble Interleukin 35 receptor (IL-35R) complex of claim 1,
wherein a) said first polypeptide comprises the extracellular
domain of SEQ ID NO:3, an active fragment thereof, or a sequence
having at least 80% sequence identity to the extracellular domain
of SEQ ID NO:3; and, b) said second polypeptide comprises the
extracellular domain of SEQ ID NO:6 or an active fragment thereof
or a sequence having at least 80% sequence identity to SEQ ID
NO:6.
3. A polynucleotide comprising a nucleotide sequence encoding a
soluble Interleukin 35 receptor (IL-35R) complex comprising: a) a
first polynucleotide encoding a first polypeptide comprising the
extracellular domain a gp130 polypeptide or a biologically active
fragment or variant thereof; and, b) a second polynucleotide
encoding a second polypeptide comprising the extracellular domain
of an IL12R.beta.2 polypeptide or a biologically active variant or
fragment thereof.
4. The polynucleotide of claim 3, wherein, a) the first
polynucleotide encoding the first polypeptide comprises the
extracellular domain of SEQ ID NO:3, an active fragment thereof, or
a sequence having at least 80% sequence identity to the
extracellular domain of SEQ ID NO:3; and, b) the second
polynucleotide encoding the second polypeptide comprises the
extracellular domain of SEQ ID NO:6, an active fragment thereof, or
a sequence having at least 80% sequence identity to SEQ ID
NO:6.
5. A mixture of polynucleotides encoding a soluble Interleukin 35
receptor (IL-35R) complex comprising: a) a first nucleotide
sequence encoding a first polypeptide comprising the extracellular
domain of a gp130 polypeptide or biologically active variant or
fragment thereof; and, b) a second nucleotide sequence encoding a
second polypeptide comprising the extracellular domain of an
IL12R.beta.2 polypeptide or a biologically active variant or
fragment thereof; wherein said first and said second polypeptide
form the soluble IL-35R complex that bind IL-35.
6. The mixture of polynucleotides of claim 5, wherein a) the first
nucleotide sequence encoding the first polypeptide comprises the
extracellular domain of SEQ ID NO:3, an active fragment thereof or
a sequence having at least 80% sequence identity to the
extracellular domain of SEQ ID NO:3; and, b) the second nucleotide
sequence encoding the second polypeptide comprises the
extracellular domain of SEQ ID NO:6, an active fragment thereof, or
a sequence having at least 80% sequence identity to SEQ ID
NO:6.
7. A non-human cell comprising the polynucleotide of claim 3.
8. A transgenic animal having stably integrated into its genome the
polynucleotide of claim 3.
9. An isolated cell comprising the polynucleotide of claim 3.
10. A pharmaceutical composition comprising the polypeptide of
claim 1.
11. An isolated Interleukin 35 receptor (IL-35R) complex comprising
a) a first polypeptide comprising a gp130 polypeptide or a
biologically active variant or fragment thereof; and, b) a second
polypeptide comprising an Il12R.beta.2 polypeptide or a
biologically active variant or fragment thereof; wherein said
complex has IL-35R activity.
12. The isolated Interleukin 35 receptor (IL-35R) complex of claim
11, wherein a) the first polypeptide comprises SEQ ID NO:3 or a
sequence having at least 80% sequence identity to the extracellular
domain of SEQ ID NO:3; and, b) the second polypeptide comprises SEQ
ID NO:6 or a sequence having at least 80% sequence identity to SEQ
ID NO:6.
13. An antibody that binds substantially only to the IL-35R complex
of claim 12.
14. (canceled)
15. (canceled)
16. The antibody of claim 13, H, or 15, wherein (a) said antibody
is a monoclonal antibody; or, (b) said antibody is bispecific,
wherein a first antigen binding domain specifically interacts with
said first polypeptide and said second antigen binding domain
specifically interacts with said second polypeptide.
17. (canceled)
18. An antibody that binds substantially only to the first
polypeptide or a biologically active variant or fragment thereof of
the IL-35R complex of claim 11, wherein (a) said antibody behaves
as a specific modulating agent for IL-35R and substantially
inhibits IL-35 activation of the IL-35R complex; or, (b) wherein
said antibody behaves as a specific modulating agent for IL-35R and
substantially inhibits IL-35 activation of the IL-35R complex.
19. (canceled)
20. A mixture of a first and a second antibody comprising: a) a
first antibody having a first chemical moiety, said first antibody
binds substantially only to a first polypeptide comprising a gp130
polypeptide or an active variant or fragment thereof; and, b) a
second antibody having a second chemical moiety, said second
antibody binds substantially only to a second polypeptide
comprising a IL12R.beta.2 polypeptide or a biologically active
variant or fragment thereat c) wherein said first and said second
chemical moiety allow for the interaction of said first and said
second antibody at an IL-35R complex to be detected.
21. The mixture of the first and the second antibody of claim 20,
wherein a) said first antibody binds substantially only to the
first polypeptide comprising SEQ ID NO:3 or a sequence having at
least 80% sequence identity to the amino acid sequence of SEQ ID
NO:3; and, b) said second antibody bind substantially only to a
second polypeptide comprising SEQ ID NO:6 or the sequence having at
least 80% sequence identity to SEQ ID NO:6.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. A kit for determining the level of expression of a
polynucleotide encoding gp130 and a polynucleotide encoding
IL12R.beta.2 or a biologically active variant or fragment thereof
in a sample comprising a) a first polynucleotide capable of
specifically detecting or specifically amplifying a polynucleotide
encoding a gp130 polypeptide or a biologically active variant or
fragment thereof; and, h) a second polynucleotide capable of
specifically detecting or specifically amplifying a polynucleotide
encoding a IL12R.beta.2 polypeptide or a biologically active
variant or fragment thereof; wherein said encoded polypeptides form
a biologically active IL-35R complex.
29. The kit of claim 28, wherein a) the first polynucleotide is
capable of specifically detecting or amplifying a polynucleotide
encoding the amino acid sequence of SEQ ID NO:3 or a sequence
having at least 80% sequence identity to SEQ ID NO:3; and, b) the
second polynucleotide is capable of specifically detecting or
amplifying a polynucleotide encoding the amino acid sequence of SEQ
ID NO:6 or a sequence having at least 80% sequence identity to SEQ
ID NO:6.
30. The kit of claim 28 or 29, wherein said kit comprises a) a
first and a second primer that share sufficient sequence homology
or complementarity to said first polynucleotide to specifically
amplify said first polynucleotide; and, b) a third and a forth
primer that share sufficient sequence homology or complementarity
to said second polynucleotide to specifically amplify said second
polynucleotide.
31. The kit of claim 28, wherein said kit comprises a) a first
polynucleotide that can specifically detect said first
polynucleotide, wherein said first polynucleotide comprises at
least one DNA molecule of a sufficient length of contiguous
nucleotides identical or complementary to SEQ ID NO:3; and, b) a
second polynucleotide that can specifically detect said second
polynucleotide, wherein said second polynucleotide comprises at
least one DNA molecule of a sufficient length of contiguous
nucleotides identical or complementary to SEQ ID NO:6.
32. The kit of claim 28, wherein said kit comprises a) a first
polynucleotide that hybridizes under stringent conditions to the
sequence of SEQ ID NO:3; and, b) a second polynucleotide that
hybridizes under stringent conditions to the sequence of SEQ ID
NO:6.
33. A kit for determining the presence of Interleukin 35 Receptor
(IL-35R) in a sample comprising an antibody of claim 13.
34. (canceled)
35. (canceled)
36. (canceled)
37. A method for detecting an IL-35R complex comprising a)
contacting a sample with a compound which selectively binds to a
IL-35R complex; and b) detecting a complex comprising the IL-35R
complex and the compound; and thereby detecting said IL-35R
complex.
38. The method of claim 37, wherein the compound which binds to the
polypeptide is an antibody.
39. A method for (a) of modulating the activity of an Interleukin
35 receptor (IL-35R) complex or increasing an immune response, or
increasing the activity of an effector T cell function in a
subject, comprising administering to the subject a therapeutically
effective amount of a soluble IL-35R complex of claim 1; or, (b)
treating a subject having a cancer or a chronic inflammatory
disease, comprising administering to the subject a therapeutically
effective amount of a soluble Interleukin 35 receptor (IL-35R)
complex of claim 1; or, (c) increasing the activity of an effector
T cell function in a subject, comprising administering to the
subject a therapeutically effective amount of a soluble IL-35R
complex of claim 1.
40. (canceled)
41. (canceled)
42. A method to identify an Interleukin 35 receptor (IL-35R)
binding agent comprising the steps of: a) contacting the IL-35R
complex, or a cell expressing the IL-35R complex with a candidate
compound; and b) determining whether the IL-35R complex binds to
the candidate compound.
43. The method of claim 42, wherein the binding of the candidate
compound to the IL-35R complex is detected by a method selected
from the group consisting of: a) detection of binding by direct
detecting of candidate compound/IL-35R binding; or b) detection of
binding using a competition binding assay.
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. (canceled)
50. A method forte (a) screening screen for an Interleukin-35R
(IL-35R) modulating agent comprising contacting IL-35R with a
candidate compound and determining the effect of the test compound
on the activity of the IL-3 5R complex to thereby identify a
compound which modulates the activity of the IL-35R complex; or,
(b) modulating the activity of an Interleukin 35 Receptor (IL-35R)
comprising contacting a cell expressing said IL-35R with an IL-35R
specific binding/modulating agent; or, (c) modulating the activity
of an effector T cell function in a subject, comprising
administering to the subject a therapeutically effective amount of
an Interleukin 35 Receptor (IL-35R) specific binding/modulating
agent or, (d) treating a subject having a cancer or a chronic
inflammatory disease, comprising administering to the subject a
therapeutically effective amount of an antagonistic Interleukin 35
Receptor (IL-35R) specific binding/modulating agent or, (e)
treating a subject having an autoimmune or inflammatory disorder
comprising administering to the subject a therapeutically effective
amount of an agonist Interleukin 35 Receptor (IL-35R) specific
binding/modulating agent.
51. (canceled)
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. (canceled)
58. (canceled)
59. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for regulating T
cell function in a subject, particularly effector T cell
activity.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The official copy of the sequence listing is submitted
concurrently with the specification as a text file via EFS-Web, in
compliance with the American Standard Code for Information
Interchange (ASCII), with a file name of 397057SEQLIST.txt, a
creation date of Nov. 16, 2010, and a size of 61 KB. The sequence
listing filed via EFS-Web is part of the specification and is
hereby incorporated in its entirety by reference herein.
BACKGROUND OF THE INVENTION
[0003] The immune system provides the human body with a means to
recognize and defend itself against microorganisms, viruses, and
substances recognized as foreign and potentially harmful. Classical
immune responses are initiated when antigen-presenting cells
present an antigen to CD4+ T helper (Th) lymphocytes resulting in T
cell activation, proliferation, and differentiation of effector T
lymphocytes. Following exposure to antigens, such as that which
results from infection or the grafting of foreign tissue, naive T
cells differentiate into Th1 and Th2 cells with differing
functions. Th1 cells produce interferon gamma (IFN-.gamma.) and
interleukin 2 (IL-2) (both associated with cell-mediated immune
responses). Th1 cells play a role in immune responses commonly
involved in the rejection of foreign tissue grafts as well as many
autoimmune diseases. Th2 cells produce cytokines such as
interleukin-4 (IL-4), and are associated with antibody-mediated
immune responses such as those commonly involved in allergies and
allergic inflammatory responses such as allergic rhinitis and
asthma. Th2 cells may also contribute to the rejection of foreign
grafts. In numerous situations, this immune response is desirable,
for example, in defending the body against bacterial or viral
infection, inhibiting the proliferation of cancerous cells and the
like. However, in other situations, such effector T cells are
undesirable, e.g., in a graft recipient.
[0004] Whether the immune system is activated by or tolerized to an
antigen depends upon the balance between T effector cell activation
and T regulatory cell activation. T regulatory cells are
responsible for the induction and maintenance of immunological
tolerance. Regulatory T cells actively suppress the proliferation
and cytokine production of Th1, Th2, or naive cells which have been
stimulated in culture with an activating signal (e.g., antigen and
antigen presenting cells or with a signal that mimics antigen in
the context of MHC, e.g., anti-CD3 antibody, plus anti-CD28
antibody).
[0005] Undesirable immune responses have generally been treated
with immunosuppressive drugs, which inhibit the entire immune
system, i.e., both desired and undesired immune responses. General
immunosuppressants must be administered frequently, for prolonged
periods of time, and have numerous harmful side effects. Withdrawal
of these drugs generally results in relapse of disease. Thus, there
is a need for agents that preferentially modulate either the
effector or the regulatory arm of the immune system.
SUMMARY OF THE INVENTION
[0006] The receptor for Interleukin 35 (IL-35) is provided. The
Interleukin-35 Receptor (IL-35R) comprises a heterodimeric complex
of the Interleukin-12R.beta.2 receptor (Il12rb2) and the gp130
receptor (also known as Interleukin-6 signal transducer, Il6st).
Various compositions comprising the IL-35R complex, along with
polynucleotides encoding the same and kits and methods for the
detection of the same are provided.
[0007] Methods of modulating the activity of IL-35R or modulating
effector T cell functions are provided. Such methods employ various
IL-35R antagonists and agonists that modulate the activity of the
IL-35R complex and, in some embodiments, modulate effector T cell
function. Further provided are methods for screening for IL-35R
binding agents and for IL-35R modulating agents. Various methods of
treatment are further provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 demonstrates that T-cells that lack both the
Interluekin12R.beta.2 receptor (IL12R.beta.2) and the gp130
receptor are completely resistant to suppression mediated by IL-35
or iTr35, an induced regulatory T cell population that suppresses
via IL-35.
[0009] FIG. 2 demonstrates that IL35R deficient T.sub.conv are
resistant to IL-35 mediated suppression in vivo. Homeostatic
expansion was monitored by i.v. injection of Thy1.2.sup.+
T.sub.conv cells from wild-type (C57BL/6), gp130.sup..DELTA.T
(gp130 deficient in T cells), Il12rb2.sup.-/- or IL35RAT
(gp130.sup..DELTA.T/Il12rb2.sup.-/-) mice alone or with
Thy1.1.sup.+ iT.sub.R35 cells (as regulatory cells) into
Rag1.sup.-/- mice. Seven days after transfer, splenic T cell
numbers were determined by flow cytometry. (B) Rag1.sup.-/- mice
received CD4.sup.+ and CD8.sup.+ T cells from wild-type (C57BL/6),
gp130.sup..DELTA.T, Il12rb2.sup.-/- or IL35R.sup..DELTA.T mice
alone or with iT.sub.R.sup.35 cells via the tail vein on day -1 of
the experiment. On day 0, all were injected with 120,000 B16 cells
i.d. in the right flank. Tumor diameter was measured daily for 14
days and is reported as mm.sup.3. Data represent the mean.+-.SEM of
5-12 mice per group.
[0010] FIG. 3 demonstrates that IL35 is a target of the IL35
signaling pathway. (A) T.sub.conv purified by FACS from wild-type
(C57BL/6), gp130.sup..DELTA.T (gp130 deficient in T cells),
Il12rb2.sup.-/- or IL35R.sup..DELTA.T (gp130.sup..DELTA.T
Il12rb2.sup.-/-) mice were activated with
anti-CD3-+anti-CD28-coated latex beads for 18 hours in the presence
of IL-35. RNA was extracted, cDNA generated and qPCR performed.
Relative Ebi3 (left panel) and Il12a (right panel) mRNA expression.
(B) T.sub.conv from wild-type (C57BL/6), gp130.sup..DELTA.T,
Il12rb2.sup.-/- or IL35R.sup..DELTA.T mice were activated in the
presence of IL-35 or control protein at 25% of total culture
volume, for 72 hours to generate iT.sub.R.sup.35 or iT.sub.Rcon
cells, respectively. Cells were re-purified and mixed at indicated
ratios (T.sub.conv: suppressor) and proliferation was determined by
[.sup.3H]-thymidine incorporation. Counts per minute of T.sub.conv
cells activated alone were 29,000-48,000 (b). Data represent the
mean.+-.SEM of 3-5 independent experiments.
DETAILED DESCRIPTION OF THE INVENTION
I. Compositions
[0011] The receptor for Interleukin 35 (IL-35) is provided. As
demonstrated herein, the Interleukin 35 Receptor (IL-35R) comprises
a heterodimeric complex of the Interluekin12R.beta.2 receptor
(IL12R.beta.2) and the gp130 receptor.
[0012] As used herein, the Interleukin 35 receptor (IL-35R) refers
to any intramolecular complex or single molecule comprising at
least one gp130 polypeptide component or biologically active
variant or fragment thereof and at least one IL12R.beta.2
polypeptide component or biologically active variant or fragment
thereof. Typically, in vivo, gp130 and IL12R.beta.2 associate via a
non-covalent association. For purposes of the present invention,
the IL12R.beta.2 and gp130 components may be associated with one
another either covalently or non-covalently. In some examples,
gp130 and IL12R.beta.2 can be co-expressed as a fusion protein.
[0013] Biologically active fragments and variants of the IL-35R
complex are also provided. Such IL-35R complexes comprise an active
variant or fragment of gp130 and/or an active variant or fragment
of IL12R.beta.2 and will retain at least one activity of the IL-35R
complex.
[0014] The phrase "biological activity of IL-35R" refers to one or
more of the biological activities of IL-35R, including but not
limited to, (1) interacting with its ligand, IL-35; (2) activating
any of the STAT pathways including the STAT1 and/or STAT4 pathways;
(3) IL-35 dependent suppression of effector T-cell function,
including for example, suppression of proliferation, cytokine
secretion and/or differentiation; and/or (4) autocrine induction of
IL-35 expression by IL-35. Such assays can be found, for example,
in Collison et al. (2007) Nature 450:566-569; Collison et al.
(2010) Nature Immunology 11: 1093-1101.
[0015] As discussed above, the IL-35R complex interacts with the
ligand, IL-35. As used herein, "Interleukin 35" or "IL-35" refers
to any intramolecular complex or single molecule comprising at
least one EBI3 polypeptide component and at least one p35
polypeptide component. See, for example, International Patent
Application No. PCT/US2007/079310, filed Sep. 24, 2007, herein
incorporated by reference in its entirety. EBI3 and p35 are known
in the art. The human EBI3 gene encodes a protein of about 33 kDa.
The protein shares about 27% sequence identity to the p40 subunit
of human IL12. Nucleic acid and amino acid sequences for EBI3 are
known. See, for example, SEQ ID NOs:1 and 2 of WO97/13859 (human)
and GenBank Accession Numbers NM015766 and BC046112 (mouse).
Nucleic acid and amino acid sequences for p35 are also known in the
art and include SEQ ID NOs:3 and 4 of WO97/13859 (human) and
GenBank Accession Numbers NM.sub.--000882 and M86672 (mouse). The
term IL-35 encompasses naturally occurring variants of IL-35, e.g.,
splice variants, allelic variants, and other isoforms. The term
also encompasses fragments or variants of a native IL-35 such that
the active variants and fragment continue to bind and allow for the
activation of IL-35R.
[0016] It is recognized that an IL-35R complex can be encoded on a
single polynucleotide. For example, in one embodiment, a
polynucleotide comprising a nucleotide sequence encoding an
Interleukin 35 receptor (IL-35R) complex is provided and comprises
a first sequence encoding the gp130 polypeptide or an active
fragment or variant thereof; and a second sequence encoding the
IL12R.beta.2 polypeptide or an active fragment or variant thereof,
wherein said encoded polypeptides form a biologically active IL-35R
complex. In another embodiment, the IL-35R complex is encoded on
distinct polynucleotides. Thus, a mixture of recombinant expression
constructs encoding the various components of the IL-35R complex
are further provided.
[0017] i. IL12R.beta.2 Polynucleotides and Polypeptides
[0018] The polypeptides that interact to form the IL-35R complex
are known in the art. As used herein, the terms
"Interleukin12R.beta.2 receptor", "IL12R.beta.2" or "IL-12R-beta2"
can be used interchangeably and refer to a family of cell surface
receptors which can homodimerize or heterodimerize and display
interleukin receptor activity and have now been shown herein to be
a subunit of the IL-35R complex. Non-limiting examples of
IL12R.beta.2 polypeptides comprise the human IL12R.beta.2
polynucleotide as set forth in SEQ ID NO:4, 5, and 6 (from GenBank
Accession No. NM.sub.--001559.2) and can be found in GenBank
Accession No. P40189.
[0019] IL12R.beta.2 polypeptides comprise a variety of conserved
structural motifs. For ease of reference, such motifs will be
discussed as they relate to the human IL12R.beta.2 isoform 1 which
is set forth in SEQ ID NO:6. IL12R.beta.2 polypeptides comprise an
extracellular domain (from about amino acids 24-522 of SEQ ID
NO:6); a transmembrane domain (from about amino acids 623-643 of
SEQ ID NO:3), and an intercellular domain (from about amino acids
644-862 of SEQ ID NO:6). Additional conserved domains and motifs
that have been characterized in the IL12R.beta.2 polypeptides
include a signal peptide (from about amino acids 1-23 of SEQ ID
NO:6), a Fibronectin type III I domain (from about amino acids
124-218 of SEQ ID NO:6), a Fibronectin type III 2 domain (from
about amino acids 224-316 of SEQ ID NO:6), a Fibronectin type III 3
domain (from about amino acids 317-415 of SEQ ID NO:6), a
Fibronectin type III 4 domain (from about amino acids 420-517 of
SEQ ID NO:6), a Fibronectin type III 5 domain (from about amino
acids 521-617 of SEQ ID NO:6), a motif involved with STAT4 binding
(from about amino acids 796-801 of SEQ ID NO:6), a WSXWS motif
(from about amino acids 305-309 of SEQ ID NO:6) which appears to be
involved in proper protein folding and thereby efficient
intracellular transport and cell-surface receptor binding, and a
Box 1 motif (from about amino acids 662-670 of SEQ ID NO:6) which
is involved in JAK interaction and/or activation. Glycosylation can
occur at amino acid positions 48, 129, 166, 195, 271, 347, 376, and
480 of SEQ ID NO:6.
[0020] It is recognized that biologically active variants and
fragments of the IL12R.beta.2 polypeptide can be employed in the
various methods and compositions of the invention. Such active
variants and fragments will contain an IL-35R receptor activity
when complexed with the gp130 partner. Variants of IL12R.beta.2 are
known including, but not limited to, an alternative sequence from
aa 650-659 of SEQ ID NO:6 replacing VFVLLAALRP with RRHSCPWTGS; an
alternative sequence from aa 660-862 of SEQ ID NO:6 is missing; the
Rat aa 149 of SEQ ID NO:6 is replaced with Q; the I at aa 185 of
SEQ ID NO:6 is replaced with V; the T at aa 201 of SEQ ID NO:6 is
replaced with I; the R at aa 313 of SEQ ID NO:6 is replaced with G;
the G at aa 420 of SEQ ID NO:6 is replaced with R; the Q at aa 426
of SEQ ID NO:6 is replaced with H; the G at aa 465 of SEQ ID NO:6
is replaced with D; the A at aa 625 of SEQ ID NO:6 is replaced with
V; the H at aa 720 of SEQ ID NO:6 is replaced with R; the L at aa
808 of SEQ ID NO:6 is replaced with R; the Y at aa 678 of SEQ ID
NO:6 is replaced with F; or the Y at aa 767 of SEQ ID NO:6 is
replaced with F.
[0021] In specific embodiments, fragments of IL12R.beta.2 are
employed which comprise the extracellular domain of the
IL12R.beta.2 polypeptide or a biologically active fragment or
variant of the extracellular domain of IL12R.beta.2. Such
biologically active variants and fragments of the IL12R.beta.2
extracellular domain will retain the ability to complex with the
gp130 binding partner's extracellular domain and upon complex
formation, the gp130/IL12R.beta.2 complex will interact with the
IL-35 ligand. Methods to assay for such binding are known.
[0022] Thus, in one embodiment, the IL12R.beta.2 polypeptide
comprises the amino acid sequence as shown in SEQ ID NO:6 or a
biologically active variant or fragment thereof. Further provided
are polynucleotides comprising the nucleotide sequence encoding a
IL12R.beta.2 polypeptide including the nucleotide sequence set
forth in SEQ ID NO:4 or 5 or a biologically active variant or
fragment thereof.
[0023] ii. Gp130 Polynucleotides and Polypeptides
[0024] As used herein, the terms "gp130", "Interleukin-6-receptor
subunit beta", "IL-6R-beta", "Interleukin-6-signal transducer",
"membrane glycoprotein 130", "CDW130",
[0025] "Oncostatin-M receptor alpha subunit", "CD_antigen=CD130" or
IL6ST" can be used interchangeably and refer to a family of cell
surface receptors which can homodimerize or heterodimerize and
display interleukin receptor activity and have now been shown
herein to be a subunit of the IL-35R complex. Non-limiting examples
of gp130 polypeptides comprising the human gp130 polynucleotide and
polypeptide are set forth in SEQ ID NOs: 1, 2, and 3 (GenBank
Accession No. NP.sub.--002184.2) or also in GenBank Accession No.
P40189.
[0026] The gp130 polypeptide comprises a variety of conserved
structural motifs and belongs to the type I cytokine receptor
family. For ease of reference, such motifs will be discussed as
they relate to the human gp130 isoform 1 which is set forth in SEQ
ID NO:3. gp130 polypeptides comprise an extracellular domain (from
about amino acids 23-619 of SEQ ID NO:3); a transmembrane domain
(from about amino acids 260-641 of SEQ ID NO:3), and an
intercellular domain (from about amino acids 642-918 of SEQ ID
NO:3). Additional conserved domains and motifs have been
characterized in the gp130 polypeptides include a signal peptide
(from about amino acids 1-22 of SEQ ID NO:3), an IG-like C2-type
domain (from about amino acids 26-120 of SEQ ID NO:3), a
Fibronectin type III I domain (from about amino acids 125-216 of
SEQ ID NO:3), a Fibronectin type III 2 domain (from about amino
acids 222-321 of SEQ ID NO:3), a Fibronectin type III 3 domain
(from about amino acids 326-418 of SEQ ID NO:3), a Fibronectin type
III 4 domain (from about amino acids 423-514 of SEQ ID NO:3), a
Fibronectin type III 5 domain (from about amino acids 518-610 of
SEQ ID NO:3), a WSXWS motif (from about amino acids 310-314 of SEQ
ID NO:3) which appears to be involved in proper protein folding and
thereby efficient intracellular transport and cell-surface receptor
binding, a Box 1 motif (from about amino acids 651-659 of SEQ ID
NO:3) which is involved in JAK interaction and/or activation, and a
compositional bias that is Ser-rich (from about amino acids 725-755
of SEQ ID NO:3). Glycosylation can occur at amino acid positions
43, 83, 131, 157, 227, 379, 383, 553, 564 of SEQ ID NO:3. Modified
phosphoserine residues can occur at amino acids 667, 782, 820, and
829 of SEQ ID NO:3. Disulfide bonds can occur between amino acids
28 and 54, 48 and 103, 134 and 144, 172 and 182, and 458 and 466 of
SEQ ID NO:3.
[0027] It is recognized that biologically active variants and
fragments of the gp130 polypeptide can be employed in the various
methods and compositions of the invention. Such active variants and
fragments will continue to retain an IL-35R activity when complexed
with the IL12R.beta.2 partner. Variants and fragments of gp130
polypeptides and polynucleotides are known including, but not
limited to, an alternative sequence from aa 325-329 of SEQ ID NO:3
replacing RPSKA with NIASF; an alternative sequence from aa 330-918
of SEQ ID NO:3 is missing; the T at aa 415 of SEQ ID NO:3 is
replaced with I; or the S at aa 782 of SEQ ID NO:3 is replaced with
A.
[0028] In specific embodiments, fragments of the gp130 are employed
which comprise the extracellular domain of the gp130 polypeptide or
a biologically active fragment or variant of the extracellular
domain of gp130. Such biologically active variants and fragments of
the extracellular domain of gp130 will retain the ability to
complex with the IL12R.beta.2 binding partner's extracellular
domain and upon complex formation, the gp130/IL12R.beta.2 complex
can interact with the IL-35 ligand. Methods to assay for such
binding are known.
[0029] Thus, in one embodiment, the gp130 polypeptide comprises the
amino acid sequence as shown in SEQ ID NO:3 or a biologically
active variant or fragment thereof. Further provided are
polynucleotides comprising the nucleotide sequence encoding a gp130
polypeptide including the nucleotide sequence set forth in SEQ ID
NO:1 or 2 or a biologically active variant or fragment thereof
[0030] iii. Variants and Fragments
[0031] Fragments and variants of the polynucleotides encoding the
gp130 and IL12R.beta.2 polypeptides can be employed in the various
methods and compositions of the invention. By "fragment" is
intended a portion of the polynucleotide and hence the protein
encoded thereby or a portion of the polypeptide. Fragments of a
polynucleotide may encode protein fragments that retain the
biological activity of the native protein and hence have IL-35R
activity when complexed with the appropriate binding partner. Thus,
fragments of a polynucleotide may range from at least about 20
nucleotides, about 50 nucleotides, about 100 nucleotides, about
150, about 200, about 250, about 300, about 350, about 400, about
450, about 500, about 550, about 600 and up to the full-length
polynucleotide encoding the gp130 or IL12R.beta.2 polypeptide.
[0032] A fragment of a polynucleotide that encodes a biologically
active portion of a gp130 or IL12R.beta.2 polypeptide will encode
at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino
acids, or up to the total number of amino acids present in a
full-length gp130 and IL12R.beta.2 polypeptide.
[0033] A biologically active portion of a gp130 or IL12R.beta.2
polypeptide can be prepared by isolating a portion of one of the
polynucleotides encoding the portion of the gp130 or IL12R.beta.2
polypeptide and expressing the encoded portion of the polypeptide
(e.g., by recombinant expression in vitro), and assessing the
activity of the portion of the gp130 or IL12R.beta.2 polypeptide.
Polynucleotides that encode fragments of a gp130 or IL12R.beta.2
polypeptide can comprise nucleotide sequence comprising at least
16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or 1,400
nucleotides, or up to the number of nucleotides present in a
full-length gp130 and IL12R.beta.2 nucleotide sequence disclosed
herein.
[0034] "Variant" sequences have a high degree of sequence
similarity. For polynucleotides, conservative variants include
those sequences that, because of the degeneracy of the genetic
code, encode the amino acid sequence of one of the gp130 or
IL12R.beta.2 polypeptides. Variants such as these can be identified
with the use of well-known molecular biology techniques, as, for
example, polymerase chain reaction (PCR) and hybridization
techniques. Variant polynucleotides also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode a gp130
or a IL12R.beta.2 polypeptide. Generally, variants of a particular
polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more sequence identity to that particular
polynucleotide as determined by sequence alignment programs and
parameters described elsewhere herein.
[0035] Variants of a particular polynucleotide can also be
evaluated by comparison of the percent sequence identity between
the polypeptide encoded by a variant polynucleotide and the
polypeptide encoded by the reference polynucleotide. Thus, for
example, isolated polynucleotides that encode a polypeptide with a
given percent sequence identity to the gp130 and IL12R.beta.2
polypeptides set forth herein. Percent sequence identity between
any two polypeptides can be calculated using sequence alignment
programs and parameters described. Where any given pair of
polynucleotides is evaluated by comparison of the percent sequence
identity shared by the two polypeptides they encode, the percent
sequence identity between the two encoded polypeptides is at least
about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity.
[0036] By "variant" protein is intended a protein derived from the
native protein by deletion (so-called truncation) or addition of
one or more amino acids to the N-terminal and/or C-terminal end of
the native protein; deletion or addition of one or more amino acids
at one or more sites in the native protein; or substitution of one
or more amino acids at one or more sites in the native protein.
Variant proteins are biologically active, that is they continue to
possess the desired biological activity of the native protein, that
is, IL-35R activity. Such variants may result from, for example,
genetic polymorphism or from human manipulation. Biologically
active variants of a gp130 or IL12R.beta.2 polypeptides will have
at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to the amino acid sequence for the native protein as
determined by sequence alignment programs and parameters described
elsewhere herein. A biologically active variant of a protein may
differ from that protein by as few as 1-15 amino acid residues, as
few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even
1 amino acid residue.
[0037] Proteins may be altered in various ways including amino acid
substitutions, deletions, truncations, and insertions. Methods for
such manipulations are generally known in the art. For example,
amino acid sequence variants of the gp130 or IL12R.beta.2 proteins
can be prepared by mutations in the DNA. Methods for mutagenesis
and nucleotide sequence alterations are well known in the art. See,
for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492;
Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No.
4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular
Biology (MacMillan Publishing Company, New York) and the references
cited therein. Guidance as to appropriate amino acid substitutions
that do not affect biological activity of the protein of interest
may be found in the model of Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington,
D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be preferable.
[0038] Thus, the polynucleotides used in the invention can include
the naturally occurring sequences, the "native" sequences, as well
as mutant forms. Likewise, the proteins used in the methods of the
invention encompass naturally occurring proteins as well as
variations and modified forms thereof. Such variants will continue
to possess the ability to implement a recombination event.
Generally, the mutations made in the polynucleotide encoding the
variant polypeptide should not place the sequence out of reading
frame, and/or create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication
No. 75,444.
[0039] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays.
[0040] Variant polynucleotides and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different gp130 or IL12R.beta.2 coding sequences can be manipulated
to create a new gp130 or IL12R.beta.2 polypeptides possessing the
desired properties. In this manner, libraries of recombinant
polynucleotides are generated from a population of related sequence
polynucleotides comprising sequence regions that have substantial
sequence identity and can be homologously recombined in vitro or in
vivo. Strategies for such DNA shuffling are known in the art. See,
for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA
91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al.
(1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol.
Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA
94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S.
Pat. Nos. 5,605,793 and 5,837,458.
[0041] iv. IL-35R Binding and/or Modulating Agents
[0042] 1. Modulating Agents
[0043] As used herein, the term "modulating" includes "inducing",
"inhibiting", "potentiating", "elevating", "increasing",
"decreasing" or the like. Each of these terms denote a quantitative
difference between two states and in particular, refer to at least
a statistically significant difference between the two states.
[0044] The term "IL-35R agonist" refers to an agent which
potentiates, induces or otherwise enhances one or more of the
biological properties of the IL-35R complex. The activity increases
by a statistically significant amount including, for example, an
increase of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95% or 100% of the
activity of the IL-35R complex compared to an appropriate
control.
[0045] The term "IL-35R antagonist" refers to an agent which
reduces, inhibits, or otherwise diminishes one or more of the
biological activities of the IL-35R complex. Antagonism using the
IL-35R antagonist does not necessarily indicate a total elimination
of the IL-35R activity. Instead, the activity could decrease by a
statistically significant amount including, for example, a decrease
of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 95% or 100% of the activity of
the IL-35R complex compared to an appropriate control.
[0046] By "specific modulating agent" is intended an agent that
modulates the activity of a defined target. Thus, an IL-35R
specific modulating agent modulates the biological activity of
IL-35R by a statically significant amount (i.e., at least 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater) and the
agent does not modulate the biological activity of any monomeric
subunits, homodimeric complexes or non-IL-35R heterodimeric
complexes which comprise either IL12R.beta.2 or gp130 by a
statistically significant amount (i.e., the activity is modulated
by less than 5%, 4%, 3%, 2% or 1%). One of skill will be aware of
the proper controls that are needed to carry out such a
determination. An IL-35R specific modulating agent may or may not
be an IL-35R specific binding agent.
[0047] In one non-limiting embodiment, the IL-35R modulating agent
comprises a soluble IL-35R complex. Such a soluble complex is an
IL-35R. As used herein, a "soluble IL-35R complex" comprises an
IL-35R polypeptide that is incapable of anchoring itself in a
membrane. Such soluble IL-35R polypeptides include, for example, a
complex of gp130 and/or IL12R.beta.2 polypeptides that lack a
sufficient portion of their membrane spanning domain to anchor the
IL-35R to the membrane or such polypeptides are modified such that
the membrane spanning domain is non-functional. For example, a
soluble fragment of a gp130 polypeptide comprises the extracellular
domain of gp130, including a fragment of the extracellular domain
that is at least 20, 30, 40, 50, 60, 70, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 590 or greater consecutive amino
acids of gp130. A soluble fragment of an IL12R.beta.2 polypeptide
comprises the extracellular domain of IL12R.beta.2, including a
fragment of the extracellular domain that is at least 20, 30, 40,
50, 60, 70, 90, 100, 150, 200, 250, 300, 350, 400, 450, 495 or
greater consecutive amino acids of IL12R.beta.2. In specific
embodiments, the soluble IL-35R complex binds IL-35. In other
embodiments, the extracellular domains of gp130 and IL12R.beta.2
that are present in the soluble form of the IL-35R complex share at
least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% to the amino acid sequence or the polynucleotide sequences
as set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6. The soluble IL-35R
complex find further use in stabilizing IL-35 (increasing
half-life) or acting to concentrate IL-35.
[0048] A soluble IL-35R complex can additionally include a second
moiety. The second moiety can be any chemical compound. In specific
embodiments, the second moiety adds in the detection of the soluble
complex or promotes the overall solubility of the complex. Such
moieties include, but are not limited to, an immunoglobulin chain,
a GST, Lex-A or MBP polypeptide sequence. For example, a fusion
protein can includes at least a fragment of an IL-35R complex,
which is capable of binding IL-35, wherein the IL-35R complex
comprises a soluble fragment of a gp130 (e.g., a fragment of gp130
comprising the extracellular domain of gp130) and a soluble
fragment of IL12R.beta.2 (e.g., a fragment of IL12R.beta.2
comprising the extracellular domain of IL12R.beta.2) wherein at
least one of the gp130 fragment, the IL12R.beta.2 fragment, or both
are fused to a second moiety. In specific embodiments, the second
moiety comprises an immunoglobulin chain, an Fc fragment (CH.sub.2,
hinge, CH.sub.3 constant region domains), a heavy chain constant
region domain of the various isotypes, including: IgG1, IgG2, IgG3,
IgG4, IgM, IgA1, IgA2, IgD, and IgE).
[0049] A soluble form of the IL-35R complex can be generated using
various protein motifs that assist in complex formation. One such
motif comprises a leucine zipper motif. Leucine zipper domains are
peptides that promote oligomerization of the polypeptides in which
they are found. Leucine zippers were originally identified in
several DNA-binding polypeptides (Landschulz et al. (1988) Science
240:1759), and have since been found in a variety of different
polypeptides. Among the known leucine zippers are naturally
occurring peptides and derivatives thereof that dimerize or
trimerize. The zipper domain (also referred to herein as an
oligomerizing, or oligomer-forming, domain) comprises a repetitive
heptad repeat, often with four or five leucine residues
interspersed with other amino acids. Use of leucine zippers and
preparation of oligomers using leucine zippers are well known in
the art. Any other method which assists in the stabilization of the
soluble complex can be employed.
[0050] It is recognized that a soluble IL-35R complex can be
encoded on a single polynucleotide. For example, in one embodiment,
a polynucleotide comprising a nucleotide sequence encoding a
soluble Interleukin 35 receptor (IL-35R) complex is provides and
comprise a first sequence encoding the extracellular domain of
gp130, an active fragment or variant thereof; and a second sequence
encoding the extracellular domain of IL12R.beta.2, active fragment
or variant thereof, the encoded soluble polypeptide complex bind
IL-35. In another embodiment, the soluble IL-35R complex is encoded
on distinct polynucleotides.
[0051] In another embodiment, a "specific modulating agent" can
comprise an agent, such as an antibody, which modulates the ability
of the IL-35R to be activated by IL-35 but permits the IL-35R
complex to be activated by other non-IL-35 ligands. Such agents can
be IL-35R binding agents, a gp130 binding agent, or an IL12R.beta.2
binding agent.
[0052] 2. IL-35R Binding Agents As used herein, an "IL-35R binding
agent" refers to any compound that directly interacts with or binds
to the IL-35R complex. By "specific binding agent" is intended an
agent that binds substantially only to a defined target. Thus, an
IL-35R specific binding agent interacts directly with IL-35R and
binds substantially only to epitopes which are formed upon the
interaction of IL12R.beta.2 and gp130 to form the biologically
active IL-35R. Thus, an IL-35R specific binding agent will not
substantially interact with monomeric protein subunits comprising
IL12R.beta.2 or gp130 and the agent will not substantially interact
with homodimeric or non-IL-35R heterodimeric complexes which
comprise IL12R.beta.2 or gp130 in a statistically significant
amount. By "specifically or selectively binds to an IL-35R complex"
is intended that the binding agent has a binding affinity for a
non-IL-35R epitope which is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2% or 1% of the binding affinity for the unique IL-35R epitope.
One of skill will be aware of the proper controls that are needed
to carry out such a determination. An IL-35R specific binding agent
may or may not modulate the activity of IL-35R.
[0053] By "IL-35R specific binding/modulating agent" is intended an
agent that possesses the properties of both an IL-35R specific
binding agent and an IL-35R specific modulating agent. The IL-35R
specific binding and/or modulating agent can be an IL-35R agonist
or an IL-35R antagonist.
[0054] By "IL-35 activation" is intended any activity resulting
from the binding of IL-35 to the IL-35R complex. As used herein, an
agent that "specifically inhibits" IL-35 activity of the IL-35R
complex will substantially block the activity of IL-35R by IL-35,
but will not significantly block the activity of IL-35R by a
non-IL-35 ligand.
[0055] In one embodiment, the IL-35R binding and/or modulating
agent is a small molecule. For example, such small molecules
include, but are not limited to, peptides, peptidomimetics, amino
acids, amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e., including heterorganic and organometallic compounds).
[0056] 3. Anti-IL-35R Antibodies
[0057] As noted herein, the invention includes antibodies that
specifically bind to the IL-35R complex. Antibodies, including
monoclonal antibodies (mAbs), can be made by standard protocols.
See, for example, Harlow and Lane, Using Antibodies: A Laboratory
Manual, CSHL, New York, 1999. Briefly, a mammal such as a mouse,
hamster or rabbit can be immunized with an immunogenic form of a
peptide. Techniques for conferring immunogenicity on a protein or
peptide include conjugation to carriers or other techniques, well
known in the art. In preferred embodiments, the subject antibodies
are immunospecific for the unique antigenic determinants of
IL-35R.
[0058] As discussed herein, these antibodies are collectively
referred to as "anti-IL-35R antibodies". Thus, by "anti-IL-35R
antibodies" is intended antibodies specific for IL-35R. All of
these antibodies are encompassed by the discussion herein. The
respective antibodies can be used alone or in combination in the
methods of the invention.
[0059] By "antibodies that specifically bind" is intended that the
antibodies will not substantially cross react with another
polypeptide. By "not substantially cross react" is intended that
the antibody or fragment has a binding affinity for a
non-homologous protein which is less than 10%, less than 5%, or
less than 1%, of the binding affinity for the IL-35R complex.
[0060] In specific embodiments, the anti-IL-35R antibody binds
specifically to IL-35R and further modulates the activity of the
IL-35R complex. Thus, in specific embodiments, the anti-IL-35R
antibody is an IL-35R agonist or is an IL-35R antagonist.
[0061] The anti-IL-35R antibodies disclosed herein and for use in
the methods of the present invention can be produced using any
antibody production method known to those of skill in the art.
Thus, polyclonal sera may be prepared by conventional methods. In
general, a solution containing the IL-35R complex or an active
variant or fragment thereof is first used to immunize a suitable
animal, preferably a mouse, rat, rabbit, or goat. Rabbits or goats
are preferred for the preparation of polyclonal sera due to the
volume of serum obtainable, and the availability of labeled
anti-rabbit and anti-goat antibodies.
[0062] Polyclonal sera can be prepared in a transgenic animal,
preferably a mouse bearing human immunoglobulin loci. In a
preferred embodiment, Sf9 (Spodoptera frugiperda) cells expressing
IL-35R are used as the immunogen Immunization can also be performed
by mixing or emulsifying the antigen-containing solution in saline,
preferably in an adjuvant such as Freund's complete adjuvant, and
injecting the mixture or emulsion parenterally (generally
subcutaneously or intramuscularly). A dose of 50-200
.mu.g/injection is typically sufficient Immunization is generally
boosted 2-6 weeks later with one or more injections of the protein
in saline, preferably using Freund's incomplete adjuvant. One may
alternatively generate antibodies by in vitro immunization using
methods known in the art, which for the purposes of this invention
is considered equivalent to in vivo immunization. Polyclonal
antisera are obtained by bleeding the immunized animal into a glass
or plastic container, incubating the blood at 25.degree. C. for one
hour, followed by incubating at 4.degree. C. for 2-18 hours. The
serum is recovered by centrifugation (e.g., 1,000.times.g for 10
minutes). About 20-50 ml per bleed may be obtained from
rabbits.
[0063] Production of the Sf9 cells is disclosed in U.S. Pat. No.
6,004,552. Briefly, sequences encoding IL-35R complex are
recombined into a baculovirus using transfer vectors. The plasmids
are co-transfected with wild-type baculovirus DNA into Sf9 cells.
Recombinant baculovirus-infected Sf9 cells are identified and
clonally purified.
[0064] Preferably the antibody is monoclonal in nature. By
"monoclonal antibody" is an antibody obtained from a population of
substantially homogeneous antibodies, that is, the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. The term is not limited regarding the species or source of
the antibody. The term encompasses whole immunoglobulins as well as
fragments such as Fab, F(ab')2, Fv, and others which retain the
antigen binding function of the antibody. Monoclonal antibodies are
highly specific, being directed against a single antigenic site on
the target polypeptide. Furthermore, in contrast to conventional
(polyclonal) antibody preparations that typically include different
antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the
antigen. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein (Nature 256:495-97, 1975), or may be made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
"monoclonal antibodies" may also be isolated from phage antibody
libraries using the techniques described in, for example, Clackson
et al. (Nature 352:624-28, 1991), Marks et al. (J. Mol. Biol.
222:581-97, 1991) and U.S. Pat. No. 5,514,548.
[0065] By "epitope" is the part of an antigenic molecule to which
an antibody is produced and to which the antibody will bind.
Epitopes can comprise linear amino acid residues (i.e., residues
within the epitope are arranged sequentially one after another in a
linear fashion), nonlinear amino acid residues (referred to herein
as "nonlinear epitopes"--these epitopes are not arranged
sequentially), or both linear and nonlinear amino acid
residues.
[0066] As discussed herein, mAbs can be prepared using the method
of Kohler and Milstein, or a modification thereof. Typically, a
mouse is immunized with a solution containing an antigen.
Immunization can be performed by mixing or emulsifying the
antigen-containing solution in saline, preferably in an adjuvant
such as Freund's complete adjuvant, and injecting the mixture or
emulsion parenterally. Any method of immunization known in the art
may be used to obtain the monoclonal antibodies of the invention.
After immunization of the animal, the spleen (and optionally,
several large lymph nodes) are removed and dissociated into single
cells. The spleen cells may be screened by applying a cell
suspension to a plate or well coated with the antigen of interest.
The B cells expressing membrane bound immunoglobulin specific for
the antigen bind to the plate and are not rinsed away. Resulting B
cells, or all dissociated spleen cells, are then induced to fuse
with myeloma cells to form hybridomas, and are cultured in a
selective medium. The resulting cells are plated by serial dilution
and are assayed for the production of antibodies that specifically
bind the antigen of interest (and that do not bind to unrelated
antigens). The selected mAb-secreting hybridomas are then cultured
either in vitro (e.g., in tissue culture bottles or hollow fiber
reactors), or in vivo (as ascites in mice).
[0067] Where the anti-IL-35R antibodies of the invention are to be
prepared using recombinant DNA methods, the DNA encoding the
monoclonal antibodies is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that
are capable of binding specifically to genes encoding the heavy and
light chains of murine antibodies). The hybridoma cells described
herein serve as a preferred source of such DNA. Once isolated, the
DNA can be placed into expression vectors, which are then
transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, to obtain the
synthesis of monoclonal antibodies in the recombinant host cells.
Review articles on recombinant expression in bacteria of DNA
encoding an antibody includes Skerra, A. (Curr. Opinion in Immunol.
5:256-62, 1993) and Phickthun, A. (Immunol. Revs. 130:151-88,
1992). Alternatively, antibody can be produced in a cell line such
as a CHO cell line, as disclosed in U.S. Pat. Nos. 5,545,403;
5,545,405 and 5,998,144. Briefly the cell line is transfected with
vectors capable of expressing a light chain and a heavy chain,
respectively. By transfecting the two proteins on separate vectors,
chimeric antibodies can be produced. Another advantage is the
correct glycosylation of the antibody.
[0068] Additionally, the term "anti-IL-35R antibody" as used herein
encompasses chimeric and humanized anti-IL-35R antibodies. By
"chimeric" antibodies is intended antibodies that are most
preferably derived using recombinant deoxyribonucleic acid
techniques and which comprise both human (including immunologically
"related" species, e.g., chimpanzee) and non-human components.
Thus, the constant region of the chimeric antibody is most
preferably substantially identical to the constant region of a
natural human antibody; the variable region of the chimeric
antibody is most preferably derived from a non-human source and has
the desired antigenic specificity to the IL-35R antigen. The
non-human source can be any vertebrate source that can be used to
generate antibodies to a human IL-35R antigen or material
comprising a human IL-35R antigen. Such non-human sources include,
but are not limited to, rodents (e.g., rabbit, rat, mouse, etc.;
see, e.g., U.S. Pat. No. 4,816,567) and non-human primates (e.g.,
Old World Monkeys, Apes, etc.; see, e.g., U.S. Pat. Nos. 5,750,105
and 5,756,096). As used herein, the phrase "immunologically active"
when used in reference to chimeric/humanized anti-IL-35R antibodies
means chimeric/humanized antibodies that bind human IL-35R.
[0069] By "humanized" is intended forms of anti-IL-35R antibodies
that contain minimal sequence derived from non-human immunoglobulin
sequences. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region (also known as complementarity determining
region or CDR) of the recipient are replaced by residues from a
hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit, or nonhuman primate having the desired
specificity, affinity, and capacity. The phrase "complementarity
determining region" refers to amino acid sequences which together
define the binding affinity and specificity of the natural Fv
region of a native immunoglobulin binding site. See, for example,
Chothia et al. (J. Mol. Biol. 196:901-17, 1987) and Kabat et al.
(U.S. Dept. of Health and Human Services, NIH Publication No.
91-3242, 1991). The phrase "constant region" refers to the portion
of the antibody molecule that confers effector functions.
[0070] Humanization can be essentially performed following the
methods described by Jones et al. (Nature 321:522-25, 1986),
Riechmann et al. (Nature 332:323-27, 1988) and Verhoeyen et al.
(Science 239:1534-36, 1988), by substituting rodent or mutant
rodent CDRs or CDR sequences for the corresponding sequences of a
human antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089;
5,693,761; 5,693,762; and 5,859,205. In some instances, residues
within the framework regions of one or more variable regions of the
human immunoglobulin are replaced by corresponding non-human
residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;
5,693,762; and 6,180,370). Furthermore, humanized antibodies may
comprise residues that are not found in the recipient antibody or
in the donor antibody. These modifications are made to further
refine antibody performance (e.g., to obtain desired affinity). In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
framework regions are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. Accordingly, such "humanized" antibodies may
include antibodies wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species.
[0071] Also encompassed by the term "anti-IL-35R antibodies" are
xenogeneic or modified anti-IL-35R antibodies produced in a
non-human mammalian host, more particularly a transgenic mouse,
characterized by inactivated endogenous immunoglobulin loci. In
such transgenic animals, competent endogenous genes for the
expression of light and heavy subunits of host immunoglobulins are
rendered non-functional and substituted with the analogous human
immunoglobulin loci. These transgenic animals produce human
antibodies in the substantial absence of light or heavy host
immunoglobulin subunits. See, for example, U.S. Pat. Nos. 5,877,397
and 5,939,598. Preferably, fully human antibodies to IL-35 can be
obtained by immunizing transgenic mice. One such mouse is disclosed
in U.S. Pat. Nos. 6,075,181; 6,091,001; and 6,114,598.
[0072] Fragments of the anti-IL-35R antibodies are suitable for use
in the methods of the invention so long as they retain the desired
affinity of the full-length antibody. Thus, a fragment of an
anti-IL-35R antibody will retain the ability to specifically bind
to IL-35R. Such fragments are characterized by properties similar
to the corresponding full-length anti-IL-35R antibody; that is, the
fragments will specifically bind IL-35R. Such fragments are
referred to herein as "antigen-binding" fragments.
[0073] Suitable antigen-binding fragments of an antibody comprise a
portion of a full-length antibody, generally the antigen-binding or
variable region thereof. Examples of antibody fragments include,
but are not limited to, Fab, F(ab').sub.2, and Fv fragments and
single-chain antibody molecules. By "Fab" is intended a monovalent
antigen-binding fragment of an immunoglobulin that is composed of
the light chain and part of the heavy chain. By F(ab').sub.2 is
intended a bivalent antigen-binding fragment of an immunoglobulin
that contains both light chains and part of both heavy chains. By
"single-chain Fv" or "sFv" antibody fragments is intended fragments
comprising the V.sub.H and V.sub.L domains of an antibody, wherein
these domains are present in a single polypeptide chain. See, for
example, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; and
5,856,456. Generally, the Fv polypeptide further comprises a
polypeptide linker between the V.sub.H and V.sub.L domains that
enables the sFv to form the desired structure for antigen binding.
For a review of sFv see Pluckthun, A. (1994) in The Pharmacology of
Monoclonal Antibodies, Vol. 113, ed. Rosenburg and Moore
(Springer-Verlag, New York), pp. 269-315.
[0074] Antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described
in, for example, McCafferty et al. (Nature 348:552-54, 1990) and
U.S. Pat. No. 5,514,548. Clackson et al. (Nature 352:624-28, 1991)
and Marks et al. (J. Mol. Biol. 222:581-97, 1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology 10:779-83, 1992), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nucleic. Acids Res.
21:2265-66, 1993). Thus, these techniques are viable alternatives
to traditional monoclonal antibody hybridoma techniques for
isolation of monoclonal antibodies.
[0075] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., J. Biochem. Biophys. Methods 24:107-17, 1992 and Brennan et
al., Science 229:81-3, 1985). However, these fragments can now be
produced directly by recombinant host cells. For example, the
antibody fragments can be isolated from the antibody phage
libraries discussed above. Alternatively, Fab fragments can be
directly recovered from E. coli and chemically coupled to form
F(ab').sub.2 fragments (Carter et al., Bio/Technology 10:163-67,
1992). According to another approach, F(ab').sub.2 fragments can be
isolated directly from recombinant host cell culture. Other
techniques for the production of antibody fragments will be
apparent to the skilled practitioner.
[0076] A representative assay to detect anti-IL-35R antibodies
specific to the unique epitopes form upon complex formation of
IL-35R is a "competitive binding assay." Competitive binding assays
are serological assays in which unknowns are detected and
quantitated by their ability to inhibit the binding of a labeled
known ligand to its specific antibody. Antibodies employed in such
immunoassays may be labeled or unlabeled. Unlabeled antibodies may
be employed in agglutination; labeled antibodies may be employed in
a wide variety of assays, employing a wide variety of labels.
Detection of the formation of an antibody-antigen complex between
an anti-IL-35R antibody and an epitope of interest can be
facilitated by attaching a detectable substance to the antibody.
Suitable detection means include the use of labels such as
radionucleotides, enzymes, coenzymes, fluorescers,
chemiluminescers, chromogens, enzyme substrates or co-factors,
enzyme inhibitors, prosthetic group complexes, free radicals,
particles, dyes, and the like. Such labeled reagents may be used in
a variety of well-known assays, such as radioimmunoassays, enzyme
immunoassays, e.g., ELISA, fluorescent immunoassays, and the like.
See, for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;
and 4,233,402.
[0077] In still further embodiments, the antibody is bispecific,
wherein a first antigen binding domain specifically interacts with
an epitope of gp130 and said second antigen binding domain
specifically interacts with an epitope of IL12R.beta.2.
[0078] Further provided is a mixture of a first and a second
antibody. The mixture comprise a first antibody having a first
chemical moiety and the first antibody binds substantially only to
gp130 and a second antibody having a second chemical moiety and the
second antibody bind substantially only to a second polypeptide
comprising IL12R.beta.2. The first and the second chemical moiety
allow for the interaction of said first and said second antibody at
an IL-35R complex to be detected. Methods for such forms of
detection and chemical moieties of interest are discussed elsewhere
herein.
[0079] 4. Anti-gp130 and Anti-IL12R.beta.2Antibodies
[0080] The compositions further include antibodies that
specifically bind to the constituents of the IL-35R complex: gp130
and IL12R.beta.2. As described above, techniques for conferring
immunogenicity on a protein or peptide include conjugation to
carriers or other techniques, well known in the art. In preferred
embodiments, anti-gp130 and anti-IL12R.beta.2 antibodies are
immunospecific for the unique antigenic determinants of gp130 and
IL12R.beta.2, respectively. The term "anti-gp130 antibody" as used
herein encompasses an antibody that binds substantially only to a
gp130 polypeptide or a biologically active variant or fragment
thereof, where the antibody behaves as a specific modulating agent
for IL-35R and substantially inhibits IL-35 activation of the
IL-35R complex. The term "anti-IL12R.beta.2_antibody" as used
herein encompasses an antibody that binds substantially only to an
IL12R.beta.2 polypeptide or a biologically active variant or
fragment thereof, where such an antibody behaves as a specific
modulating agent for IL-35R and substantially inhibits IL-35
activation of the IL-35R complex. In some embodiments, anti-gp130
and anti-IL12R.beta.2_antibodies can be "specific modulating
agents" which modulate the ability of the IL-35R to be activated by
IL-35 but permit the IL-35R complex to be activated by other
non-IL-35 ligands. Thus, in some embodiments, an anti-gp130
antibody can bind substantially only to a gp130 polypeptide, or a
biologically active variant or fragment thereof, and act as a
specific modulating agent for IL-35R by substantially and
specifically inhibiting IL-35 activation of the IL-35R complex. In
such cases, IL-35R activation by non-IL-35 ligands can include, for
example, the binding of Interleukin 27 (IL-27) or interleukin 12
(IL-12) to the IL-35R complex. Methodologies provided herein for
the production and use of anti-IL-35R antibodies can be adapted to
make and use anti-gp130 and anti-IL12R.beta.2 antibodies,
[0081] vi. Expression Cassettes and Host Cells
[0082] The various polynucleotides of the invention can be
expressed in an expression cassette. An expression cassette
comprises one or more regulatory sequences, selected on the basis
of the cells to be used for expression, operably linked to the
desired polynucleotide. "Operably linked" is intended to mean that
the desired polynucleotide (i.e., gp130 and/or IL12R.beta.2 or
active variants and fragments thereof) is linked to the regulatory
sequence(s) in a manner that allows for expression of the
nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a cell when the expression cassette or vector is
introduced into a cell). "Regulatory sequences" include promoters,
enhancers, and other expression control elements (e.g.,
polyadenylation signals). See, for example, Goeddel (1990) in Gene
Expression Technology: Methods in Enzymology 185 (Academic Press,
San Diego, Calif.). Regulatory sequences include those that direct
constitutive expression of a nucleotide sequence in many types of
host cells, those that direct expression of the nucleotide sequence
only in certain host cells (e.g., tissue-specific regulatory
sequences), or those that direct expression of the polynucleotide
in the presence of an appropriate inducer (inducible promoter). It
will be appreciated by those skilled in the art that the design of
the expression cassette can depend on such factors as the choice of
the host cell to be transformed, the level of expression of the
polynucleotide that is desired, and the like. Such expression
cassettes typically include one or more appropriately positioned
sites for restriction enzymes, to facilitate introduction of the
nucleic acid into a vector.
[0083] As used herein, "heterologous" in reference to a sequence is
a sequence that originates from a foreign species, or, if from the
same species, is substantially modified from its native form in
composition and/or genomic locus by deliberate human intervention.
For example, a promoter operably linked to a heterologous
polynucleotide is from a species different from the species from
which the polynucleotide was derived, or, if from the
same/analogous species, one or both are substantially modified from
their original form and/or genomic locus, or the promoter is not
the native promoter for the operably linked polynucleotide.
Alternatively, a sequence that is heterologous to a cell is a
sequence that originates from a foreign species, or, if from the
same species, is substantially modified in the cell from its native
form in composition and/or genomic locus by deliberate human
intervention.
[0084] It will further be appreciated that appropriate promoter
and/or regulatory elements can readily be selected to allow
expression of the relevant transcription units in the cell of
interest. In certain embodiments, the promoter utilized to direct
intracellular expression of a silencing element is a promoter for
RNA polymerase III (Pol III). References discussing various Pol III
promoters, include, for example, Yu et al. (2002) Proc. Natl. Acad.
Sci. 99(9), 6047-6052; Sui et al. (2002) Proc. Natl. Acad. Sci.
99(8), 5515-5520 (2002); Paddison et al. (2002) Genes and Dev. 16,
948-958; Brummelkamp et al. (2002) Science 296, 550-553; Miyagashi
(2002) Biotech. 20, 497-500; Paul et al. (2002) Nat. Biotech. 20,
505-508; Tuschl et al. (2002) Nat. Biotech. 20, 446-448. According
to other embodiments, a promoter for RNA polymerase I, e.g., a tRNA
promoter, can be used. See McCown et al. (2003) Virology
313(2):514-24; Kawasaki (2003) Nucleic Acids Res. 31 (2):700-7.
[0085] The regulatory sequences can also be provided by viral
regulatory elements. For example, commonly used promoters are
derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells, see Chapters 16 and 17 of
Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See,
Goeddel (1990) in Gene Expression Technology: Methods in Enzymology
185 (Academic Press, San Diego, Calif.).
[0086] Various constitutive promoters are known. For example, in
various embodiments, the human cytomegalovirus (CMV) immediate
early gene promoter, the SV40 early promoter, the Rous sarcoma
virus long terminal repeat, rat insulin promoter and
glyceraldehyde-3-phosphate dehydrogenase can be used to obtain
high-level expression of the coding sequence of interest. The use
of other viral or mammalian cellular or bacterial phage promoters
which are well-known in the art to help achieve expression of a
coding sequence of interest. Promoters which may be used include,
but are not limited to, the long terminal repeat as described in
Squinto et al. (1991) Cell 65:1 20); the SV40 early promoter region
(Bernoist and Chambon (1981) Nature 290:304 310), the CMV promoter,
the M-MuLV 5' terminal repeat the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell
22:787 797), the herpes thymidine kinase promoter (Wagner et al.
(1981) Proc. Natl. Acad. Sci. U.S.A. 78:144 1445), the regulatory
sequences of the metallothionine gene (Brinster et al. (1982)
Nature 296:39 42); the following animal transcriptional control
regions, which exhibit tissue specificity and have been utilized in
transgenic animals: elastase I gene control region which is active
in pancreatic acinar cells (Swift et al. (1984) Cell 38:639 646;
Ornitz et al. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399
409; MacDonald, 1987, Hepatology Z:425 515); insulin gene control
region which is active in pancreatic beta cells (Hanahan (1985)
Nature 315:115 122), immunoglobulin gene control region which is
active in lymphoid cells (Grosschedl et al. (1984) Cell 38:647 658;
Adames et al (1985) Nature 318:533 538; Alexander et al. (1987)
Mol. Cell. Biol. 7:1436 1444), mouse mammary tumor virus control
region which is active in testicular, breast, lymphoid and mast
cells (Leder et al. (1986) Cell 45:485 495).
[0087] Inducible promoters are also known. Non-limiting examples of
inducible promoters and their inducer inlcude MT II/Phorbol Ester
(TPA) (Palmiter et al. (1982) Nature 300:611) and heavy metals
(Haslinger and Karin (1985) Proc. Nat'l Acad. Sci. USA. 82:8572;
Searle et al. (1985) Mol. Cell. Biol. 5:1480; Stuart et al. (1985)
Nature 317:828; Imagawa et al. (1987) Cell 51:251; Karin et al.
(1987) Mol. Cell. Biol. 7:606; Angel et al. (1987) Cell 49:729;
McNeall et al. (1989) Gene 76:8); MMTV (mouse mammary tumor
virus)/Glucocorticoids (Huang et al. (1981) Cell 27:245; Lee et al.
(1981) Nature 294:228; Majors and Varmus (1983) Proc. Nat'l Acad.
Sci. USA. 80:5866; Chandler et al. (1983) Cell 33:489; Ponta et al.
(1985) Proc. Nat'l Acad. Sci. USA. 82:1020; Sakai et al. (1988)
Genes and Dev. 2:1144); .beta.-Interferon/poly(rI)X and poly(rc)
(Tavernier et al. (1983) Nature 301:634); Adenovirus 5 E2/E1A
(Imperiale and Nevins (1984) Mol. Cell. Biol. 4:875); c-jun/Phorbol
Ester (TPA), H.sub.2O.sub.2; Collagenase/Phorbol Ester (TPA) (Angel
et al. (1987) Mol. Cell. Biol. 7:2256); Stromelysin/Phorbol Ester
(TPA), IL-1 (Angel et al. (1987) Cell 49:729); SV40/Phorbol Ester
(TPA) (Angel et al. (1987) Cell 49:729); Murine MX Gene/Interferon,
Newcastle Disease Virus; GRP78 Gene/A23187 (Resendez Jr. et al.
(1988) Mol. Cell. Biol. 8:4579); .alpha.-2-Macroglobulin/IL-6;
Vimentin/Serum (Kunz et al. (1989) Nucl. Acids Res. 17:1121); MHC
Class I Gene H-2 kB/Interferon (Blanar et al. (1989) EMBO J.
8:1139); HSP70/E1a, SV40 Large T Antigen (Taylor and Kingston
(1990) Mol. Cell. Biol. 10:165; Taylor and Kingston (1990) Mol.
Cell. Biol. 10:176; Taylor et al. (1989) J. Biol. Chem. 264:15160);
Proliferin/Phorbol Ester-TPA (Mordacq and Linzer (1989) Genes and
Dev. 3:760); Tumor Necrosis Factor/PMA (Hensel et al. (1989)
Lymphokine Res. 8:347); Thyroid Stimulating Hormone a Gene/Thyroid
Hormone (Chatterjee et al. (1989) Proc. Nat'l Acad. Sci. USA.
86:9114); and, Insulin E Box/Glucose.
[0088] Such expression cassettes can be contained in a vector which
allow for the introduction of the expression cassette into a cell.
In specific embodiments, the vector allows for autonomous
replication of the expression cassette in a cell or may be
integrated into the genome of a cell. Such vectors are replicated
along with the host genome (e.g., nonepisomal mammalian vectors).
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids (vectors). However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses, and adeno-associated viruses). See, for
example, U.S. Publication 2005214851, herein incorporated by
reference.
[0089] Any expression cassette can further comprise a selection
marker. As used herein, the term "selection marker" comprises any
polynucleotide, which when expressed in a cell allows for the
selection of the transformed cell with the vector. For example, a
selection marker can confer resistance to a drug, a nutritional
requirement, or a cytotoxic drug. A selection marker can also
induce a selectable phenotype such as fluorescence or a color
deposit. A "positive selection marker" allows a cell expressing the
marker to survive against a selective agent and thus confers a
positive selection characteristic onto the cell expressing that
marker. Positive selection marker/agents include, for example,
Neo/G418, Neo/Kanamycin, Hyg/Hygromycin, hisD/Histidinol,
Gpt/Xanthine, Ble/Bleomycin, HPRT/Hypoxanthine. Other positive
selection markers include DNA sequences encoding membrane bound
polypeptides. Such polypeptides are well known to those skilled in
the art and can comprise, for example, a secretory sequence, an
extracellular domain, a transmembrane domain and an intracellular
domain. When expressed as a positive selection marker, such
polypeptides associate with the cell membrane. Fluorescently
labeled antibodies specific for the extracellular domain may then
be used in a fluorescence activated cell sorter (FACS) to select
for cells expressing the membrane bound polypeptide. FACS selection
may occur before or after negative selection.
[0090] A "negative selection marker" allows the cell expressing the
marker to not survive against a selective agent and thus confers a
negative selection characteristic onto the cell expressing the
marker. Negative selection marker/agents include, for example,
HSV-tk/Acyclovir or Gancyclovir or FIAU, Hprt/6-thioguanine,
Gpt/6-thioxanthine, cytosine deaminase/5-fluoro-cytosine,
diphtheria toxin or the ricin toxin. See, for example, U.S. Pat.
No. 5,464,764, herein incorporated by reference.
[0091] In preparing an expression cassette or a homologous
recombination cassette, the various DNA fragments may be
manipulated, so as to provide for the DNA sequences in the proper
orientation and, as appropriate, in the proper reading frame.
Toward this end, adapters or linkers may be employed to join the
DNA fragments or other manipulations may be involved to provide for
convenient restriction sites, removal of superfluous DNA, removal
of restriction sites, or the like. For this purpose, in vitro
mutagenesis, primer repair, restriction, annealing,
resubstitutions, e.g., transitions and transversions, may be
involved.
[0092] An "isolated" or "purified" polynucleotide or protein, or
biologically active portion thereof, is substantially or
essentially free from components that normally accompany or
interact with the polynucleotide or protein as found in its
naturally occurring environment. Thus, an isolated or purified
protein is substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. An "isolated" polynucleotide is free of
sequences (optimally protein encoding sequences) that naturally
flank the polynucleotide (i.e., sequences located at the 5' and 3'
ends of the polynucleotide) in the genomic DNA of the organism from
which the polynucleotide is derived. For example, in various
embodiments, the isolated polynucleotide can contain less than
about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide
sequence that naturally flank the polynucleotide in genomic DNA of
the cell from which the polynucleotide is derived. A protein that
is substantially free of cellular material includes preparations of
protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry
weight) of contaminating protein. When the protein of the invention
or biologically active portion thereof is recombinantly produced,
optimally culture medium represents less than about 30%, 20%, 10%,
5%, or 1% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
[0093] The use of the term "polynucleotide" is not intended to
limit the present invention to polynucleotides comprising DNA.
Those of ordinary skill in the art will recognize that
polynucleotides, can comprise ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides
and ribonucleotides include both naturally occurring molecules and
synthetic analogues. The polynucleotides of the invention also
encompass all forms of sequences including, but not limited to,
single-stranded forms, double-stranded forms, hairpins,
stem-and-loop structures, and the like.
[0094] Any cell can be used in the methods and compositions. In
specific embodiments, the cell is from a mammal, a primate, a
human, a domestic animal or an agricultural animal. In specific
embodiment, the cell is a non-human cell. Non-limiting animals that
the cell can be derived from include cattle, sheep, goats, pigs,
horses, rabbits, dogs, monkeys, cats, large felines (lions, tigers,
etc.), wolves, mice, rats, rabbits, deer, mules, bears, cows, pigs,
horses, oxen, zebras, elephants, and so on. The cell can further be
from a plant, a worm (e.g., C. elegans), an insect, a fish, a
reptile, an amphibian, a bird (including, but not limited to
chickens, turkeys, ducks, geese and the like), a marsupial, etc.
The cells can be derived from any tissue (diseased or healthy) from
any of these organisms. Expression of IL-35R can be engineered to
occur in any cell type that one would want to control growth or
proliferation of, especially tumor cells or tissues/cells that are
common targets of autoimmune diseases. Such host cells include
cultured cells (in vitro), explants and primary cultures (in vitro
and ex vivo).
[0095] The present invention further provides transgenic animals
expressing a first heterologous polynucleotide encoding gp130 or an
active variant or fragment thereof and a second heterologous
polynucleotide encoding an IL12R.beta.2 polypeptide or an active
variant or fragment thereof.
[0096] Such animals are useful as animal models having modulated
IL-35R activity, including for example, animal models having
modulated effector T cell function. In general, methods of
generating transgenic animals and transformed cell lines are well
known in the art (for example, see Grosveld et al., Transgenic
Animals, Academic Press Ltd., San Diego, Calif. (1992)). Using the
nucleotide sequences disclosed herein encoding gp130 and
IL12R.beta.2, a skilled artisan can readily generate transgenic
animals and transformed cell lines which contain and express both
heterologous sequences. Such animals serve as models for the
development of alternative therapies for therapies that modulate
effector T cell function.
[0097] Such methods of the invention involve introducing a
polypeptide or polynucleotide into a cell. "Introducing" is
intended to mean presenting to the cell the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell of the plant. The methods of the invention do
not depend on a particular method for introducing a sequence into a
cell, only that the polynucleotide or polypeptides gains access to
the interior of a cell. Methods for introducing polynucleotide or
polypeptides into various cell types are known in the art
including, but not limited to, stable transformation methods,
transient transformation methods, and virus-mediated methods.
[0098] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a cell integrates into the DNA
of the cell and is capable of being inherited by the progeny
thereof "Transient transformation" is intended to mean that a
polynucleotide is introduced into the cell and does not integrate
into the genome of the cell or a polypeptide is introduced into a
cell. Transformation protocols as well as protocols for introducing
polypeptides or polynucleotide sequences into cell may vary
depending on the type of cell targeted for transformation.
[0099] Exemplary art-recognized techniques for introducing foreign
polynucleotides into a host cell, include, but are not limited to,
calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, particle gun, or
electroporation and viral vectors. Suitable methods for
transforming or transfecting host cells can be found in U.S. Pat.
No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No.
4,897,355, Sambrook et al. (1989) Molecular Cloning: A Laboratory
Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview,
N.Y.) and other standard molecular biology laboratory manuals.
Various transfection agents can be used in these techniques. Such
agent are known, see for example, WO 2005012487. One of skill will
recognize that depending on the method by which a polynucleotide is
introduced into a cell, the silencing element can be stably
incorporated into the genome of the cell, replicated on an
autonomous vector or plasmid, or present transiently in the
cell.
[0100] Viral vector delivery systems include DNA and RNA viruses,
which have either episomal or integrated genomes after delivery to
the cell. For a review of viral vector procedures, see Anderson
(1992) Science 256:808-813; Haddada et al. (1995) Current Topics in
Microbiology and Immunology Doerfler and Bohm (eds); and Yu et al.
(1994) Gene Therapy 1:13-26. Conventional viral based systems for
the delivery of polynucleotides could include retroviral,
lentivirus, adenoviral, adeno-associated and herpes simplex virus
vectors for gene transfer. Integration in the host genome is
possible with the retrovirus, lentivirus, and adeno-associated
virus gene transfer methods, often resulting in long term
expression of the inserted transgene.
II. Uses, Methods, and Kits
[0101] The polynucleotide(s) encoding the IL-35R complex and active
variants and fragments thereof, the IL-35R complex and active
variants and fragments thereof, the soluble form of the IL-35R
complex, the IL-35R specific binding and/or modulating agents, and
the IL-35R agonist and antagonists disclosed herein can be used in
one or more of the following methods: (a) screening assays; (b)
detection assays; (c) predictive medicine (e.g., diagnostic assays,
prognostic assays, monitoring clinical trials, and
pharmacogenomics); and (d) methods of treatment (e.g., therapeutic
and prophylactic).
[0102] i. Methods to Screen for IL-35R Binding and/or
Modulating
[0103] The invention provides a method (also referred to herein as
a "screening assay") for identifying binding and/or modulating
agents of IL-35R. As discussed above, identification of various
IL-35R binding agents are of interest including agonist IL-35R
binding agents, antagonist IL-35R binding agents, and IL-35R
specific binding agents. Similarly, identification of various
IL-35R modulating agents are of interest including, for example,
IL-35R agonist and antagonists.
[0104] The test compounds employed in the various screening assays
can include any candidate compound including, for example,
peptides, peptidomimetics, small molecules, antibodies, or other
drugs. Such test compounds can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including biological libraries, spatially addressable parallel
solid phase or solution phase libraries, synthetic library methods
requiring deconvolution, the "one-bead one-compound" library
method, and synthetic library methods using affinity chromatography
selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to
peptide, nonpeptide oligomer, or small molecule libraries of
compounds (Lam (1997) Anticancer Drug Des. 12:145).
[0105] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678;
Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem.
37:1233.
[0106] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992)
Proc. Natl. Acad. Sci. USA 89:1865-1869), or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science 249:404-406;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and
Felici (1991) J. Mol. Biol. 222:301-310).
[0107] Determining the ability of the test compound to bind to the
IL-35R complex can be accomplished, for example, by coupling the
test compound with a radioisotope or enzymatic label such that
binding of the test compound to the IL-35R complex or a
biologically active portion thereof can be determined by detecting
the labeled compound in a complex. For example, test compounds can
be labeled with .sup.125I, .sup.35S, .sup.14C, or .sup.3H, either
directly or indirectly, and the radioisotope detected by direct
counting of radioemmission or by scintillation counting.
Alternatively, test compounds can be enzymatically labeled with,
for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
[0108] In one embodiment, an assay is a cell-free assay comprising
contacting an IL-35R complex or biologically active fragment or
variant thereof with a test compound and determining the ability of
the test compound to bind to the IL-35R complex or the biologically
active variant or fragment thereof. Binding of the test compound to
the IL-35R complex can be determined either directly or indirectly.
An indirect assay could include assaying for a modulation in IL-35R
activity. In a further embodiment, the test or candidate compound
specifically binds to or selectively binds to the IL-35R
complex.
[0109] In another embodiment, the assay includes contacting the
IL-35R complex or biologically active variant or fragment thereof
with a known compound that binds to the IL-35R complex (such as its
ligand, IL-35) to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to preferentially bind to IL-35R complex or
biologically active fragment or variant thereof as compared to the
known compound.
[0110] In another embodiment, an assay comprises contacting the
IL-35R complex or biologically active portion thereof with a test
compound and determining the ability of the test compound to
modulate (e.g., stimulate or inhibit, act as an agonist or
antagonist) the activity of the IL-35R complex or biologically
active portion thereof. Determining the ability of the test
compound to modulate the activity of an IL-35R complex can be
accomplished, for example, by determining the ability of the IL-35R
complex to bind to its ligand, IL-35, as described above, for
determining direct binding. In an alternative embodiment,
determining the ability of the test compound to modulate the
activity of an IL-35R complex can be accomplished by determining
the ability of the IL-35R complex to further modulate intercellular
downstream pathways modulated by IL-35R. Such activities are
discussed elsewhere herein.
[0111] In some assays, it may be desirable to immobilize either an
IL-35R complex or a biologically active fragment or variant thereof
or the test compound to facilitate separation of complexed from
uncomplexed forms of the IL-35R complex, as well as to accommodate
automation of the assay. In one embodiment, a fusion protein can be
provided that adds a domain that allows the IL-35R complex or
active fragment or variant thereof or the test agent to be bound to
a matrix. For example, glutathione-S-transferase/IL-35R complex
fusion proteins or glutathione-S-transferase/IL-35R complex fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma
Chemical, St. Louis, Mo.) or glutathione-derivatized microtitre
plates, which are then combined with the test compound, and the
mixture incubated under conditions conducive to complex formation
(e.g., at physiological conditions for salt and pH). Following
incubation, the beads or microtitre plate wells are washed to
remove any unbound components and complex formation is measured
either directly or indirectly, for example, as described above.
[0112] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either the IL-35R complex or active fragment thereof or the test
compound can be immobilized utilizing conjugation of biotin and
streptavidin. Biotinylated IL-35R complexes or active fragments
thereof or test agents can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96-well plates
(Pierce Chemicals).
[0113] In yet another aspect of the invention, the IL-35R complex
can be used as "bait proteins" in a two-hybrid assay or
three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication
No. WO 94/10300), to identify other proteins, which bind to or
interact with the IL-35R complex or active fragments and variants
thereof and, in some embodiments, modulate IL-35R complex
activity.
[0114] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0115] ii. Methods for Detecting
[0116] Various methods and compositions for detecting and/or
determining the level of expression of a polynucleotide encoding
gp130 and IL12R.beta.2 in a sample are provided. A biological
sample can comprise any sample in which one desires to determine
the level of expression of a polynucleotide encoding gp130 and
IL12R.beta.2 or one desires to detect or quantitate the level of
the IL-35R complex. The term "biological sample" is intended to
include tissues, cells, and biological fluids isolated from a
subject, as well as tissues, cells, and fluids present within a
subject. Detection of the expression of IL-35R in any cell type
that expresses IL-35R can be performed, including expression levels
in either diseased verses healthy tissue. That is, the detection
method of the invention can be used to detect gp130 mRNA or genomic
DNA, IL12R.beta.2 mRNA or genomic DNA, or the IL-35R complex in a
biological sample in vitro, as well as, in vivo. For example, in
vitro techniques for detection of include Northern hybridizations
and in situ hybridizations. In vitro techniques for detection of
the IL-35R complex include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations, and
immunofluorescence. In vitro techniques for detection of genomic
DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of the IL-35R complex include introducing
into a subject a labeled anti-IL-35R specific antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
a. Detecting Polynucleotides
[0117] In one embodiment, a method for detecting the level of
expression of a polynucleotide encoding gp130 or active variants
and fragments thereof and IL12R.beta.2 or active variants and
fragments thereof in a sample comprises contacting the sample with
a) a first and a second primer capable of specifically amplifying a
first amplicon comprising a polynucleotide encoding a gp130
polypeptide or an active variant or fragment thereof; and, b) a
third and a fourth primer capable of specifically amplifying a
second amplicon comprising a polynucleotide encoding an
IL12R.beta.2 polypeptide or an active variant or fragment thereof;
wherein the encoded polypeptides form a biologically active IL-35R
complex. The first and the second amplicon is amplified and then
detected.
[0118] In other embodiments, a method for detecting the level of
expression of a polynucleotide encoding gp130 or active variants
and fragments thereof and IL12R.beta.2 or active variants and
fragments thereof in a sample comprises contacting the sample with
a) a first polynucleotide capable of specifically detecting a
polynucleotide encoding a gp130 polypeptide or an active variant or
fragment thereof; and, b) a second polynucleotide capable of
specifically detecting a polynucleotide encoding an IL12R.beta.2
polypeptide or an active variant or fragment thereof; wherein the
encoded polypeptides form a biologically active IL-35R complex; and
detecting the polynucleotide encoding the gp130 polypeptide or an
active variant or fragment thereof and detecting the polynucleotide
encoding the IL12R.beta.2 polypeptide or an active variant or
fragment thereof.
[0119] In specific embodiments, the sample is contacted with a
polynucleotide probe that hybridizes under stringent hybridization
conditions to the target sequences to be detected. The sample and
probes are then subjected the sample and probe to stringent
hybridization conditions and the hybridization of the probe to the
target sequences is detected.
[0120] Primers and probes are based on the sequence of the
polynucleotides encoded by gp130 and IL12R.beta.2 or active
variants and fragments thereof. Any conventional nucleic acid
hybridization or amplification method can be used to identify the
presence of the polynucleotides encoded by gp130 and IL12R.beta.2
in a sample. By "specifically detect" is intended that the
polynucleotide can be used either as a primer to specifically
amplify an amplicon of a polynucleotide encoding gp130 or
IL12R.beta.2 or the polynucleotide can be used as a probe that
hybridizes under stringent conditions to a polynucleotide encoding
gp130 or IL12R.beta.2. The level or degree of hybridization which
allows for the specific detection or the specific amplification of
a polynucleotide encoding gp130 or IL12R.beta.2 is sufficient to
distinguish the polynucleotide encoding gp130 or IL12R.beta.2 from
a polynucleotide that does not encode the recited polypeptide. By
"shares sufficient sequence identity or complementarity to allow
for the amplification of a polynucleotide encoding gp130 or
IL12R.beta.2" is intended the sequence shares at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity
or complementarity to a fragment or across the full length of the
polynucleotide encoding gp130 or IL12R.beta.2.
[0121] Regarding the amplification of a target polynucleotide
(e.g., by PCR) using a particular amplification primer pair,
"stringent conditions" are conditions that permit the primer pair
to hybridize to the target polynucleotide to which a primer having
the corresponding wild-type sequence (or its complement) would bind
and preferably to produce an identifiable amplification product
(the amplicon) in a DNA thermal amplification reaction. In a PCR
approach, oligonucleotide primers can be designed for use in PCR
reactions to amplify a polynucleotide encoding gp130 or
IL12R.beta.2. Methods for designing PCR primers and PCR cloning are
generally known in the art and are disclosed in Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring
Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al.,
eds. (1990) PCR Protocols: A Guide to Methods and Applications
(Academic Press, New York); Innis and Gelfand, eds. (1995) PCR
Strategies (Academic Press, New York); and Innis and Gelfand, eds.
(1999) PCR Methods Manual (Academic Press, New York). Methods of
amplification are further described in U.S. Pat. Nos. 4,683,195,
4,683,202 and Chen et al. (1994) PNAS 91:5695-5699. These methods
as well as other methods known in the art of DNA amplification may
be used in the practice of the embodiments of the present
invention. It is understood that a number of parameters in a
specific PCR protocol may need to be adjusted to specific
laboratory conditions and may be slightly modified and yet allow
for the collection of similar results. These adjustments will be
apparent to a person skilled in the art.
[0122] The amplified polynucleotide (amplicon) can be of any length
that allows for the detection of the polynucleotide encoding gp130
or IL12R.beta.2. For example, the amplicon can be about 10, 50,
100, 200, 300, 500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in
length or longer.
[0123] Any primer can be employed in the methods of the invention
that allows a polynucleotide encoding gp130 or IL12R.beta.2 to be
amplified and/or detected. For example, in specific embodiments,
the first primer pair comprises primers comprising a fragment of a
polynucleotide encoding gp130, wherein the first primer pair shares
sufficient sequence identity or complementarity to the
polynucleotide to amplify the polynucleotide encoding gp130; and,
the second primer pair comprises primers comprising a fragment of a
polynucleotide encoding IL12R.beta.2, wherein the first primer pair
shares sufficient sequence identity or complementarity to the
polynucleotide to amplify the polynucleotide encoding IL12R.beta.2.
In specific embodiments, the primer can comprise at least 8, 10,
15, 20, 25, 30, 40 or greater consecutive nucleotide of SEQ ID NO:
1, 2, 4 or 5. In order for a nucleic acid molecule to serve as a
primer or probe it need only be sufficiently complementary in
sequence to be able to form a stable double-stranded structure
under the particular solvent and salt concentrations employed.
[0124] In hybridization techniques, all or part of a polynucleotide
that selectively hybridizes to a target polynucleotide encoding
gp130 or IL12R.beta.2 is employed. By "stringent conditions" or
"stringent hybridization conditions" when referring to a
polynucleotide probe is intended conditions under which a probe
will hybridize to its target sequence to a detectably greater
degree than to other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences that are 100% complementary to the probe can be
identified (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of identity are detected (heterologous
probing). Generally, a probe is less than about 1000 nucleotides in
length or less than 500 nucleotides in length.
[0125] As used herein, a substantially identical or complementary
sequence is a polynucleotide that will specifically hybridize to
the complement of the nucleic acid molecule to which it is being
compared under high stringency conditions. Appropriate stringency
conditions which promote DNA hybridization, for example, 6Xsodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.times.SSC at 50.degree. C., are known to those skilled in
the art or can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically,
stringent conditions for hybridization and detection will be those
in which the salt concentration is less than about 1.5 M Na ion,
typically about 0.01 to 1.0 M Na ion concentration (or other salts)
at pH 7.0 to 8.3 and the temperature is at least about 30.degree.
C. for short probes (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes (e.g., greater than 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Optionally, wash
buffers may comprise about 0.1% to about 1% SDS. Duration of
hybridization is generally less than about 24 hours, usually about
4 to about 12 hours. The duration of the wash time will be at least
a length of time sufficient to reach equilibrium.
[0126] In hybridization reactions, specificity is typically the
function of post-hybridization washes, the critical factors being
the ionic strength and temperature of the final wash solution. For
DNA-DNA hybrids, the T.sub.m can be approximated from the equation
of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:
T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61 (%
form)-500/L; where M is the molarity of monovalent cations, % GC is
the percentage of guanosine and cytosine nucleotides in the DNA, %
form is the percentage of formamide in the hybridization solution,
and L is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. T.sub.m is reduced by about 1.degree. C. for each 1% of
mismatching; thus, T.sub.m, hybridization, and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity.
For example, if sequences with >90% identity are sought, the
T.sub.m can be decreased 10.degree. C. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence and its
complement at a defined ionic strength and pH. However, severely
stringent conditions can utilize a hybridization and/or wash at 1,
2, 3, or 4.degree. C. lower than the thermal melting point
(T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9, or 10.degree. C. lower
than the thermal melting point (T.sub.m); low stringency conditions
can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or
20.degree. C. lower than the thermal melting point (T.sub.m). Using
the equation, hybridization and wash compositions, and desired
T.sub.m, those of ordinary skill will understand that variations in
the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a T.sub.m of less than 45.degree. C. (aqueous solution) or
32.degree. C. (formamide solution), it is optimal to increase the
SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2
(Elsevier, New York); and Ausubel et al., eds. (1995) Current
Protocols in Molecular Biology, Chapter 2 (Greene Publishing and
Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Plainview, N.Y.) and Haymes et al. (1985) In: Nucleic Acid
Hybridization, a Practical Approach, IRL Press, Washington,
D.C.
[0127] A polynucleotide is said to be the "complement" of another
polynucleotide if they exhibit complementarity. As used herein,
molecules are said to exhibit "complete complementarity" when every
nucleotide of one of the polynucleotide molecules is complementary
to a nucleotide of the other. Two molecules are said to be
"minimally complementary" if they can hybridize to one another with
sufficient stability to permit them to remain annealed to one
another under at least conventional "low-stringency" conditions.
Similarly, the molecules are said to be "complementary" if they can
hybridize to one another with sufficient stability to permit them
to remain annealed to one another under conventional
"high-stringency" conditions.
[0128] b. Detecting the IL-35R Complex
[0129] One aspect of the present invention relates to assays for
detecting IL-35R complexes in the context of a biological sample.
An exemplary method for detecting the presence or absence or the
quantity of the IL-35R complex in a biological sample involves
obtaining a biological sample and contacting the biological sample
with a compound or an agent capable of specifically binding and
detecting an IL-35R complex, such that the presence of the IL-35R
complex is detected in the biological sample. Results obtained with
a biological sample from a test subject may be compared to results
obtained with a biological sample from a control subject.
[0130] Detection of IL-35R with an IL-35R specific binding agent
allows for the detection, purification, and/or isolation of cell
populations expressing IL-35R. Such methods find use in determining
cell populations that are sensitive or resistant to the effects of
IL-35. Information gained by such techniques can then be used when
designing IL-35 treatments or therapies.
[0131] In one embodiment, an agent for detecting the IL-35R complex
is an antibody capable of specifically binding to the IL-35R
complex, preferably an antibody with a detectable label. Antibodies
can be polyclonal, or more preferably, monoclonal. An intact
antibody, or a fragment thereof (e.g., Fab or F(abN).sub.2) can be
used. The term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary
antibody.
[0132] iii. Kits
[0133] As used herein, "kit" refers to a set of reagents for the
identification and/or the detection of the polynucleotide encoding
gp130 or IL12R.beta.2 or detection and/or quantitation of the
IL-35R complex in biological samples. The terms "kit" and "system,"
as used herein are intended to refer to at least one or more
detection reagents which, in specific embodiments, are in
combination with one or more other types of elements or components
(e.g., other types of biochemical reagents, containers, packages,
such as packaging intended for commercial sale, substrates to which
detection reagents are attached, electronic hardware components,
instructions of use, and the like).
[0134] In one embodiment, a kit for determining the level of
expression of a polynucleotide encoding gp130 and IL12R.beta.2 in a
sample is provided. The kit comprises a) a first polynucleotide
capable of specifically detecting or amplifying a polynucleotide
encoding a first polypeptide encoding gp130 or a biologically
active variant or fragment thereof; and, b) a second polynucleotide
capable of specifically detecting or amplifying a polynucleotide
encoding IL12R.beta.2 or a biologically active variant or fragment
thereof, wherein the encoded polypeptides form a biologically
active IL-35R complex.
[0135] In specific embodiments, the kit comprises a) a first and a
second primer that share sufficient sequence homology or
complementarity to the polynucleotide encoding gp130 or the active
variant or fragment thereof to specifically amplify the
polynucleotide encoding gp130; and, b) a third and a forth primer
that share sufficient sequence homology or complementarity to a
polynucleotide encoding IL12R.beta.2 or an active variant or
fragment thereof to specifically amplify the polynucleotide
encoding IL12R.beta.2.
[0136] In still other embodiments, the kit comprises a) a first
probe that can specifically detect the polynucleotide encoding
gp130 or the active variant or fragment thereof, wherein the first
probe comprises at least one polynucleotide of a sufficient length
of contiguous nucleotides identical or complementary to the
polynucleotide encoding gp130 or the active variant thereof; and,
b) a second probe that can specifically detect a second
polynucleotide encoding IL12R.beta.2 or an active variant or
fragment thereof, wherein the second probe comprises at least one
polynucleotide of a sufficient length of contiguous nucleotides
identical or complementary to a polynucleotide encoding
IL12R.beta.2 or an active variant or fragment thereof. In still
further embodiments, the first polynucleotide hybridizes under
stringent conditions to the sequence encoding the gp130 polypeptide
or active variant thereof; and, the second polynucleotide
hybridizes under stringent conditions to the sequence encoding
IL12R.beta.2 or an active variant or fragment thereof.
[0137] In still other embodiments, a kit for determining the
presence of Interleukin 35 Receptor (IL-35R) in a sample is
provided. Such a kit can comprises any IL-35R specific binding
and/or IL-35R specific binding/modulating agent disclosed herein,
including, but not limited to one or more of the IL-35R specific
antibodies disclosed herein or any mixture thereof
[0138] iv. Methods for Modulating the Activity of the IL-35R
Complex
[0139] Methods for modulating (i.e., inducing, inhibiting,
potentiating, elevating, increasing, decreasing) the activity of
the IL-35R complex or modulating effector T-cell function are
provided. Such methods can comprise contacting a cell expressing
the IL-35R complex with an IL-35R antagonists or agonists.
[0140] As used herein, "responder T cells" or "effector T cells"
refer to a subpopulation of mature T cells that facilitate an
immune response through cell activation and/or the secretion of
cytokines. As used herein, "effector T cells" include cytotoxic T
cells (Tc), including for example, CD8+ cells, and helper T cells
(Th1 cells, Th2 cells, CD4+, Th17, Th9, and gamma delta T-cells).
As used herein, "effector T cell function" includes an activity
exerted by an effector T cell, as determined in vitro or in vivo,
according to standard techniques. In one embodiment, the effector T
cell function includes the elimination of an antigen by, for
example, the production of cytokines preferentially associated with
effector T cells, which modulate the activation of other cells, or
by cytotoxic activity. In one embodiment, an effector T cell
function is a cytotoxic (or cytolytic) T cell (Tc or CTL) function,
such as, for example, cytolysis of cells infected with microbes. In
another embodiment, an effector T cell function is a Th1 cell
function, e.g., mediation of delayed type hypersensitivity
responses and macrophage activation. In yet another embodiment, an
effector T cell function is a Th2 cell function, e.g., help to B
cells (Mosmann et al. (1989) Annu. Rev. Immunol. 7, 145-173; Paul
et al. (1994) Cell 76, 241-251; Arthur and Mason (1986) J Exp. Med.
163, 774-786; Paliard et al. (1988) J. Immunol. 141, 849-855;
Finkelman et al., (1988) J. Immunol. 141, 2335-2341). In another
embodiment, an effector T cell function includes an inflammatory
response, the suppression of immunological tolerance, or "tipping
the balance" toward a proliferative/stimulatory environment. For
purposes of the invention, effector T cell function is enhanced or
inhibited by a statistically significant amount, for example, by at
least 5%, at least 10%, at least 15%, at least 20%, at least 30%,
at least 50%, at least 60%, at least 70%, at least 80%, or at least
90% as compared to an appropriate control cells.
[0141] An IL-35R agonist will act to suppress or inhibit effector
T-cell activity. The agonist can be, for example, an IL-35R
specific binding/modulating agent or an IL-35R specific modulating
agent. By "inhibiting or suppressing an effector T cell function in
a subject" is intended reducing and/or blocking of one or more of
the functions mediated by effector T cells. Thus, in one
embodiment, a method of suppressing an effector T cell function is
provided and comprises administering to the subject a
therapeutically effective amount of an Interleukin 35 Receptor
(IL-35R) agonist.
[0142] For example, an IL-35R agonist promotes immune tolerance,
which can find use, for example, in treating a subject having an
autoimmune or an inflammatory disorder, including but not limited
to, graft rejections and allergies. Thus, in one embodiment, a
method of treating a subject having an autoimmune or inflammatory
disorder is provided. Such a method comprises administering to the
subject a therapeutically effective amount of an agonist
Interleukin 35 Receptor (IL-35R) agent. Various agonist Interleukin
35 Receptor (IL-35R) agents and method for preparing such agents
are discuses elsewhere herein. In specific embodiments, the agonist
agent is an antibody or a small molecule. Examples of autoimmune
diseases include, for example, type 1 diabetes, rheumatoid
arthritis and multiple sclerosis. Inflammatory disorders that may
be treated include, for example, asthma and inflammatory bowel
disease. In addition, limiting IL-35 by IL-35 antagonist could help
enhance anti-tumor immunity elicited by effector T cells.
[0143] In other embodiments, the IL-35R agonist can be used in
combination with a therapeutic agent to reduce the immune response
to the agent (i.e., protein). For example, the IL-35R agonist can
be used in combination with a therapeutic protein which must be
chronically administered to a subject. Thus, in a specific
embodiment, the method comprises includes administering to the
subject at least one additional therapeutic agent in combination
with an IL-35R agonist. Such therapeutic agents, include but are
not limited to, a cytokine, a glucocorticoid, an anthracycline
(e.g., doxorubicin or epirubicin), a fluoroquinolone (e.g.,
ciprofloxacin), an antifolate (e.g., methotrexate), an
antimetabolite (e.g., fluorouracil), a topoisomerase inhibitor
(e.g., camptothecin, irinotecan or etoposide), an alkylating agent
(e.g., cyclophosphamide, ifosfamide, mitolactol, or melphalan), an
antiandrogen (e.g., flutamide), an antiestrogen (e.g., tamoxifen),
a platinum compound (e.g., cisplatin), a vinca alkaloid (e.g.,
vinorelbine, vinblastine or vindesine), a mitotic inhibitor (e.g.,
paclitaxel or docetaxel), an inhibitor of the PI3K/Akt/mTOR
pathway, such as rapamycin, and/or an inhibitor of calcineurin.
[0144] An IL-35R antagonist will act to enhance or promote effector
T-cell activity. The antagonist can be, for example, an IL-35R
specific binding/modulating agent or an IL-35R specific modulating
agent. By "enhancing an effector T cell function in a subject" is
intended reducing and/or blocking one or more of the functions
mediated by effector T cells. For example, an IL-35R antagonist
will act to increase or potentiate at least one effector T cell
function and thereby increase the immune response. Thus, in one
embodiment, a method of increasing an effector T cell function is
provided and comprises administering to the subject a
therapeutically effective amount of an antagonistic Interleukin 35
Receptor (IL-35R) agent.
[0145] Such IL-35R antagonists find use in treating any conditions
in which the IL-35 mediated activity of the T regulatory cells is
shown to be blocking or limiting disease resolution. For example,
the IL-35R antagonists find use when activation of effector
responses is desired such as in cases of acute infection, vaccine
response, anti-tumor immunity or treating cancer. Thus, in one
embodiment, a method of treating a subject having a cancer or acute
infection is provided. Such a method comprises administering to the
subject a therapeutically effective amount of an antagonistic
Interleukin 35 Receptor (IL-35R) agent. Various antagonistic
Interleukin 35 Receptor (IL-35R) agents and method for preparing
such agents are discuses elsewhere herein. In specific embodiments,
the antagonist agent is an antibody or a small molecule.
[0146] It is further recognized that the various IL-35R antagonists
can be used in combination with an antigen to enhance the immune
response to the antigen. For example, T effector cell responses
employing an IL-35R antagonist can be used to enhance a vaccine
preparation. Thus, the various IL-35R antagonist are useful for
increasing the efficacy of anti-cancer vaccines or for vaccines
that are poorly immunogenic.
[0147] Thus, further provided are methods for enhancing the
efficacy or immunogenicity of a vaccine in a subject, or overcoming
a suppressed immune response to a vaccine in a subject, including
(i) administering to the subject a therapeutically effective amount
of an antagonist IL-35R agent and (ii) administering to the subject
a vaccine. In specific embodiments, the antagonist is an IL-35R
specific binding/modulating agent. In one embodiment, the vaccine
is a cancer vaccine. For example, immune responses are suppressed
in cancer and chronic infections and thus combining IL-35R agonists
with therapeutic cancer vaccines or vaccines against chronic
infections such as HCV, HIV and TB could improve efficacy.
[0148] By "vaccine" is intended a composition useful for
stimulating a specific immune response (or immunogenic response) in
a subject. In some embodiments, the immunogenic response is
protective or provides protective immunity. For example, in the
case of a disease-causing organism the vaccine enables the subject
to better resist infection with or disease progression from the
organism against which the vaccine is directed. Alternatively, in
the case of a cancer, the vaccine strengthens the subject's natural
defenses against cancers that have already developed. These types
of vaccines may also prevent the further growth of existing
cancers, prevent the recurrence of treated cancers, and/or
eliminate cancer cells not killed by prior treatments.
[0149] Representative vaccines include, but are not limited to,
vaccines against diphtheria, tetanus, pertussis, polio, measles,
mumps, rubella, hepatitis B, Haemophilus influenzae type b,
varicella, meningitis, human immunodeficiency virus, tuberculosis,
Epstein Barr virus, malaria, hepatitis E, dengue, rotavirus,
herpes, human papillomavirus, and cancers. Vaccines of interest
include the two vaccines that have been licensed by the U.S. Food
and Drug Administration to prevent virus infections that can lead
to cancer: the hepatitis B vaccine, which prevents infection with
the hepatitis B virus, an infectious agent associated with liver
cancer (MMWR Morb. Mortal. Wkly. Rep. 46:107-09, 1997); and
Gardasil.TM. which prevents infection with the two types of human
papillomavirus that together cause 70 percent of cervical cancer
cases worldwide (Speck and Tyring, Skin Therapy Lett. 11:1-3,
2006). Other treatment vaccines of interest include therapeutic
vaccines for the treatment of cervical cancer, follicular B cell
non-Hodgkin's lymphoma, kidney cancer, cutaneous melanoma, ocular
melanoma, prostate cancer, and multiple myeloma.
[0150] By "enhancing the efficacy" or "enhancing the
immunogenicity" with regard to a vaccine is intended improving an
outcome, for example, as measured by a change in a specific value,
such as an increase or a decrease in a particular parameter of an
activity of a vaccine associated with protective immunity. In one
embodiment, enhancement refers to at least a 25%, 50%, 100% or
greater than 100% increase in a particular parameter. In another
embodiment, enhancement refers to at least a 25%, 50%, 100% or
greater than 100% decrease in a particular parameter. In one
example, enhancement of the efficacy/immunogenicity of a vaccine
refers to an increase in the ability of the vaccine to inhibit or
treat disease progression, such as at least a 25%, 50%, 100%, or
greater than 100% increase in the effectiveness of the vaccine for
that purpose. In a further example, enhancement of the
efficacy/immunogenicity of a vaccine refers to an increase in the
ability of the vaccine to recruit the subject's natural defenses
against cancers that have already developed, such as at least a
25%, 50%, 100%, or greater than 100% increase in the effectiveness
of the vaccine for that purpose.
[0151] Similarly, by "overcoming a suppressed immune response" with
regard to a vaccine is intended improving an outcome, for example,
as measured by a change in a specific value, such as a return to a
formerly positive value in a particular parameter of an activity of
a vaccine associated with protective immunity. In one embodiment,
overcoming refers to at least a 25%, 50%, 100% or greater than 100%
increase in a particular parameter. In one example, overcoming a
suppressed immune response to a vaccine refers to a renewed ability
of the vaccine to inhibit or treat disease progression, such as at
least a 25%, 50%, 100%, or greater than 100% renewal in the
effectiveness of the vaccine for that purpose. In a further
example, overcoming a suppressed immune response to a vaccine
refers to a renewed ability of the vaccine to recruit the subject's
natural defenses against cancers that have already developed, such
as at least a 25%, 50%, 100%, or greater than 100% renewal in the
effectiveness of the vaccine for that purpose.
[0152] A therapeutically effective amount of an IL-35R antagonist
or agonist can be administered to a subject. By "therapeutically
effective amount" is intended an amount that is useful in the
treatment, prevention or diagnosis of a disease or condition. As
used herein, a therapeutically effective amount of an IL-R35
agonist or antagonist is an amount which, when administered to a
subject, is sufficient to achieve a desired effect, such as
modulating (inhibiting or promoting) effector T cell function in a
subject being treated with that composition without causing a
substantial cytotoxic effect in the subject. The effective amount
of an IL-35R-- agonist or antagonist useful for modulating effector
T-cell function will depend on the subject being treated, the
severity of the affliction, and the manner of administration of the
IL-35R-agonist or antagonist.
[0153] By "subject" is intended mammals, e.g., primates, humans,
agricultural and domesticated animals such as, but not limited to,
dogs, cats, cattle, horses, pigs, sheep, and the like. Preferably
the subject undergoing treatment with the pharmaceutical
formulations of the invention is a human.
[0154] When administration is for the purpose of treatment,
administration may be for either a prophylactic or therapeutic
purpose. When provided prophylactically, the substance is provided
in advance of any symptom. The prophylactic administration of the
substance serves to prevent or attenuate any subsequent symptom.
When provided therapeutically, the substance is provided at (or
shortly after) the onset of a symptom. The therapeutic
administration of the substance serves to attenuate any actual
symptom.
[0155] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of an IL-35R
agonist or antagonist can include a single treatment or,
preferably, can include a series of treatments. It will also be
appreciated that the effective dosage of an IL-35R agonist or
antagonist used for treatment may increase or decrease over the
course of a particular treatment. Changes in dosage may result and
become apparent from the results of diagnostic assays as described
herein.
[0156] It is understood that appropriate doses of such active
compounds depends upon a number of factors within the knowledge of
the ordinarily skilled physician, veterinarian, or researcher. The
dose(s) of the active compounds will vary, for example, depending
upon the identity, size, and condition of the subject or sample
being treated, further depending upon the route by which the
composition is to be administered, if applicable, and the effect
which the practitioner desires the active compound to have upon the
IL-35R complex. Exemplary doses include milligram or microgram
amounts of the small molecule per kilogram of subject or sample
weight (e.g., about 1 microgram per kilogram to about 500
milligrams per kilogram, about 100 micrograms per kilogram to about
5 milligrams per kilogram, or about 1 microgram per kilogram to
about 50 micrograms per kilogram. It is furthermore understood that
appropriate doses of an active agent depend upon the potency of the
active agent with respect to the expression or activity to be
modulated. Such appropriate doses may be determined using the
assays described herein. When one or more of these small molecules
is to be administered to an animal (e.g., a human) in order to
modulate activity of the IL-35R complex, a physician, veterinarian,
or researcher may, for example, prescribe a relatively low dose at
first, subsequently increasing the dose until an appropriate
response is obtained. In addition, it is understood that the
specific dose level for any particular animal subject will depend
upon a variety of factors including the activity of the specific
compound employed, the age, body weight, general health, gender,
and diet of the subject, the time of administration, the route of
administration, the rate of excretion, any drug combination, and
the degree of expression or activity to be modulated.
[0157] Therapeutically effective amounts of an IL-35R-specific
binding and/or modulating agent can be determined by animal
studies. When animal assays are used, a dosage is administered to
provide a target tissue concentration similar to that which has
been shown to be effective in the animal assays. It is recognized
that the method of treatment may comprise a single administration
of a therapeutically effective amount or multiple administrations
of a therapeutically effective amount of the IL-35R agonist or
antagonist.
[0158] Any delivery system or treatment regimen that effectively
achieves the desired effect of modulating effector T cell function
can be used. Thus, for example, formulations comprising an
effective amount of a pharmaceutical composition of the invention
comprising IL-35R agonists or antagonists can be used for the
purpose of treatment, prevention, and diagnosis of a number of
clinical indications related to the activity of the IL-35R
complex.
[0159] v. Pharmaceutical Compositions
[0160] The IL-35R complexes or active fragments and variants
thereof, soluble forms of the IL-35R complex or active variants and
fragments thereof, the IL-35R specific binding agents, and/or the
IL-35R antagonist or agonists (also referred to herein as "active
compounds") disclosed herein can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the nucleic acid molecule, protein,
or antibody and a pharmaceutically acceptable carrier. As used
herein the language "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0161] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration. In
addition, it may be desirable to administer a therapeutically
effective amount of the pharmaceutical composition locally to an
area in need of treatment (e.g., to an area of the body where
inhibiting a regulatory T (T.sub.R) cell function is desired). This
can be achieved by, for example, local or regional infusion or
perfusion during surgery, topical application, injection, catheter,
suppository, or implant (for example, implants formed from porous,
non-porous, or gelatinous materials, including membranes, such as
sialastic membranes or fibers), and the like. In one embodiment,
administration can be by direct injection at the site (or former
site) of a cancer that is to be treated. In another embodiment, the
therapeutically effective amount of the pharmaceutical composition
is delivered in a vesicle, such as liposomes (see, e.g., Langer,
Science 249:1527-33, 1990 and Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez Berestein and
Fidler (eds.), Liss, N.Y., pp. 353-65, 1989).
[0162] In yet another embodiment, the therapeutically effective
amount of the pharmaceutical composition can be delivered in a
controlled release system. In one example, a pump can be used (see,
e.g., Langer, Science 249:1527-33, 1990; Sefton, Crit. Rev. Biomed.
Eng. 14:201-40, 1987; Buchwald et al., Surgery 88:507-16, 1980;
Saudek et al., N Engl. J. Med. 321:574-79, 1989). In another
example, polymeric materials can be used (see, e.g., Levy et al.,
Science 228:190-92, 1985; During et al., Ann. Neurol. 25:351-56,
1989; Howard et al., J. Neurosurg. 71:105-12, 1989). Other
controlled release systems, such as those discussed by Langer
(Science 249:1527-33, 1990), can also be used.
[0163] Solutions or suspensions used for parenteral, intradermal,
or subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes, or multiple dose vials made of glass
or plastic.
[0164] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.differential. (BASF; Parsippany,
N.J.), or phosphate buffered saline (PBS). In all cases, the
composition must be sterile and should be fluid to the extent that
easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride, in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0165] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying, which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0166] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth, or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser that contains a suitable propellant, e.g., a
gas such as carbon dioxide, or a nebulizer.
[0167] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. The compounds can also be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0168] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0169] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated with each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0170] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
IV. Sequence Identity
[0171] As used herein, "sequence identity" or "identity" in the
context of two polynucleotides or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0172] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0173] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using GAP Version 10
using the following parameters: % identity and % similarity for a
nucleotide sequence using GAP Weight of 50 and Length Weight of 3,
and the nwsgapdna.cmp scoring matrix; % identity and % similarity
for an amino acid sequence using GAP Weight of 8 and Length Weight
of 2, and the BLOSUM62 scoring matrix; or any equivalent program
thereof. By "equivalent program" is intended any sequence
comparison program that, for any two sequences in question,
generates an alignment having identical nucleotide or amino acid
residue matches and an identical percent sequence identity when
compared to the corresponding alignment generated by GAP Version
10.
[0174] As used herein, the singular terms "a," "an," and "the"
include plural referents unless context clearly indicates
otherwise. Similarly, the word "or" is intended to include "and"
unless the context clearly indicates otherwise. It is further to be
understood that all base sizes or amino acid sizes, and all
molecular weight or molecular mass values, given for nucleic acids
or polypeptides are approximate, and are provided for
description.
[0175] The subject matter of the present disclosure is further
illustrated by the following non-limiting examples.
EXPERIMENTAL
Example 1
IL-35 Signaling and Suppression Mediated by IL-35 Require the
Expression of the IL-35R
Materials and Methods:
[0176] Mice. C57BL/6 (wild type), CD4.cre, and IL12R.beta.2.sup.-/-
mice were purchased from the Jackson Laboratory. Gp130 floxed
knockin mice were provided by Rodger McEver at Oklahoma Medical
Research Foundation. gp130 fl.times.CD4.cre.times.IL12Rb2.sup.-/-
were obtained by breeding the three mouse strains listed. All
animal experiments were performed in American Association for the
Accreditation of Laboratory Animal Care-accredited,
specific-pathogen-free facilities in the St. Jude Animal Resource
Center following national, state and institutional guidelines.
Animal protocols were approved by the St Jude Animal Care and Use
Committee.
[0177] T.sub.conv Cell Purification.
T.sub.eff(CD4.sup.+CD25.sup.-CD45RB.sup.hi) from the spleens and
lymph nodes of C57BL/6 or knockout age-matched
gp130.fl.times.CD4.cre.times.IL12Rb2.sup.-/- mice were positively
sorted by FACS. After red blood cell lysis, cells were stained with
antibodies against CD4, CD25 and CD45RB and purified on a MoFlo
cell sorter.
[0178] Transfection of HEK293T cells for IL-35 protein generation.
IL-35 constructs were generated by recombinant PCR and cloned into
pPlGneo, a pClneo-based vector (Promega) that we have modified to
include an IRES-GFP cassette. HEK293T cells were transfected using
10 .mu.g plasmid per 2.times.10.sup.6 cells using Trans IT
transfection reagent. Cells were sorted for equivalent GFP
expression and were cultured for 36 hours to facilitate protein
secretion. Dialyzed, filtered supernatant from cells was used as
the source of IL-35 in IL-35 mediated suppression assays.
[0179] iT.sub.R35 conversion. iTr35 are an induced regulatory T
cell population that is generated by treatment with IL-35 and
suppress via IL-35. See, U.S. Provisional Application No.
61/156,995, herein incorporated by reference in its entirety.
Purified murine T.sub.eff cells were activated by
anti-CD3-+anti-CD28-coated latex beads in the presence of IL-35
supernatant, at 25% of total culture medium, to induce "conversion"
of T.sub.eff cells into iT.sub.R35. Following conversion,
iT.sub.R35 were purified for use in suppression assays.
[0180] In Vitro Suppression by IL-35 and iT.sub.R35. T.sub.eff were
activated for 72 hours with anti-CD3-+anti-CD28-coated latex beads
in the presence of IL-35 supernatant as 25%, 12.5%, or 6.25% of
culture media. In parallel, iT.sub.R35 were purified and assayed
for their capacity to suppress freshly sorted T.sub.eff cell
proliferation. Cultures were pulsed with 1 mCi [.sup.3H]-thymidine
for the final 8 hours of the 72 hour assay, and were harvested with
a Packard Micromate cell harvester. Counts per minute were
determined using a Packard Matrix 96 direct counter. Percent
suppression was calculated using the following formula: ((cpm of
T.sub.eff cells alone-cpm of T.sub.eff cells treated with IL-35 or
iT.sub.R35)/cpm of T.sub.eff cells alone)*100.
Discussion/Conclusion:
[0181] FIG. 1 shows that wild-type T.sub.eff proliferation is
potently suppressed by IL-35 in a titratable manner. However, IL-35
is unable to suppress the proliferation of T.sub.eff cells that
lack the IL-35R (gp130.fl.times.CD4.cre.times.IL12Rb2.sup.-/-).
iT.sub.R35 suppression of T.sub.eff cell proliferation is dependent
upon IL-35. As such, IL-35R deficient T.sub.eff cells are resistant
to suppression mediated by iT.sub.R35. These results demonstrate
that IL-35 signaling and suppression mediated by IL-35 require the
expression of the IL-35R.
Example 2
IL-35 Signals Primarily Through Two Different STAT Proteins, STAT1
and STAT4
Materials and Methods:
[0182] Mice. Spleens and lymph nodes from Il12rb1.sup.-/- mice were
provided by D. Fairweather and J. A. Frisancho (Johns Hopkins
University), CD4.sup.cre.times.gp130.sup.fl/fl mice were provided
by M. Karin and S. Grivennikov (University of California at San
Diego), IL27ra.sup.-/- mice were provided by C. Hunter and J.
Stumhofer (University of Pennsylvania), Stat1.sup.-/- mice were
provided by A. Satoskar and P. Reville (Ohio State University), and
Stat3.sup.-/- mice were provided by C. Drake and H.R. Yen (Johns
Hopkins University). IL12rb2.sup.-/-, Stat4.sup.-/-, Rag1.sup.-/-,
C57BL/6, B6.PL and Balb/c mice were purchased from the Jackson
Laboratory. All animal experiments were performed in American
Association for the Accreditation of Laboratory Animal
Care-accredited, specific-pathogen-free facilities in the St. Jude
Animal Resource Center following national, state and institutional
guidelines. Animal protocols were approved by the St. Jude Animal
Care and Use Committee.
[0183] Neutralizing IL-35 mAb. Neutralizing IL-35 mAb was developed
by immunization with recombinant murine Ebi3 protein. Briefly,
recombinant murine Ebi3 was cloned and expressed in a proprietary
E. coli expression system developed by Mike Jones (Shenandoah
Biotechnology) and used for immunization of Ebi3.sup.-/- mice.
Clones V1.4F5.29, V1.4H6.25, and V1.4C4.22 were subsequently chosen
for their capacity to IP, blot, and specifically neutralize IL-35
bioactivity.
[0184] Transfection of HEK293T cells for IL-35 and control protein
generation. IL-35 constructs were generated by recombinant PCR and
cloned into pPlGneo, a pClneo-based vector (Promega) that has been
modified to include an IRES-GFP cassette. A construct containing
Ebi3 and 1112a linked by a flexible glycine-serine linker was used
for IL-35 generation and an empty pPlGneo vector was used as a
control. HEK293T cells were transfected using 10 .mu.g plasmid per
2.times.10.sup.6 cells using TransIT transfection reagent (Mirus).
Transfection media was exchanged for fresh culture media after 24
hours and were cultured for an additional 36 hours to facilitate
protein secretion. Dialyzed, filtered supernatant from cells was
used at 25% of total culture medium to induce conversion of
T.sub.conv cells into iT.sub.R35 or iT.sub.Rcontrol cells.
[0185] Anti-CD3/CD28-coated latex beads. 4 .mu.M sulfate latex
beads (Molecular Probes) were incubated overnight at room
temperature with rotation in a 1:4 dilution of anti-CD3+ anti-CD28
antibody mix (13.3 .mu.g/ml anti-CD3 (murine clone #145-2c11, human
clone #OKT3) (eBioscience) and 26.6 .mu.g/ml anti-CD28 (murine
clone #37.51, human clone #CD28.6) (eBioscience). Beads were washed
3 times with 5 mM phosphate buffer pH 6.5 and resuspended at
5.times.10.sup.7/ml in sterile phosphate buffer with 2 mM BSA.
[0186] Recombinant IL-35 beads. Beads were generated that presented
IL-35 to cells in a manner that excluded use of 293T supernatants.
Anti-p35 mAb clone 25806 (R&D Systems) or isotype control (rat
IgG2) mAb was added to 1 ml of IL-35 supernatant or control
supernatant and rotated at 4.degree. C. for 4 hours. Protein G
beads were added and rotated for an additional 12-18 hours. To
ensure the protein was attached to the beads, the beads were boiled
to release bound protein, resolved by SDS-PAGE and probed with
anti-Ebi3 mAb. Both the beads and post IP supernatant were tested
for functional activity in a standard suppression assay. Beads were
cultured with T.sub.conv in medium containing anti-CD3+ anti-CD28
conjugated beads as indicated for 3 days. Proliferation was
determined by [.sup.3H]-thymidine incorporation.
[0187] T.sub.conv purification, iT.sub.R35 conversion and
suppressed T.sub.conv, cell generation. T.sub.conv
(CD4.sup.+CD25.sup.-CD45RB.sup.hi) and
T.sub.reg)(CD4.sup.+CD25.sup.+CD45RB.sup.lo cells from the spleens
and lymph nodes of C57BL/6 or knockout age-matched mice were
positively sorted by FACS. After red blood cell lysis, cells were
stained with antibodies against CD4, CD25 and CD45RB (Biolegend)
and sorted on a MoFlo (Dako) or Reflection (i-Cyt). Murine
iT.sub.R35 cells were generated. Briefly, purified murine
T.sub.conv cells from wild-type or indicated knockout mice were
activated by anti-CD3-+anti-CD28-coated latex beads in the presence
of 25% culture medium from control or IL-35 transfected 293T cells
(dialyzed against media and filtered) to generate murine
iT.sub.R35. To generate suppressed T.sub.conv, purified T.sub.conv
cells were activated in the presence of anti-CD3-+anti-CD28-coated
latex beads and T.sub.regs at a 4:1 (T.sub.conv:T.sub.reg ratio)
for 72 hours. Suppressed T.sub.conv from the co-culture were
re-sorted on the basis of congenic markers and used for qPCR
analysis of receptor expression.
[0188] Immunoprecipitation and Western Blotting. Following 18 hour
activation with anti-CD3+anti-CD28 coated beads, cells were treated
with 100 ng/ml IL12, IL27 or IL35 for indicated times. Whole cell
lysates were lysed in cold RIPA buffer and subjected to
immunoblotting with antibodies for pSTAT1, pSTAT3, pSTAT4 and
pSTATS (Cell Signaling Technology and Santa Cruz Biotechnology).
Blots were developed using ECL (Amersham Biosciences) and
autoradiography.
[0189] In vitro proliferation and suppression assays. To determine
proliferative capacity of cells generated as described above,
2.5.times.10.sup.4 cells were activated with
anti-CD3-+anti-CD28-coated latex beads for 72 hours. Cultures were
pulsed with 1 mCi [.sup.3H]-thymidine for the final 8 hours of the
72 hour assay, and were harvested with a Packard Micromate cell
harvester. Counts per minute were determined using a Packard Matrix
96 direct counter (Packard Biosciences). For suppression assays,
IL-35 supernatants, IL-35 beads or iT.sub.R35 were titrated into
T.sub.conv cell proliferation assays as indicated. Cultures were
pulsed and harvested as described for proliferation assays.
[0190] iT.sub.R35-mediated control of homeostatic expansion.
Homeostasis assays were performed. Naive Thy1.2.sup.+ T.sub.conv
cells were isolated by FACS from wild-type or knockout mice (as
indicated) and used as "responder" cells in adoptive transfer.
Thy1.1.sup.+ iT.sub.R35 were generated as described above and used
as "suppressor" cells in adoptive transfer. T.sub.conv cells
(2.times.10.sup.6) with or without suppressor cells
(5.times.10.sup.5) were resuspended in 0.5 ml of PBS plus 2% FBS,
and were injected intravenously through the tail vein into
Rag1.sup.-/- mice. Mice were euthanized seven days post transfer,
and splenocytes were counted, stained and analyzed by flow
cytometry using antibodies against Thy1.1 and Thy1.2 (BD
Bioscience). For each group, 5-10 mice were analyzed.
[0191] B16 tumor model. For T cell adoptive transfer experiments
using the B16 melanoma model, Rag1.sup.-/- mice received indicated
cells via the tail vein on day -1 of experiment. Wild type or
receptor deficient naive CD4.sup.+CD25.sup.-
(9.times.10.sup.6/mouse) and CD8.sup.+ T cells
(6.times.10.sup.6/mouse) alone or in combination with iT.sub.R35
cells (10.sup.6/mouse) were adoptively transferred into mice.
B16-F10 melanoma was a gift from Mary Jo Turk (Dartmouth College,
Hanover, N.H.) and was passaged intradermally (i.d.) in C57/B16
mice 5 times to ensure reproducible growth. B16 cells were cultured
in RPMI 1640 containing 7.5% FBS and washed three times with RPMI
prior to injections if viability exceeded 96%. Rag1.sup.-/- mice
were injected with 120,000 cells on the right flank i.d. B16 tumor
diameters were measured daily with calipers and reported as
mm.sup.3 (a.sup.2.times.b/2, where "a" is the smaller caliper
measurement and "b" the larger). For all experiments, B16 tumors
were excised at day 14 when tumor size was 5-10 mm in diameter. For
each group, 4-5 mice were analyzed.
The IL35 receptor comprises IL12R.beta.2 and gp130.
[0192] To determine which IL12 family receptor chains are required
for IL-35 mediated suppression, three approaches were utilized, all
of which yielded similar results. The use of genetically deficient
mice to determine functions of proteins has been extremely useful
in defining protein activity. Therefore, it was first assessed
whether IL-35 could suppress the proliferation of CD4.sup.+
T.sub.conv cells that lacked expression of each of the IL12 family
receptor chains. T.sub.conv cells purified by FACS from wild-type
(C57BL/6), CD4.sup.crc.times.gp130.sup.fl/fl (abbreviated
gp130.sup..DELTA.T), Il27ra.sup.-/-, Il12rb1.sup.-/-,
Il12rb2.sup.-/-, or
Il12rb2.sup.-/-.times.CD4.sup.crc.times.gp130.sup.fl/fl
(abbreviated IL35R.sup..DELTA.T) mice were activated with
anti-CD3-+anti-CD28-coated latex beads for 3 days in the presence
of indicated concentrations of IL-35 or iT.sub.R.sup.35 in
combination with neutralizing IL-35 mAb or isotype control mAb.
Proliferation was determined by [.sup.3H]-thymidine incorporation.
T.sub.conv were treated with or without rIL-27 for 18 hours prior
to analysis of receptor expression and proliferation. RNA was
extracted, cDNA generated and qPCR performed. Cytokine treated
cells were mixed at indicated concentrations of IL-35 for 3 days.
Proliferation was determined by [.sup.3H]-thymidine
incorporation.
[0193] IL-35 can suppress the proliferation of both Il27ra.sup.-/-
and Il12rb1.sup.-/- T.sub.covnv cells to a degree similar to that
seen in wild-type T.sub.conv cells (data not shown). However,
T.sub.conv cells that lack expression of either IL12R.beta.2
(Il12rb2.sup.-/-) or gp130 (CD4.sup.crc.times.gp130.sup.fl/fl;
referred to herein as gp130.sup..DELTA.T) are partially resistant
to IL-35 mediated suppression. Generation of Il12rb2.sup.-/- and
CD4.sup.crc.times.gp130'' mice (referred to herein as
IL35R.sup..DELTA.T) results in T.sub.conv cells that are completely
resistant to IL-35 mediated suppression. Many cytokines that signal
through the gp130 chain, including LIF, OncM and CNTF, require
leukocyte inhibitory factor-.beta. (LIFR.beta.) in addition to
gp130 and the specificity-determining receptor chain. To determine
whether IL-35 might also utilize LIFR.beta., LIFR.beta. expression
in T.sub.conv cells was examined. Quantitative real-time PCR
analysis suggests that T.sub.conv cells, the targets of IL-35
signaling, do not express LIFR.beta., therefore it doesn't appear
to be important for IL-35 signaling.
[0194] Second, IL-35 conjugated via an anti-p35 specific mAb, or
isotype control mAb, to Protein G beads was utilized as suppressors
of T.sub.conv cell proliferation. Isotype control or
non-neutralizing anti-IL35 mAb were incubated with IL-35
supernatant and then coupled with protein G beads. The protein G
coupled beads were then incubated with T.sub.conv cells activated
in presence of a CD3 and a CD28. T.sub.conv sorted from indicated
wild-type or receptor deficient T.sub.conv cells were activated in
the presence of wild-type Tregs and proliferation determined by
[.sup.3H]-thymidine incorporation. For a media alone control,
nTreg, or T.sub.conv cells mixed at a 4:1 ratio were activated in
the presence of anti-CD3-CD28-coated beads in the top chamber of a
Transwell.TM. culture plate. Responder T.sub.conv were activated
with anti-CD3-CD28-coated beads in the bottom chamber of the
plates. Proliferation of the responder T.sub.conv cells in the
bottom chambers was determined. No suppression of proliferation was
detected in isotype control beads, regardless of genotype. However,
as seen with both IL-35 protein and iT.sub.R35, suppression was
limited in IL12R.beta.2 and gp 130 deficient T.sub.conv cells and
completely absent in T.sub.conv cells that lack both IL12R.beta.2
and gp130. It was previously shown that natural T.sub.regs that
lack IL-35 expression (Ebi3.sup.-/- or Il12a.sup.-/-) are partially
defective both in vitro and in vivo (Collison et al. (2007) Nature
450:566-569). Therefore, it was expected that T.sub.conv cells that
lack the IL-35R and, thus, are unable to respond to IL-35, would be
partially resistant to T.sub.reg-mediated suppression. Indeed,
gp130.sup..DELTA.T, Il12rb2.sup.-/- and
IL35R.sup..DELTA.TT.sub.conv cells are all partially resistant to
T.sub.reg mediated suppression of proliferation (data not shown).
It was previously shown that T.sub.conv cells activated in the
presence of T.sub.reg are potently suppressive across a permeable
membrane in an IL-35-dependent manner (Collison et al. (2009). J.
Immunol. 182:6121-6128). Therefore, in addition to a standard
suppression assay, it was also determined whether
IL35R.sup..DELTA.TT.sub.conv were suppressed across a permeable
membrane. While wild-type T.sub.conv cells were potently suppressed
by co-cultured T.sub.conv and T.sub.reg,
IL35R.sup..DELTA.TT.sub.conv cells were completely resistant to
iT.sub.R.sup.35 mediated suppression (data not shown).
[0195] Third, a novel induced T.sub.reg population,
iT.sub.R.sup.35, has been described that suppresses the
proliferation of T.sub.conv cells exclusively via IL-35 (Collison
et al. (2010) Nature Immunology 11: 1093-1101). Both exogenously
added IL-35 and T.sub.reg cells induce conversion of T.sub.conv
cells to iT.sub.R.sup.35 in vitro, and in vivo, under inflammatory
conditions. Given that their mode of suppression is by way of
IL-35, they represent a perfect tool for determining cell-mediated
suppression via IL-35. Therefore, it was assessed whether
iT.sub.R.sup.35 suppressed the proliferation of each of the
receptor deficient T.sub.conv cells. While iT.sub.R.sup.35
suppressed wild type, Il27ra.sup.-/- and Il12rb1.sup.-/- T.sub.conv
cells equally well, T.sub.conv cells that lacked expression of
either IL12R.beta.2 or gp130 were partially resistant and cells
that lacked both IL12R.beta.2 and gp130 were completely resistant
to iT.sub.R35 mediated suppression (data not shown). Moreover,
neutralizing mAb to IL-35, but not an isotype control, completely
blocked the suppressive capacity of iT.sub.R35 of wild-type
T.sub.conv cells.
IL35R-Deficient T.sub.conv Cells are Resistant to IL-35 Mediated
Suppression In Vivo.
[0196] In the absence of their respective cytokine signaling
receptor chains, in vivo cellular effects of IL-12 and IL-27 are
completely abolished. Therefore, it was determined whether loss of
the IL35R in vivo renders T cells refractory to IL35-mediated
suppression. Given that IL-35 is central to the suppression
mediated by iT.sub.R35, iT.sub.R35 was utilized in two different in
vivo models to address this question. First, iT.sub.R35 can control
the homeostatic expansion of T.sub.conv cells in the lymphopenic
environment of the recombination activating gene-1 (Rag1).sup.-/-
mouse. Therefore, purified wild-type, gp130.sup..DELTA.T,
Il27ra.sup.-/-, Il12rb1.sup.-/-,Il12rb2.sup.-/- or
IL35R.sup..DELTA.TThy1.2.sup.+ T.sub.conv cells, either alone or in
the presence of Thy1.1.sup.+ iT.sub.R.sup.35 cells were adoptively
transferred into Rag1.sup.-/- mice. Seven days post transfer,
suppression of T.sub.conv cell expansion was monitored by
determining the Thy1.2.sup.+ T.sub.conv cell numbers.
iT.sub.R.sup.35 cells significantly limited the proliferation of
wild-type, gp130.sup..DELTA.T, Il27ra.sup.-/-, Il12rb1.sup.-/- and
Il12rb2.sup.-/-Thy1.2.sup.+T.sub.conv cells. However,
iT.sub.R.sup.35 cells failed to block the expansion of
IL35R.sup..DELTA.TThy1.1.sup.+T.sub.conv cells (FIG. 2A). In the
absence of only one receptor chain, in vivo biological activity of
both IL-12 and IL-27 is lost, it appears that IL-35 signaling in
vivo is abrogated only by loss of both IL12R.beta.2 and gp130
expression.
[0197] Second, it was previously shown that, like natural
T.sub.regs, iT.sub.R.sup.35 can block the anti-tumor CD8.sup.+ T
cell response against B16 melanoma. Wild-type, gp130.sup..DELTA.T,
Il27ra.sup.-/-, Il12rb1.sup.-/-, Il12rb2.sup.-/- or
IL35R.sup..DELTA.TCD4.sup.+ and CD8.sup.+ T cells, either alone or
in the presence of iT.sub.R.sup.35 cells were adoptively
transferred into Rag1.sup.-/- mice. The following day, mice were
inoculated intradermally with B 16 melanoma cells and tumor size
was monitored daily and reported after 14 days. In the absence of
iT.sub.R.sup.35, tumor burden was similar between mice receiving
all CD4.sup.+ and CD8.sup.+ T cells, regardless of genotype (FIG.
2B). Tumor size was exacerbated in mice receiving iT.sub.R.sup.35
cells in combination with wild-type, gp130.sup..DELTA.T,
Il27ra.sup.-/-, Il12rb1.sup.-/-, and Il12rb2.sup.-/-CD4.sup.+ and
CD8.sup.+ T cells. However, IL35R.sup..DELTA.T recipients were
completely resistant to iT.sub.R.sup.35 mediated prevention of
tumor immunity. Collectively, these data clearly demonstrate that
IL35R.sup..DELTA.T cells are resistant to IL-35 mediated
suppression in vivo.
IL-35 Signals Through STAT1 and STAT4
[0198] Given that IL12R.beta.2 and gp130 constitute the IL-35
receptor, it was hypothesized that the IL-35 signaling pathway
might also overlap with that of other cytokines that utilize these
receptor chains. T.sub.conv cells were activated in the presence of
T.sub.reg at a 4:1 ratio (responder:suppressor) for 72 hours. RNA
was extracted and cDNA generated from resting or activated
T.sub.conv cells or from suppressed T.sub.conv cells from
T.sub.conv:T.sub.reg co-cultures (resorted based on differential
Thy1 markers). Relative gp130, Il27ra, Il12rb1 and Il12rb2 mRNA
expression was determined. Consistent with previous reports ref,
IL-12 treatment of T.sub.conv cells resulted in phosphorylation of
STAT4 and IL-27 signaling induced STAT1 and STAT3 phosphorylation
(data not shown). Interestingly, wild-type T.sub.conv cells, which
are responsive to IL-35 mediated suppression, demonstrated
phosphorylation of both STAT1 and STAT4, but no activation of
either STAT3 or STAT5. Moreover, no induction of p-STAT1 or p-STAT4
was seen in T.sub.conv cells that lack the IL-35 receptor
(Il12rb2.sup.-/-.times.gp130.sup..DELTA.T T.sub.conv, hence forth
referred to as IL35R.sup..DELTA.T) (data not shown). To better
determine the kinetics of STAT phosphorylation in response to IL-35
treatment, T.sub.conv cells were activated for 24 hours with
anti-CD3+anti-CD28 coated beads and treated with IL-35 for
indicated times. Western blot analysis demonstrated that p-STAT1
was the most dramatic, with maximal phosphorylation evident at 30
minutes. Similar to STAT3 and STAT5, STAT4 phosphorylation was less
pronounced, but was sustained over the course of time analyzed. To
determine which STATs were most critical to IL-35 signaling,
T.sub.conv cells that lack STAT1, STAT3, or STAT4 were utilized.
Whereas IL-35 can suppress the proliferation of Stat3.sup.-/-
T.sub.conv cells to a degree similar to that seen in wild-type
T.sub.conv cells, suppression of Stat1.sup.-/- and Stat4.sup.-/-
T.sub.conv, cells was reduced (data not shown). Similarly,
T.sub.reg-mediated suppression of both Stat1.sup.-/- and
Stat4.sup.-/- T.sub.conv cells was impaired (data not shown).
Reduced signaling in Stat1.sup.-/- and Stat4.sup.-/- T.sub.conv
cells is not due to lack of receptor expression as mRNA expression
of receptor chains is similar in Stat1.sup.-/-, Stat4.sup.-/- and
wild-type T.sub.conv cells (data not shown). Collectively, these
data suggest that STAT1 and STAT4 are critical for IL-35 mediated
signal transduction.
IL-35 is a Target of the IL-35 Signaling Pathway.
[0199] It has been previously shown that IL-35 can convert
proliferative, IL35 T.sub.conv cells into hypo-responsive, strongly
suppressive iT.sub.R35 which express and mediate suppression via
IL-35 (Collison et al. (2010) Nature Immunology 11: 1093-1101).
Activation of wild-type T.sub.conv in the presence of IL-35
significantly upregulated Ebi3 and 1112a mRNA, the two components
of IL-35 (Ebi3 and p35, respectively). Interestingly,
gp130.sup..DELTA.T, Il12rb2.sup.-/- and IL35R.sup..DELTA.T
T.sub.conv cells are all resistant to induction of Ebi3 expression
(FIG. 3A). However, gp130.sup..DELTA.T T.sub.conv cells retain the
ability to upregulate 1112a expression in response to IL-35
treatment, suggesting that p35 expression may be downstream of
IL12R.beta.2 signaling. Induction of IL-35 expression in response
to IL-35 treatment is critical for conversion of T.sub.conv cells
into iT.sub.R.sup.35. Therefore, the ability of receptor deficient
mice to be converted into iT.sub.R.sup.35 was assessed. To
determine whether IL-35 treated T.sub.conv cells had acquired
regulatory capacity, they were co-cultured as suppressors with
freshly purified responder T.sub.conv cells (FIG. 3B). T.sub.conv
cells treated with control protein, regardless of genotype, were
incapable of suppressing responder T.sub.conv cell proliferation.
Furthermore, wild-type, but not gp130.sup..DELTA.T, Il12rb2.sup.-/-
or IL35R.sup..DELTA.TT.sub.conv9, cells were capable of suppressing
T.sub.conv cell proliferation. In addition, both Stat1.sup.-/-,
Stat4.sup.-/- T.sub.conv cells fail to upregulate expression of
Ebi3 and 1112a to the same degree as wild-type T.sub.conv cells
(data not shown). Moreover, early induction of Ebi3 and 1112a mRNA
expression, which peak at 3 hours and 1 hour, respectively, suggest
that IL-35 is a direct target of IL-35 signaling (data not shown).
Together, these results suggest that cells that lack the IL-35
receptor or signaling components are unable to induce IL-35
expression.
Discussion
[0200] Important similarities and interesting differences between
IL-12, IL-27 and IL-35 signaling have been illuminated by this
study. Not surprisingly, the IL-35 receptor and signaling pathway
overlap with that of IL-12 and IL-27. However, unlike its siblings,
IL-35 appears to be able to signal, in part, through each of the
receptor chains, IL-12R.beta.2 and gp130. This is likely due to the
fact that each of these chains is the signal transducing subunit of
their respective cytokine receptors. In addition, like IL-27, IL-35
signals primarily through two different STAT proteins, STAT1 and
STAT4. However, STAT3 appears dispensable for IL-35 signaling, an
interesting observation given its importance for IL-27 signaling,
which is downstream of gp130 engagement. In addition, STAT1/STAT3
heterodimers have been previously described yet there is no
precedent for STAT1/STAT 4 heterodimerization.
[0201] The expression pattern of the IL-35 receptor also provides
insight into potential IL-35 target cell types. While gp130 is
fairly ubiquitously expressed, both IL-12R chains are expressed
mainly by activated T cells and NK cells. In T cells, the
expression of IL-12R.beta.2 is confined to Th1 cells, and its
expression correlates with responsiveness to IL-12 and presumably
IL-35. Expression of IL-12R.beta.2 has also been shown on other
cell types, such as dendritic cells which would vastly affect the
scope of IL-35 bioactivity in the immune system. IL-12R.beta.2 is
undetectable on most resting T cells, but can be rapidly
upregulated by exposure to IL-12, IL-27, IFN-.gamma.,
tumor-necrosis factor (TNF) and co-stimulation through CD28. Thus,
IL35 might have biological effects on a variety of cellular targets
and under a variety of disease conditions.
[0202] Since IL-35 appears to utilize receptor chains and STATs
that are similar to those used by other IL-12 family members,
another important question is how a T cell can translate
potentially similar signals into such distinct biological outcomes.
Given the opposing activities of IL-35 and IL-12, IL-23, and IL-27,
it is possible that different kinetics, binding affinities, or
potentially as yet unidentified heterodimerization patterns may
differentiate the signaling pathways in such a way to mediate such
diverse biological consequences.
TABLE-US-00001 TABLE 1 Summary of SEQ ID NOS SEQ ID NO Description
Type of sequence 1 gp130 Full length cDNA 2 gp130 DNA coding region
3 gp130 Amino acid 4 IL12R.beta.2 Full length cDNA 5 IL12R.beta.2
DNA coding region 6 IL12R.beta.2 Amino acid
[0203] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0204] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
613242DNAHomo sapiensCDS(256)...(3010)misc_feature(0)...(0)cDNA for
gp130 1gagcagccaa aaggcccgcg gagtcgcgct gggccgcccc ggcgcagctg
aaccgggggc 60cgcgcctgcc aggccgacgg gtctggccca gcctggcgcc aaggggttcg
tgcgctgtgg 120agacgcggag ggtcgaggcg gcgcggcctg agtgaaaccc
aatggaaaaa gcatgacatt 180tagaagtaga agacttagct tcaaatccct
actccttcac ttactaattt tgtgatttgg 240aaatatccgc gcaag atg ttg acg
ttg cag act tgg cta gtg caa gcc ttg 291 Met Leu Thr Leu Gln Thr Trp
Leu Val Gln Ala Leu 1 5 10ttt att ttc ctc acc act gaa tct aca ggt
gaa ctt cta gat cca tgt 339Phe Ile Phe Leu Thr Thr Glu Ser Thr Gly
Glu Leu Leu Asp Pro Cys 15 20 25ggt tat atc agt cct gaa tct cca gtt
gta caa ctt cat tct aat ttc 387Gly Tyr Ile Ser Pro Glu Ser Pro Val
Val Gln Leu His Ser Asn Phe 30 35 40act gca gtt tgt gtg cta aag gaa
aaa tgt atg gat tat ttt cat gta 435Thr Ala Val Cys Val Leu Lys Glu
Lys Cys Met Asp Tyr Phe His Val45 50 55 60aat gct aat tac att gtc
tgg aaa aca aac cat ttt act att cct aag 483Asn Ala Asn Tyr Ile Val
Trp Lys Thr Asn His Phe Thr Ile Pro Lys 65 70 75gag caa tat act atc
ata aac aga aca gca tcc agt gtc acc ttt aca 531Glu Gln Tyr Thr Ile
Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr 80 85 90gat ata gct tca
tta aat att cag ctc act tgc aac att ctt aca ttc 579Asp Ile Ala Ser
Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe 95 100 105gga cag
ctt gaa cag aat gtt tat gga atc aca ata att tca ggc ttg 627Gly Gln
Leu Glu Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu 110 115
120cct cca gaa aaa cct aaa aat ttg agt tgc att gtg aac gag ggg aag
675Pro Pro Glu Lys Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly
Lys125 130 135 140aaa atg agg tgt gag tgg gat ggt gga agg gaa aca
cac ttg gag aca 723Lys Met Arg Cys Glu Trp Asp Gly Gly Arg Glu Thr
His Leu Glu Thr 145 150 155aac ttc act tta aaa tct gaa tgg gca aca
cac aag ttt gct gat tgc 771Asn Phe Thr Leu Lys Ser Glu Trp Ala Thr
His Lys Phe Ala Asp Cys 160 165 170aaa gca aaa cgt gac acc ccc acc
tca tgc act gtt gat tat tct act 819Lys Ala Lys Arg Asp Thr Pro Thr
Ser Cys Thr Val Asp Tyr Ser Thr 175 180 185gtg tat ttt gtc aac att
gaa gtc tgg gta gaa gca gag aat gcc ctt 867Val Tyr Phe Val Asn Ile
Glu Val Trp Val Glu Ala Glu Asn Ala Leu 190 195 200ggg aag gtt aca
tca gat cat atc aat ttt gat cct gta tat aaa gtg 915Gly Lys Val Thr
Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val205 210 215 220aag
ccc aat ccg cca cat aat tta tca gtg atc aac tca gag gaa ctg 963Lys
Pro Asn Pro Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu 225 230
235tct agt atc tta aaa ttg aca tgg acc aac cca agt att aag agt gtt
1011Ser Ser Ile Leu Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val
240 245 250ata ata cta aaa tat aac att caa tat agg acc aaa gat gcc
tca act 1059Ile Ile Leu Lys Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala
Ser Thr 255 260 265tgg agc cag att cct cct gaa gac aca gca tcc acc
cga tct tca ttc 1107Trp Ser Gln Ile Pro Pro Glu Asp Thr Ala Ser Thr
Arg Ser Ser Phe 270 275 280act gtc caa gac ctt aaa cct ttt aca gaa
tat gtg ttt agg att cgc 1155Thr Val Gln Asp Leu Lys Pro Phe Thr Glu
Tyr Val Phe Arg Ile Arg285 290 295 300tgt atg aag gaa gat ggt aag
gga tac tgg agt gac tgg agt gaa gaa 1203Cys Met Lys Glu Asp Gly Lys
Gly Tyr Trp Ser Asp Trp Ser Glu Glu 305 310 315gca agt ggg atc acc
tat gaa gat aga cca tct aaa gca cca agt ttc 1251Ala Ser Gly Ile Thr
Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe 320 325 330tgg tat aaa
ata gat cca tcc cat act caa ggc tac aga act gta caa 1299Trp Tyr Lys
Ile Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln 335 340 345ctc
gtg tgg aag aca ttg cct cct ttt gaa gcc aat gga aaa atc ttg 1347Leu
Val Trp Lys Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu 350 355
360gat tat gaa gtg act ctc aca aga tgg aaa tca cat tta caa aat tac
1395Asp Tyr Glu Val Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn
Tyr365 370 375 380aca gtt aat gcc aca aaa ctg aca gta aat ctc aca
aat gat cgc tat 1443Thr Val Asn Ala Thr Lys Leu Thr Val Asn Leu Thr
Asn Asp Arg Tyr 385 390 395cta gca acc cta aca gta aga aat ctt gtt
ggc aaa tca gat gca gct 1491Leu Ala Thr Leu Thr Val Arg Asn Leu Val
Gly Lys Ser Asp Ala Ala 400 405 410gtt tta act atc cct gcc tgt gac
ttt caa gct act cac cct gta atg 1539Val Leu Thr Ile Pro Ala Cys Asp
Phe Gln Ala Thr His Pro Val Met 415 420 425gat ctt aaa gca ttc ccc
aaa gat aac atg ctt tgg gtg gaa tgg act 1587Asp Leu Lys Ala Phe Pro
Lys Asp Asn Met Leu Trp Val Glu Trp Thr 430 435 440act cca agg gaa
tct gta aag aaa tat ata ctt gag tgg tgt gtg tta 1635Thr Pro Arg Glu
Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu445 450 455 460tca
gat aaa gca ccc tgt atc aca gac tgg caa caa gaa gat ggt acc 1683Ser
Asp Lys Ala Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr 465 470
475gtg cat cgc acc tat tta aga ggg aac tta gca gag agc aaa tgc tat
1731Val His Arg Thr Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr
480 485 490ttg ata aca gtt act cca gta tat gct gat gga cca gga agc
cct gaa 1779Leu Ile Thr Val Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser
Pro Glu 495 500 505tcc ata aag gca tac ctt aaa caa gct cca cct tcc
aaa gga cct act 1827Ser Ile Lys Ala Tyr Leu Lys Gln Ala Pro Pro Ser
Lys Gly Pro Thr 510 515 520gtt cgg aca aaa aaa gta ggg aaa aac gaa
gct gtc tta gag tgg gac 1875Val Arg Thr Lys Lys Val Gly Lys Asn Glu
Ala Val Leu Glu Trp Asp525 530 535 540caa ctt cct gtt gat gtt cag
aat gga ttt atc aga aat tat act ata 1923Gln Leu Pro Val Asp Val Gln
Asn Gly Phe Ile Arg Asn Tyr Thr Ile 545 550 555ttt tat aga acc atc
att gga aat gaa act gct gtg aat gtg gat tct 1971Phe Tyr Arg Thr Ile
Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser 560 565 570tcc cac aca
gaa tat aca ttg tcc tct ttg act agt gac aca ttg tac 2019Ser His Thr
Glu Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr 575 580 585atg
gta cga atg gca gca tac aca gat gaa ggt ggg aag gat ggt cca 2067Met
Val Arg Met Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro 590 595
600gaa ttc act ttt act acc cca aag ttt gct caa gga gaa att gaa gcc
2115Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu
Ala605 610 615 620ata gtc gtg cct gtt tgc tta gca ttc cta ttg aca
act ctt ctg gga 2163Ile Val Val Pro Val Cys Leu Ala Phe Leu Leu Thr
Thr Leu Leu Gly 625 630 635gtg ctg ttc tgc ttt aat aag cga gac cta
att aaa aaa cac atc tgg 2211Val Leu Phe Cys Phe Asn Lys Arg Asp Leu
Ile Lys Lys His Ile Trp 640 645 650cct aat gtt cca gat cct tca aag
agt cat att gcc cag tgg tca cct 2259Pro Asn Val Pro Asp Pro Ser Lys
Ser His Ile Ala Gln Trp Ser Pro 655 660 665cac act cct cca agg cac
aat ttt aat tca aaa gat caa atg tat tca 2307His Thr Pro Pro Arg His
Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser 670 675 680gat ggc aat ttc
act gat gta agt gtt gtg gaa ata gaa gca aat gac 2355Asp Gly Asn Phe
Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp685 690 695 700aaa
aag cct ttt cca gaa gat ctg aaa tca ttg gac ctg ttc aaa aag 2403Lys
Lys Pro Phe Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys 705 710
715gaa aaa att aat act gaa gga cac agc agt ggt att ggg ggg tct tca
2451Glu Lys Ile Asn Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser
720 725 730tgc atg tca tct tct agg cca agc att tct agc agt gat gaa
aat gaa 2499Cys Met Ser Ser Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu
Asn Glu 735 740 745tct tca caa aac act tcg agc act gtc cag tat tct
acc gtg gta cac 2547Ser Ser Gln Asn Thr Ser Ser Thr Val Gln Tyr Ser
Thr Val Val His 750 755 760agt ggc tac aga cac caa gtt ccg tca gtc
caa gtc ttc tca aga tcc 2595Ser Gly Tyr Arg His Gln Val Pro Ser Val
Gln Val Phe Ser Arg Ser765 770 775 780gag tct acc cag ccc ttg tta
gat tca gag gag cgg cca gaa gat cta 2643Glu Ser Thr Gln Pro Leu Leu
Asp Ser Glu Glu Arg Pro Glu Asp Leu 785 790 795caa tta gta gat cat
gta gat ggc ggt gat ggt att ttg ccc agg caa 2691Gln Leu Val Asp His
Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln 800 805 810cag tac ttc
aaa cag aac tgc agt cag cat gaa tcc agt cca gat att 2739Gln Tyr Phe
Lys Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile 815 820 825tca
cat ttt gaa agg tca aag caa gtt tca tca gtc aat gag gaa gat 2787Ser
His Phe Glu Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp 830 835
840ttt gtt aga ctt aaa cag cag att tca gat cat att tca caa tcc tgt
2835Phe Val Arg Leu Lys Gln Gln Ile Ser Asp His Ile Ser Gln Ser
Cys845 850 855 860gga tct ggg caa atg aaa atg ttt cag gaa gtt tct
gca gca gat gct 2883Gly Ser Gly Gln Met Lys Met Phe Gln Glu Val Ser
Ala Ala Asp Ala 865 870 875ttt ggt cca ggt act gag gga caa gta gaa
aga ttt gaa aca gtt ggc 2931Phe Gly Pro Gly Thr Glu Gly Gln Val Glu
Arg Phe Glu Thr Val Gly 880 885 890atg gag gct gcg act gat gaa ggc
atg cct aaa agt tac tta cca cag 2979Met Glu Ala Ala Thr Asp Glu Gly
Met Pro Lys Ser Tyr Leu Pro Gln 895 900 905act gta cgg caa ggc ggc
tac atg cct cag t gaaggactag tagttcctgc 3030Thr Val Arg Gln Gly Gly
Tyr Met Pro Gln 910 915tacaacttca gcagtaccta taaagtaaag ctaaaatgat
tttatctgtg aattcagatt 3090ttaaaaagtc ttcactctct gaagatgatc
atttgccctt aaggacaaaa atgaactgaa 3150gtttcacatg agctatttcc
attccagaat atctgggatt ctactttaag cactacataa 3210actgacttta
tcctcagaaa aaaaaaaaaa aa 324222757DNAHomo
sapiensmisc_feature(0)...(0)gp130 coding region 2atg ttg acg ttg
cag act tgg cta gtg caa gcc ttg ttt att ttc ctc 48Met Leu Thr Leu
Gln Thr Trp Leu Val Gln Ala Leu Phe Ile Phe Leu1 5 10 15acc act gaa
tct aca ggt gaa ctt cta gat cca tgt ggt tat atc agt 96Thr Thr Glu
Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser 20 25 30cct gaa
tct cca gtt gta caa ctt cat tct aat ttc act gca gtt tgt 144Pro Glu
Ser Pro Val Val Gln Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45gtg
cta aag gaa aaa tgt atg gat tat ttt cat gta aat gct aat tac 192Val
Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55
60att gtc tgg aaa aca aac cat ttt act att cct aag gag caa tat act
240Ile Val Trp Lys Thr Asn His Phe Thr Ile Pro Lys Glu Gln Tyr
Thr65 70 75 80atc ata aac aga aca gca tcc agt gtc acc ttt aca gat
ata gct tca 288Ile Ile Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp
Ile Ala Ser 85 90 95tta aat att cag ctc act tgc aac att ctt aca ttc
gga cag ctt gaa 336Leu Asn Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe
Gly Gln Leu Glu 100 105 110cag aat gtt tat gga atc aca ata att tca
ggc ttg cct cca gaa aaa 384Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser
Gly Leu Pro Pro Glu Lys 115 120 125cct aaa aat ttg agt tgc att gtg
aac gag ggg aag aaa atg agg tgt 432Pro Lys Asn Leu Ser Cys Ile Val
Asn Glu Gly Lys Lys Met Arg Cys 130 135 140gag tgg gat ggt gga agg
gaa aca cac ttg gag aca aac ttc act tta 480Glu Trp Asp Gly Gly Arg
Glu Thr His Leu Glu Thr Asn Phe Thr Leu145 150 155 160aaa tct gaa
tgg gca aca cac aag ttt gct gat tgc aaa gca aaa cgt 528Lys Ser Glu
Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175gac
acc ccc acc tca tgc act gtt gat tat tct act gtg tat ttt gtc 576Asp
Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185
190aac att gaa gtc tgg gta gaa gca gag aat gcc ctt ggg aag gtt aca
624Asn Ile Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr
195 200 205tca gat cat atc aat ttt gat cct gta tat aaa gtg aag ccc
aat ccg 672Ser Asp His Ile Asn Phe Asp Pro Val Tyr Lys Val Lys Pro
Asn Pro 210 215 220cca cat aat tta tca gtg atc aac tca gag gaa ctg
tct agt atc tta 720Pro His Asn Leu Ser Val Ile Asn Ser Glu Glu Leu
Ser Ser Ile Leu225 230 235 240aaa ttg aca tgg acc aac cca agt att
aag agt gtt ata ata cta aaa 768Lys Leu Thr Trp Thr Asn Pro Ser Ile
Lys Ser Val Ile Ile Leu Lys 245 250 255tat aac att caa tat agg acc
aaa gat gcc tca act tgg agc cag att 816Tyr Asn Ile Gln Tyr Arg Thr
Lys Asp Ala Ser Thr Trp Ser Gln Ile 260 265 270cct cct gaa gac aca
gca tcc acc cga tct tca ttc act gtc caa gac 864Pro Pro Glu Asp Thr
Ala Ser Thr Arg Ser Ser Phe Thr Val Gln Asp 275 280 285ctt aaa cct
ttt aca gaa tat gtg ttt agg att cgc tgt atg aag gaa 912Leu Lys Pro
Phe Thr Glu Tyr Val Phe Arg Ile Arg Cys Met Lys Glu 290 295 300gat
ggt aag gga tac tgg agt gac tgg agt gaa gaa gca agt ggg atc 960Asp
Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile305 310
315 320acc tat gaa gat aga cca tct aaa gca cca agt ttc tgg tat aaa
ata 1008Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys
Ile 325 330 335gat cca tcc cat act caa ggc tac aga act gta caa ctc
gtg tgg aag 1056Asp Pro Ser His Thr Gln Gly Tyr Arg Thr Val Gln Leu
Val Trp Lys 340 345 350aca ttg cct cct ttt gaa gcc aat gga aaa atc
ttg gat tat gaa gtg 1104Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile
Leu Asp Tyr Glu Val 355 360 365act ctc aca aga tgg aaa tca cat tta
caa aat tac aca gtt aat gcc 1152Thr Leu Thr Arg Trp Lys Ser His Leu
Gln Asn Tyr Thr Val Asn Ala 370 375 380aca aaa ctg aca gta aat ctc
aca aat gat cgc tat cta gca acc cta 1200Thr Lys Leu Thr Val Asn Leu
Thr Asn Asp Arg Tyr Leu Ala Thr Leu385 390 395 400aca gta aga aat
ctt gtt ggc aaa tca gat gca gct gtt tta act atc 1248Thr Val Arg Asn
Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr Ile 405 410 415cct gcc
tgt gac ttt caa gct act cac cct gta atg gat ctt aaa gca 1296Pro Ala
Cys Asp Phe Gln Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425
430ttc ccc aaa gat aac atg ctt tgg gtg gaa tgg act act cca agg gaa
1344Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu
435 440 445tct gta aag aaa tat ata ctt gag tgg tgt gtg tta tca gat
aaa gca 1392Ser Val Lys Lys Tyr Ile Leu Glu Trp Cys Val Leu Ser Asp
Lys Ala 450 455 460ccc tgt atc aca gac tgg caa caa gaa gat ggt acc
gtg cat cgc acc 1440Pro Cys Ile Thr Asp Trp Gln Gln Glu Asp Gly Thr
Val His Arg Thr465 470 475 480tat tta aga ggg aac tta gca gag agc
aaa tgc tat ttg ata aca gtt 1488Tyr Leu Arg Gly Asn Leu Ala Glu Ser
Lys Cys Tyr Leu Ile Thr Val 485 490 495act cca gta tat gct gat gga
cca gga agc cct gaa tcc ata aag gca 1536Thr Pro Val Tyr Ala Asp Gly
Pro Gly Ser Pro Glu Ser Ile Lys Ala 500 505 510tac ctt aaa caa gct
cca cct tcc aaa gga cct act gtt cgg aca aaa 1584Tyr Leu Lys Gln Ala
Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525aaa gta ggg
aaa aac gaa gct gtc tta gag tgg gac caa ctt cct gtt 1632Lys Val Gly
Lys Asn Glu Ala Val Leu Glu Trp Asp Gln Leu Pro Val 530 535 540gat
gtt cag aat gga ttt atc aga aat tat act ata ttt tat aga acc 1680Asp
Val Gln Asn Gly Phe Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr545 550
555
560atc att gga aat gaa act gct gtg aat gtg gat tct tcc cac aca gaa
1728Ile Ile Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu
565 570 575tat aca ttg tcc tct ttg act agt gac aca ttg tac atg gta
cga atg 1776Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val
Arg Met 580 585 590gca gca tac aca gat gaa ggt ggg aag gat ggt cca
gaa ttc act ttt 1824Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro
Glu Phe Thr Phe 595 600 605act acc cca aag ttt gct caa gga gaa att
gaa gcc ata gtc gtg cct 1872Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile
Glu Ala Ile Val Val Pro 610 615 620gtt tgc tta gca ttc cta ttg aca
act ctt ctg gga gtg ctg ttc tgc 1920Val Cys Leu Ala Phe Leu Leu Thr
Thr Leu Leu Gly Val Leu Phe Cys625 630 635 640ttt aat aag cga gac
cta att aaa aaa cac atc tgg cct aat gtt cca 1968Phe Asn Lys Arg Asp
Leu Ile Lys Lys His Ile Trp Pro Asn Val Pro 645 650 655gat cct tca
aag agt cat att gcc cag tgg tca cct cac act cct cca 2016Asp Pro Ser
Lys Ser His Ile Ala Gln Trp Ser Pro His Thr Pro Pro 660 665 670agg
cac aat ttt aat tca aaa gat caa atg tat tca gat ggc aat ttc 2064Arg
His Asn Phe Asn Ser Lys Asp Gln Met Tyr Ser Asp Gly Asn Phe 675 680
685act gat gta agt gtt gtg gaa ata gaa gca aat gac aaa aag cct ttt
2112Thr Asp Val Ser Val Val Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe
690 695 700cca gaa gat ctg aaa tca ttg gac ctg ttc aaa aag gaa aaa
att aat 2160Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys
Ile Asn705 710 715 720act gaa gga cac agc agt ggt att ggg ggg tct
tca tgc atg tca tct 2208Thr Glu Gly His Ser Ser Gly Ile Gly Gly Ser
Ser Cys Met Ser Ser 725 730 735tct agg cca agc att tct agc agt gat
gaa aat gaa tct tca caa aac 2256Ser Arg Pro Ser Ile Ser Ser Ser Asp
Glu Asn Glu Ser Ser Gln Asn 740 745 750act tcg agc act gtc cag tat
tct acc gtg gta cac agt ggc tac aga 2304Thr Ser Ser Thr Val Gln Tyr
Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765cac caa gtt ccg tca
gtc caa gtc ttc tca aga tcc gag tct acc cag 2352His Gln Val Pro Ser
Val Gln Val Phe Ser Arg Ser Glu Ser Thr Gln 770 775 780ccc ttg tta
gat tca gag gag cgg cca gaa gat cta caa tta gta gat 2400Pro Leu Leu
Asp Ser Glu Glu Arg Pro Glu Asp Leu Gln Leu Val Asp785 790 795
800cat gta gat ggc ggt gat ggt att ttg ccc agg caa cag tac ttc aaa
2448His Val Asp Gly Gly Asp Gly Ile Leu Pro Arg Gln Gln Tyr Phe Lys
805 810 815cag aac tgc agt cag cat gaa tcc agt cca gat att tca cat
ttt gaa 2496Gln Asn Cys Ser Gln His Glu Ser Ser Pro Asp Ile Ser His
Phe Glu 820 825 830agg tca aag caa gtt tca tca gtc aat gag gaa gat
ttt gtt aga ctt 2544Arg Ser Lys Gln Val Ser Ser Val Asn Glu Glu Asp
Phe Val Arg Leu 835 840 845aaa cag cag att tca gat cat att tca caa
tcc tgt gga tct ggg caa 2592Lys Gln Gln Ile Ser Asp His Ile Ser Gln
Ser Cys Gly Ser Gly Gln 850 855 860atg aaa atg ttt cag gaa gtt tct
gca gca gat gct ttt ggt cca ggt 2640Met Lys Met Phe Gln Glu Val Ser
Ala Ala Asp Ala Phe Gly Pro Gly865 870 875 880act gag gga caa gta
gaa aga ttt gaa aca gtt ggc atg gag gct gcg 2688Thr Glu Gly Gln Val
Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895act gat gaa
ggc atg cct aaa agt tac tta cca cag act gta cgg caa 2736Thr Asp Glu
Gly Met Pro Lys Ser Tyr Leu Pro Gln Thr Val Arg Gln 900 905 910ggc
ggc tac atg cct cag tga 2757Gly Gly Tyr Met Pro Gln 9153918PRTHomo
Sapiens 3Met Leu Thr Leu Gln Thr Trp Leu Val Gln Ala Leu Phe Ile
Phe Leu1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys
Gly Tyr Ile Ser 20 25 30 Pro Glu Ser Pro Val Val Gln Leu His Ser
Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp
Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 Ile Val Trp Lys Thr Asn
His Phe Thr Ile Pro Lys Glu Gln Tyr Thr65 70 75 80 Ile Ile Asn Arg
Thr Ala Ser Ser Val Thr Phe Thr Asp Ile Ala Ser 85 90 95 Leu Asn
Ile Gln Leu Thr Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu 100 105 110
Gln Asn Val Tyr Gly Ile Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys 115
120 125 Pro Lys Asn Leu Ser Cys Ile Val Asn Glu Gly Lys Lys Met Arg
Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn
Phe Thr Leu145 150 155 160 Lys Ser Glu Trp Ala Thr His Lys Phe Ala
Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr Ser Cys Thr Val
Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn Ile Glu Val Trp Val
Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser Asp His Ile
Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 Pro His
Asn Leu Ser Val Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu225 230 235
240 Lys Leu Thr Trp Thr Asn Pro Ser Ile Lys Ser Val Ile Ile Leu Lys
245 250 255 Tyr Asn Ile Gln Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser
Gln Ile 260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe
Thr Val Gln Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg
Ile Arg Cys Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp
Trp Ser Glu Glu Ala Ser Gly Ile305 310 315 320 Thr Tyr Glu Asp Arg
Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile 325 330 335 Asp Pro Ser
His Thr Gln Gly Tyr Arg Thr Val Gln Leu Val Trp Lys 340 345 350 Thr
Leu Pro Pro Phe Glu Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val 355 360
365 Thr Leu Thr Arg Trp Lys Ser His Leu Gln Asn Tyr Thr Val Asn Ala
370 375 380 Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala
Thr Leu385 390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala
Ala Val Leu Thr Ile 405 410 415 Pro Ala Cys Asp Phe Gln Ala Thr His
Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu
Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr
Ile Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys Ile
Thr Asp Trp Gln Gln Glu Asp Gly Thr Val His Arg Thr465 470 475 480
Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val 485
490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser Ile Lys
Ala 500 505 510 Tyr Leu Lys Gln Ala Pro Pro Ser Lys Gly Pro Thr Val
Arg Thr Lys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp
Asp Gln Leu Pro Val 530 535 540 Asp Val Gln Asn Gly Phe Ile Arg Asn
Tyr Thr Ile Phe Tyr Arg Thr545 550 555 560 Ile Ile Gly Asn Glu Thr
Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser
Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala
Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 605
Thr Thr Pro Lys Phe Ala Gln Gly Glu Ile Glu Ala Ile Val Val Pro 610
615 620 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe
Cys625 630 635 640 Phe Asn Lys Arg Asp Leu Ile Lys Lys His Ile Trp
Pro Asn Val Pro 645 650 655 Asp Pro Ser Lys Ser His Ile Ala Gln Trp
Ser Pro His Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp
Gln Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val
Glu Ile Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu
Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys Ile Asn705 710 715 720 Thr
Glu Gly His Ser Ser Gly Ile Gly Gly Ser Ser Cys Met Ser Ser 725 730
735 Ser Arg Pro Ser Ile Ser Ser Ser Asp Glu Asn Glu Ser Ser Gln Asn
740 745 750 Thr Ser Ser Thr Val Gln Tyr Ser Thr Val Val His Ser Gly
Tyr Arg 755 760 765 His Gln Val Pro Ser Val Gln Val Phe Ser Arg Ser
Glu Ser Thr Gln 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu
Asp Leu Gln Leu Val Asp785 790 795 800 His Val Asp Gly Gly Asp Gly
Ile Leu Pro Arg Gln Gln Tyr Phe Lys 805 810 815 Gln Asn Cys Ser Gln
His Glu Ser Ser Pro Asp Ile Ser His Phe Glu 820 825 830 Arg Ser Lys
Gln Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys
Gln Gln Ile Ser Asp His Ile Ser Gln Ser Cys Gly Ser Gly Gln 850 855
860 Met Lys Met Phe Gln Glu Val Ser Ala Ala Asp Ala Phe Gly Pro
Gly865 870 875 880 Thr Glu Gly Gln Val Glu Arg Phe Glu Thr Val Gly
Met Glu Ala Ala 885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu
Pro Gln Thr Val Arg Gln 900 905 910 Gly Gly Tyr Met Pro Gln 915
44040DNAHomo Sapiensmisc_feature(0)...(0)cDNA for IL12RB2
4tgcagagcac agagaaagga catctgcgag gaaagttccc tgatggctgt caacaaagtg
60ccacgtctct atggctgtga acgctgagca cacgatttta tcgcgcctat catatcttgg
120tgcataaacg cacctcacct cggtcaaccc ttgctccgtc ttatgagaca
ggctttatta 180tccgcatttt atatgagggg aaactgacgg tggagagaga
attatcttgc tcaaggcgac 240acagcagagc ccacaggtgg cagaatccca
cccgagcccg cttcgacccg cggggtggaa 300accacgggcg cccgcccggc
tgcgcttcca gagctgaact gagaagcgag tcctctccgc 360cctgcggcca
ccgcccagcc ccgacccccg ccccggcccg atcctcactc gccgccagct
420ccccgcgccc accccggagt tggtggcgca gaggcgggag gcggaggcgg
gagggcgggc 480gctggcaccg ggaacgcccg agcgccggca gagagcgcgg
agagcgcgac acgtgcggcc 540cagagcaccg gggccacccg gtccccgcag
gcccgggacc gcgcccgctg gcaggcgaca 600cgtggaagaa tacggagttc
tataccagag ttgattgttg atg gca cat act ttt 655 Met Ala His Thr Phe 1
5aga gga tgc tca ttg gca ttt atg ttt ata atc acg tgg ctg ttg att
703Arg Gly Cys Ser Leu Ala Phe Met Phe Ile Ile Thr Trp Leu Leu Ile
10 15 20aaa gca aaa ata gat gcg tgc aag aga ggc gat gtg act gtg aag
cct 751Lys Ala Lys Ile Asp Ala Cys Lys Arg Gly Asp Val Thr Val Lys
Pro 25 30 35tcc cat gta att tta ctt gga tcc act gtc aat att aca tgc
tct ttg 799Ser His Val Ile Leu Leu Gly Ser Thr Val Asn Ile Thr Cys
Ser Leu 40 45 50aag ccc aga caa ggc tgc ttt cac tat tcc aga cgt aac
aag tta atc 847Lys Pro Arg Gln Gly Cys Phe His Tyr Ser Arg Arg Asn
Lys Leu Ile 55 60 65ctg tac aag ttt gac aga aga atc aat ttt cac cat
ggc cac tcc ctc 895Leu Tyr Lys Phe Asp Arg Arg Ile Asn Phe His His
Gly His Ser Leu70 75 80 85aat tct caa gtc aca ggt ctt ccc ctt ggt
aca acc ttg ttt gtc tgc 943Asn Ser Gln Val Thr Gly Leu Pro Leu Gly
Thr Thr Leu Phe Val Cys 90 95 100aaa ctg gcc tgt atc aat agt gat
gaa att caa ata tgt gga gca gag 991Lys Leu Ala Cys Ile Asn Ser Asp
Glu Ile Gln Ile Cys Gly Ala Glu 105 110 115atc ttc gtt ggt gtt gct
cca gaa cag cct caa aat tta tcc tgc ata 1039Ile Phe Val Gly Val Ala
Pro Glu Gln Pro Gln Asn Leu Ser Cys Ile 120 125 130cag aag gga gaa
cag ggg act gtg gcc tgc acc tgg gaa aga gga cga 1087Gln Lys Gly Glu
Gln Gly Thr Val Ala Cys Thr Trp Glu Arg Gly Arg 135 140 145gac acc
cac tta tac act gag tat act cta cag cta agt gga cca aaa 1135Asp Thr
His Leu Tyr Thr Glu Tyr Thr Leu Gln Leu Ser Gly Pro Lys150 155 160
165aat tta acc tgg cag aag caa tgt aaa gac att tat tgt gac tat ttg
1183Asn Leu Thr Trp Gln Lys Gln Cys Lys Asp Ile Tyr Cys Asp Tyr Leu
170 175 180gac ttt gga atc aac ctc acc cct gaa tca cct gaa tcc aat
ttc aca 1231Asp Phe Gly Ile Asn Leu Thr Pro Glu Ser Pro Glu Ser Asn
Phe Thr 185 190 195gcc aag gtt act gct gtc aat agt ctt gga agc tcc
tct tca ctt cca 1279Ala Lys Val Thr Ala Val Asn Ser Leu Gly Ser Ser
Ser Ser Leu Pro 200 205 210tcc aca ttc aca ttc ttg gac ata gtg agg
cct ctt cct ccg tgg gac 1327Ser Thr Phe Thr Phe Leu Asp Ile Val Arg
Pro Leu Pro Pro Trp Asp 215 220 225att aga atc aaa ttt caa aag gct
tct gtg agc aga tgt acc ctt tat 1375Ile Arg Ile Lys Phe Gln Lys Ala
Ser Val Ser Arg Cys Thr Leu Tyr230 235 240 245tgg aga gat gag gga
ctg gta ctg ctt aat cga ctc aga tat cgg ccc 1423Trp Arg Asp Glu Gly
Leu Val Leu Leu Asn Arg Leu Arg Tyr Arg Pro 250 255 260agt aac agc
agg ctc tgg aat atg gtt aat gtt aca aag gcc aaa gga 1471Ser Asn Ser
Arg Leu Trp Asn Met Val Asn Val Thr Lys Ala Lys Gly 265 270 275aga
cat gat ttg ctg gat ctg aaa cca ttt aca gaa tat gaa ttt cag 1519Arg
His Asp Leu Leu Asp Leu Lys Pro Phe Thr Glu Tyr Glu Phe Gln 280 285
290att tcc tct aag cta cat ctt tat aag gga agt tgg agt gat tgg agt
1567Ile Ser Ser Lys Leu His Leu Tyr Lys Gly Ser Trp Ser Asp Trp Ser
295 300 305gaa tca ttg aga gca caa aca cca gaa gaa gag cct act ggg
atg tta 1615Glu Ser Leu Arg Ala Gln Thr Pro Glu Glu Glu Pro Thr Gly
Met Leu310 315 320 325gat gtc tgg tac atg aaa cgg cac att gac tac
agt aga caa cag att 1663Asp Val Trp Tyr Met Lys Arg His Ile Asp Tyr
Ser Arg Gln Gln Ile 330 335 340tct ctt ttc tgg aag aat ctg agt gtc
tca gag gca aga gga aaa att 1711Ser Leu Phe Trp Lys Asn Leu Ser Val
Ser Glu Ala Arg Gly Lys Ile 345 350 355ctc cac tat cag gtg acc ttg
cag gag ctg aca gga ggg aaa gcc atg 1759Leu His Tyr Gln Val Thr Leu
Gln Glu Leu Thr Gly Gly Lys Ala Met 360 365 370aca cag aac atc aca
gga cac acc tcc tgg acc aca gtc att cct aga 1807Thr Gln Asn Ile Thr
Gly His Thr Ser Trp Thr Thr Val Ile Pro Arg 375 380 385acc gga aat
tgg gct gtg gct gtg tct gca gca aat tca aaa ggc agt 1855Thr Gly Asn
Trp Ala Val Ala Val Ser Ala Ala Asn Ser Lys Gly Ser390 395 400
405tct ctg ccc act cgt att aac ata atg aac ctg tgt gag gca ggg ttg
1903Ser Leu Pro Thr Arg Ile Asn Ile Met Asn Leu Cys Glu Ala Gly Leu
410 415 420ctg gct cct cgc cag gtc tct gca aac tca gag ggc atg gac
aac att 1951Leu Ala Pro Arg Gln Val Ser Ala Asn Ser Glu Gly Met Asp
Asn Ile 425 430 435ctg gtg act tgg cag cct ccc agg aaa gat ccc tct
gct gtt cag gag 1999Leu Val Thr Trp Gln Pro Pro Arg Lys Asp Pro Ser
Ala Val Gln Glu 440 445 450tac gtg gtg gaa tgg aga gag ctc cat cca
ggg ggt gac aca cag gtc 2047Tyr Val Val Glu Trp Arg Glu Leu His Pro
Gly Gly Asp Thr Gln Val 455 460 465cct cta aac tgg cta cgg agt cga
ccc tac aat gtg tct gct ctg att 2095Pro Leu Asn Trp Leu Arg Ser Arg
Pro Tyr Asn Val Ser Ala Leu Ile470 475 480 485tca gag aac ata aaa
tcc tac atc tgt tat
gaa atc cgt gtg tat gca 2143Ser Glu Asn Ile Lys Ser Tyr Ile Cys Tyr
Glu Ile Arg Val Tyr Ala 490 495 500ctc tca ggg gat caa gga gga tgc
agc tcc atc ctg ggt aac tct aag 2191Leu Ser Gly Asp Gln Gly Gly Cys
Ser Ser Ile Leu Gly Asn Ser Lys 505 510 515cac aaa gca cca ctg agt
ggc ccc cac att aat gcc atc aca gag gaa 2239His Lys Ala Pro Leu Ser
Gly Pro His Ile Asn Ala Ile Thr Glu Glu 520 525 530aag ggg agc att
tta att tca tgg aac agc att cca gtc cag gag caa 2287Lys Gly Ser Ile
Leu Ile Ser Trp Asn Ser Ile Pro Val Gln Glu Gln 535 540 545atg ggc
tgc ctc ctc cat tat agg ata tac tgg aag gaa cgg gac tcc 2335Met Gly
Cys Leu Leu His Tyr Arg Ile Tyr Trp Lys Glu Arg Asp Ser550 555 560
565aac tcc cag cct cag ctc tgt gaa att ccc tac aga gtc tcc caa aat
2383Asn Ser Gln Pro Gln Leu Cys Glu Ile Pro Tyr Arg Val Ser Gln Asn
570 575 580tca cat cca ata aac agc ctg cag ccc cga gtg aca tat gtc
ctg tgg 2431Ser His Pro Ile Asn Ser Leu Gln Pro Arg Val Thr Tyr Val
Leu Trp 585 590 595atg aca gct ctg aca gct gct ggt gaa agt tcc cac
gga aat gag agg 2479Met Thr Ala Leu Thr Ala Ala Gly Glu Ser Ser His
Gly Asn Glu Arg 600 605 610gaa ttt tgt ctg caa ggt aaa gcc aat tgg
atg gcg ttt gtg gca cca 2527Glu Phe Cys Leu Gln Gly Lys Ala Asn Trp
Met Ala Phe Val Ala Pro 615 620 625agc att tgc att gct atc atc atg
gtg ggc att ttc tca acg cat tac 2575Ser Ile Cys Ile Ala Ile Ile Met
Val Gly Ile Phe Ser Thr His Tyr630 635 640 645ttc cag caa aag gtg
ttt gtt ctc cta gca gcc ctc aga cct cag tgg 2623Phe Gln Gln Lys Val
Phe Val Leu Leu Ala Ala Leu Arg Pro Gln Trp 650 655 660tgt agc aga
gaa att cca gat cca gca aat agc act tgc gct aag aaa 2671Cys Ser Arg
Glu Ile Pro Asp Pro Ala Asn Ser Thr Cys Ala Lys Lys 665 670 675tat
ccc att gca gag gag aag aca cag ctg ccc ttg gac agg ctc ctg 2719Tyr
Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro Leu Asp Arg Leu Leu 680 685
690ata gac tgg ccc acg cct gaa gat cct gaa ccg ctg gtc atc agt gaa
2767Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu Pro Leu Val Ile Ser Glu
695 700 705gtc ctt cat caa gtg acc cca gtt ttc aga cat ccc ccc tgc
tcc aac 2815Val Leu His Gln Val Thr Pro Val Phe Arg His Pro Pro Cys
Ser Asn710 715 720 725tgg cca caa agg gaa aaa gga atc caa ggt cat
cag gcc tct gag aaa 2863Trp Pro Gln Arg Glu Lys Gly Ile Gln Gly His
Gln Ala Ser Glu Lys 730 735 740gac atg atg cac agt gcc tca agc cca
cca cct cca aga gct ctc caa 2911Asp Met Met His Ser Ala Ser Ser Pro
Pro Pro Pro Arg Ala Leu Gln 745 750 755gct gag agc aga caa ctg gtg
gat ctg tac aag gtg ctg gag agc agg 2959Ala Glu Ser Arg Gln Leu Val
Asp Leu Tyr Lys Val Leu Glu Ser Arg 760 765 770ggc tcc gac cca aag
ccc gaa aac cca gcc tgt ccc tgg acg gtg ctc 3007Gly Ser Asp Pro Lys
Pro Glu Asn Pro Ala Cys Pro Trp Thr Val Leu 775 780 785cca gca ggt
gac ctt ccc acc cat gat ggc tac tta ccc tcc aac ata 3055Pro Ala Gly
Asp Leu Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile790 795 800
805gat gac ctc ccc tca cat gag gca cct ctc gct gac tct ctg gaa gaa
3103Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala Asp Ser Leu Glu Glu
810 815 820ctg gag cct cag cac atc tcc ctt tct gtt ttc ccc tca agt
tct ctt 3151Leu Glu Pro Gln His Ile Ser Leu Ser Val Phe Pro Ser Ser
Ser Leu 825 830 835cac cca ctc acc ttc tcc tgt ggt gat aag ctg act
ctg gat cag tta 3199His Pro Leu Thr Phe Ser Cys Gly Asp Lys Leu Thr
Leu Asp Gln Leu 840 845 850aag atg agg tgt gac tcc ctc atg ctc tga
gtggtgaggc ttcaagcctt 3249Lys Met Arg Cys Asp Ser Leu Met Leu 855
860aaagtcagtg tgccctcaac cagcacagcc tgccccaatt cccccagccc
ctgctccagc 3309agctgtcatc tctgggtgcc accatcggtc tggctgcagc
tagaggacag gcaagccagc 3369tctgggggag tcttaggaac tgggagttgg
tcttcactca gatgcctcat cttgcctttc 3429ccagggcctt aaaattacat
ccttcactgt gtggacctag agactccaac ttgaattcct 3489agtaactttc
ttggtatgct ggccagaaag ggaaatgagg aggagagtag aaaccacagc
3549tcttagtagt aatggcatac agtctagagg accattcatg caatgactat
ttctaaagca 3609cctgctacac agcaggctgt acacagcaga tcagtactgt
tcaacagaac ttcctgagat 3669gatggaaatg ttctacctct gcactcactg
tccagtacat tagacactag gcacattggc 3729tgttaatcac ttggaatgtg
tttagcttga ctgaggaatt aaattttgat tgtaaattta 3789aatcgccaca
catggctagt ggctactgta ttggagtgca cagctctaga tggctcctag
3849attattgaga gccttcaaaa caaatcaacc tagttctata gatgaagaca
taaaagacac 3909tggtaaacac caaggtaaaa gggcccccaa ggtggtcatg
actggtctca tttgcagaag 3969tctaagaatg tacctttttc tggccgggcg
tggtagctca tgcctgtaat cccagcactt 4029tgggaggctg a 404052589DNAHomo
Sapiensmisc_feature(0)...(0)coding sequence for IL12RB2 5atg gca
cat act ttt aga gga tgc tca ttg gca ttt atg ttt ata atc 48Met Ala
His Thr Phe Arg Gly Cys Ser Leu Ala Phe Met Phe Ile Ile1 5 10 15acg
tgg ctg ttg att aaa gca aaa ata gat gcg tgc aag aga ggc gat 96Thr
Trp Leu Leu Ile Lys Ala Lys Ile Asp Ala Cys Lys Arg Gly Asp 20 25
30gtg act gtg aag cct tcc cat gta att tta ctt gga tcc act gtc aat
144Val Thr Val Lys Pro Ser His Val Ile Leu Leu Gly Ser Thr Val Asn
35 40 45att aca tgc tct ttg aag ccc aga caa ggc tgc ttt cac tat tcc
aga 192Ile Thr Cys Ser Leu Lys Pro Arg Gln Gly Cys Phe His Tyr Ser
Arg 50 55 60cgt aac aag tta atc ctg tac aag ttt gac aga aga atc aat
ttt cac 240Arg Asn Lys Leu Ile Leu Tyr Lys Phe Asp Arg Arg Ile Asn
Phe His65 70 75 80cat ggc cac tcc ctc aat tct caa gtc aca ggt ctt
ccc ctt ggt aca 288His Gly His Ser Leu Asn Ser Gln Val Thr Gly Leu
Pro Leu Gly Thr 85 90 95acc ttg ttt gtc tgc aaa ctg gcc tgt atc aat
agt gat gaa att caa 336Thr Leu Phe Val Cys Lys Leu Ala Cys Ile Asn
Ser Asp Glu Ile Gln 100 105 110ata tgt gga gca gag atc ttc gtt ggt
gtt gct cca gaa cag cct caa 384Ile Cys Gly Ala Glu Ile Phe Val Gly
Val Ala Pro Glu Gln Pro Gln 115 120 125aat tta tcc tgc ata cag aag
gga gaa cag ggg act gtg gcc tgc acc 432Asn Leu Ser Cys Ile Gln Lys
Gly Glu Gln Gly Thr Val Ala Cys Thr 130 135 140tgg gaa aga gga cga
gac acc cac tta tac act gag tat act cta cag 480Trp Glu Arg Gly Arg
Asp Thr His Leu Tyr Thr Glu Tyr Thr Leu Gln145 150 155 160cta agt
gga cca aaa aat tta acc tgg cag aag caa tgt aaa gac att 528Leu Ser
Gly Pro Lys Asn Leu Thr Trp Gln Lys Gln Cys Lys Asp Ile 165 170
175tat tgt gac tat ttg gac ttt gga atc aac ctc acc cct gaa tca cct
576Tyr Cys Asp Tyr Leu Asp Phe Gly Ile Asn Leu Thr Pro Glu Ser Pro
180 185 190gaa tcc aat ttc aca gcc aag gtt act gct gtc aat agt ctt
gga agc 624Glu Ser Asn Phe Thr Ala Lys Val Thr Ala Val Asn Ser Leu
Gly Ser 195 200 205tcc tct tca ctt cca tcc aca ttc aca ttc ttg gac
ata gtg agg cct 672Ser Ser Ser Leu Pro Ser Thr Phe Thr Phe Leu Asp
Ile Val Arg Pro 210 215 220ctt cct ccg tgg gac att aga atc aaa ttt
caa aag gct tct gtg agc 720Leu Pro Pro Trp Asp Ile Arg Ile Lys Phe
Gln Lys Ala Ser Val Ser225 230 235 240aga tgt acc ctt tat tgg aga
gat gag gga ctg gta ctg ctt aat cga 768Arg Cys Thr Leu Tyr Trp Arg
Asp Glu Gly Leu Val Leu Leu Asn Arg 245 250 255ctc aga tat cgg ccc
agt aac agc agg ctc tgg aat atg gtt aat gtt 816Leu Arg Tyr Arg Pro
Ser Asn Ser Arg Leu Trp Asn Met Val Asn Val 260 265 270aca aag gcc
aaa gga aga cat gat ttg ctg gat ctg aaa cca ttt aca 864Thr Lys Ala
Lys Gly Arg His Asp Leu Leu Asp Leu Lys Pro Phe Thr 275 280 285gaa
tat gaa ttt cag att tcc tct aag cta cat ctt tat aag gga agt 912Glu
Tyr Glu Phe Gln Ile Ser Ser Lys Leu His Leu Tyr Lys Gly Ser 290 295
300tgg agt gat tgg agt gaa tca ttg aga gca caa aca cca gaa gaa gag
960Trp Ser Asp Trp Ser Glu Ser Leu Arg Ala Gln Thr Pro Glu Glu
Glu305 310 315 320cct act ggg atg tta gat gtc tgg tac atg aaa cgg
cac att gac tac 1008Pro Thr Gly Met Leu Asp Val Trp Tyr Met Lys Arg
His Ile Asp Tyr 325 330 335agt aga caa cag att tct ctt ttc tgg aag
aat ctg agt gtc tca gag 1056Ser Arg Gln Gln Ile Ser Leu Phe Trp Lys
Asn Leu Ser Val Ser Glu 340 345 350gca aga gga aaa att ctc cac tat
cag gtg acc ttg cag gag ctg aca 1104Ala Arg Gly Lys Ile Leu His Tyr
Gln Val Thr Leu Gln Glu Leu Thr 355 360 365gga ggg aaa gcc atg aca
cag aac atc aca gga cac acc tcc tgg acc 1152Gly Gly Lys Ala Met Thr
Gln Asn Ile Thr Gly His Thr Ser Trp Thr 370 375 380aca gtc att cct
aga acc gga aat tgg gct gtg gct gtg tct gca gca 1200Thr Val Ile Pro
Arg Thr Gly Asn Trp Ala Val Ala Val Ser Ala Ala385 390 395 400aat
tca aaa ggc agt tct ctg ccc act cgt att aac ata atg aac ctg 1248Asn
Ser Lys Gly Ser Ser Leu Pro Thr Arg Ile Asn Ile Met Asn Leu 405 410
415tgt gag gca ggg ttg ctg gct cct cgc cag gtc tct gca aac tca gag
1296Cys Glu Ala Gly Leu Leu Ala Pro Arg Gln Val Ser Ala Asn Ser Glu
420 425 430ggc atg gac aac att ctg gtg act tgg cag cct ccc agg aaa
gat ccc 1344Gly Met Asp Asn Ile Leu Val Thr Trp Gln Pro Pro Arg Lys
Asp Pro 435 440 445tct gct gtt cag gag tac gtg gtg gaa tgg aga gag
ctc cat cca ggg 1392Ser Ala Val Gln Glu Tyr Val Val Glu Trp Arg Glu
Leu His Pro Gly 450 455 460ggt gac aca cag gtc cct cta aac tgg cta
cgg agt cga ccc tac aat 1440Gly Asp Thr Gln Val Pro Leu Asn Trp Leu
Arg Ser Arg Pro Tyr Asn465 470 475 480gtg tct gct ctg att tca gag
aac ata aaa tcc tac atc tgt tat gaa 1488Val Ser Ala Leu Ile Ser Glu
Asn Ile Lys Ser Tyr Ile Cys Tyr Glu 485 490 495atc cgt gtg tat gca
ctc tca ggg gat caa gga gga tgc agc tcc atc 1536Ile Arg Val Tyr Ala
Leu Ser Gly Asp Gln Gly Gly Cys Ser Ser Ile 500 505 510ctg ggt aac
tct aag cac aaa gca cca ctg agt ggc ccc cac att aat 1584Leu Gly Asn
Ser Lys His Lys Ala Pro Leu Ser Gly Pro His Ile Asn 515 520 525gcc
atc aca gag gaa aag ggg agc att tta att tca tgg aac agc att 1632Ala
Ile Thr Glu Glu Lys Gly Ser Ile Leu Ile Ser Trp Asn Ser Ile 530 535
540cca gtc cag gag caa atg ggc tgc ctc ctc cat tat agg ata tac tgg
1680Pro Val Gln Glu Gln Met Gly Cys Leu Leu His Tyr Arg Ile Tyr
Trp545 550 555 560aag gaa cgg gac tcc aac tcc cag cct cag ctc tgt
gaa att ccc tac 1728Lys Glu Arg Asp Ser Asn Ser Gln Pro Gln Leu Cys
Glu Ile Pro Tyr 565 570 575aga gtc tcc caa aat tca cat cca ata aac
agc ctg cag ccc cga gtg 1776Arg Val Ser Gln Asn Ser His Pro Ile Asn
Ser Leu Gln Pro Arg Val 580 585 590aca tat gtc ctg tgg atg aca gct
ctg aca gct gct ggt gaa agt tcc 1824Thr Tyr Val Leu Trp Met Thr Ala
Leu Thr Ala Ala Gly Glu Ser Ser 595 600 605cac gga aat gag agg gaa
ttt tgt ctg caa ggt aaa gcc aat tgg atg 1872His Gly Asn Glu Arg Glu
Phe Cys Leu Gln Gly Lys Ala Asn Trp Met 610 615 620gcg ttt gtg gca
cca agc att tgc att gct atc atc atg gtg ggc att 1920Ala Phe Val Ala
Pro Ser Ile Cys Ile Ala Ile Ile Met Val Gly Ile625 630 635 640ttc
tca acg cat tac ttc cag caa aag gtg ttt gtt ctc cta gca gcc 1968Phe
Ser Thr His Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala 645 650
655ctc aga cct cag tgg tgt agc aga gaa att cca gat cca gca aat agc
2016Leu Arg Pro Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser
660 665 670act tgc gct aag aaa tat ccc att gca gag gag aag aca cag
ctg ccc 2064Thr Cys Ala Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln
Leu Pro 675 680 685ttg gac agg ctc ctg ata gac tgg ccc acg cct gaa
gat cct gaa ccg 2112Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr Pro Glu
Asp Pro Glu Pro 690 695 700ctg gtc atc agt gaa gtc ctt cat caa gtg
acc cca gtt ttc aga cat 2160Leu Val Ile Ser Glu Val Leu His Gln Val
Thr Pro Val Phe Arg His705 710 715 720ccc ccc tgc tcc aac tgg cca
caa agg gaa aaa gga atc caa ggt cat 2208Pro Pro Cys Ser Asn Trp Pro
Gln Arg Glu Lys Gly Ile Gln Gly His 725 730 735cag gcc tct gag aaa
gac atg atg cac agt gcc tca agc cca cca cct 2256Gln Ala Ser Glu Lys
Asp Met Met His Ser Ala Ser Ser Pro Pro Pro 740 745 750cca aga gct
ctc caa gct gag agc aga caa ctg gtg gat ctg tac aag 2304Pro Arg Ala
Leu Gln Ala Glu Ser Arg Gln Leu Val Asp Leu Tyr Lys 755 760 765gtg
ctg gag agc agg ggc tcc gac cca aag ccc gaa aac cca gcc tgt 2352Val
Leu Glu Ser Arg Gly Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys 770 775
780ccc tgg acg gtg ctc cca gca ggt gac ctt ccc acc cat gat ggc tac
2400Pro Trp Thr Val Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly
Tyr785 790 795 800tta ccc tcc aac ata gat gac ctc ccc tca cat gag
gca cct ctc gct 2448Leu Pro Ser Asn Ile Asp Asp Leu Pro Ser His Glu
Ala Pro Leu Ala 805 810 815gac tct ctg gaa gaa ctg gag cct cag cac
atc tcc ctt tct gtt ttc 2496Asp Ser Leu Glu Glu Leu Glu Pro Gln His
Ile Ser Leu Ser Val Phe 820 825 830ccc tca agt tct ctt cac cca ctc
acc ttc tcc tgt ggt gat aag ctg 2544Pro Ser Ser Ser Leu His Pro Leu
Thr Phe Ser Cys Gly Asp Lys Leu 835 840 845act ctg gat cag tta aag
atg agg tgt gac tcc ctc atg ctc tga 2589Thr Leu Asp Gln Leu Lys Met
Arg Cys Asp Ser Leu Met Leu 850 855 8606862PRTHomo sapiens 6Met Ala
His Thr Phe Arg Gly Cys Ser Leu Ala Phe Met Phe Ile Ile1 5 10 15
Thr Trp Leu Leu Ile Lys Ala Lys Ile Asp Ala Cys Lys Arg Gly Asp 20
25 30 Val Thr Val Lys Pro Ser His Val Ile Leu Leu Gly Ser Thr Val
Asn 35 40 45 Ile Thr Cys Ser Leu Lys Pro Arg Gln Gly Cys Phe His
Tyr Ser Arg 50 55 60 Arg Asn Lys Leu Ile Leu Tyr Lys Phe Asp Arg
Arg Ile Asn Phe His65 70 75 80 His Gly His Ser Leu Asn Ser Gln Val
Thr Gly Leu Pro Leu Gly Thr 85 90 95 Thr Leu Phe Val Cys Lys Leu
Ala Cys Ile Asn Ser Asp Glu Ile Gln 100 105 110 Ile Cys Gly Ala Glu
Ile Phe Val Gly Val Ala Pro Glu Gln Pro Gln 115 120 125 Asn Leu Ser
Cys Ile Gln Lys Gly Glu Gln Gly Thr Val Ala Cys Thr 130 135 140 Trp
Glu Arg Gly Arg Asp Thr His Leu Tyr Thr Glu Tyr Thr Leu Gln145 150
155 160 Leu Ser Gly Pro Lys Asn Leu Thr Trp Gln Lys Gln Cys Lys Asp
Ile 165 170 175 Tyr Cys Asp Tyr Leu Asp Phe Gly Ile Asn Leu Thr Pro
Glu Ser Pro 180 185 190 Glu Ser Asn Phe Thr Ala Lys Val Thr Ala Val
Asn Ser Leu Gly Ser 195 200 205 Ser Ser Ser Leu Pro Ser Thr Phe Thr
Phe Leu Asp Ile Val Arg Pro 210 215 220 Leu Pro Pro Trp Asp Ile Arg
Ile Lys Phe Gln Lys Ala Ser Val Ser225 230 235 240 Arg Cys Thr Leu
Tyr Trp Arg Asp Glu Gly Leu Val Leu Leu Asn Arg 245 250 255 Leu Arg
Tyr Arg Pro Ser Asn Ser Arg Leu Trp Asn Met Val Asn Val 260 265 270
Thr Lys Ala Lys Gly Arg His Asp Leu Leu Asp Leu Lys Pro Phe Thr 275
280 285 Glu Tyr Glu Phe Gln Ile Ser Ser Lys Leu His Leu Tyr Lys Gly
Ser 290 295 300 Trp Ser Asp Trp Ser Glu Ser Leu Arg Ala Gln Thr Pro
Glu Glu Glu305
310 315 320 Pro Thr Gly Met Leu Asp Val Trp Tyr Met Lys Arg His Ile
Asp Tyr 325 330 335 Ser Arg Gln Gln Ile Ser Leu Phe Trp Lys Asn Leu
Ser Val Ser Glu 340 345 350 Ala Arg Gly Lys Ile Leu His Tyr Gln Val
Thr Leu Gln Glu Leu Thr 355 360 365 Gly Gly Lys Ala Met Thr Gln Asn
Ile Thr Gly His Thr Ser Trp Thr 370 375 380 Thr Val Ile Pro Arg Thr
Gly Asn Trp Ala Val Ala Val Ser Ala Ala385 390 395 400 Asn Ser Lys
Gly Ser Ser Leu Pro Thr Arg Ile Asn Ile Met Asn Leu 405 410 415 Cys
Glu Ala Gly Leu Leu Ala Pro Arg Gln Val Ser Ala Asn Ser Glu 420 425
430 Gly Met Asp Asn Ile Leu Val Thr Trp Gln Pro Pro Arg Lys Asp Pro
435 440 445 Ser Ala Val Gln Glu Tyr Val Val Glu Trp Arg Glu Leu His
Pro Gly 450 455 460 Gly Asp Thr Gln Val Pro Leu Asn Trp Leu Arg Ser
Arg Pro Tyr Asn465 470 475 480 Val Ser Ala Leu Ile Ser Glu Asn Ile
Lys Ser Tyr Ile Cys Tyr Glu 485 490 495 Ile Arg Val Tyr Ala Leu Ser
Gly Asp Gln Gly Gly Cys Ser Ser Ile 500 505 510 Leu Gly Asn Ser Lys
His Lys Ala Pro Leu Ser Gly Pro His Ile Asn 515 520 525 Ala Ile Thr
Glu Glu Lys Gly Ser Ile Leu Ile Ser Trp Asn Ser Ile 530 535 540 Pro
Val Gln Glu Gln Met Gly Cys Leu Leu His Tyr Arg Ile Tyr Trp545 550
555 560 Lys Glu Arg Asp Ser Asn Ser Gln Pro Gln Leu Cys Glu Ile Pro
Tyr 565 570 575 Arg Val Ser Gln Asn Ser His Pro Ile Asn Ser Leu Gln
Pro Arg Val 580 585 590 Thr Tyr Val Leu Trp Met Thr Ala Leu Thr Ala
Ala Gly Glu Ser Ser 595 600 605 His Gly Asn Glu Arg Glu Phe Cys Leu
Gln Gly Lys Ala Asn Trp Met 610 615 620 Ala Phe Val Ala Pro Ser Ile
Cys Ile Ala Ile Ile Met Val Gly Ile625 630 635 640 Phe Ser Thr His
Tyr Phe Gln Gln Lys Val Phe Val Leu Leu Ala Ala 645 650 655 Leu Arg
Pro Gln Trp Cys Ser Arg Glu Ile Pro Asp Pro Ala Asn Ser 660 665 670
Thr Cys Ala Lys Lys Tyr Pro Ile Ala Glu Glu Lys Thr Gln Leu Pro 675
680 685 Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr Pro Glu Asp Pro Glu
Pro 690 695 700 Leu Val Ile Ser Glu Val Leu His Gln Val Thr Pro Val
Phe Arg His705 710 715 720 Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu
Lys Gly Ile Gln Gly His 725 730 735 Gln Ala Ser Glu Lys Asp Met Met
His Ser Ala Ser Ser Pro Pro Pro 740 745 750 Pro Arg Ala Leu Gln Ala
Glu Ser Arg Gln Leu Val Asp Leu Tyr Lys 755 760 765 Val Leu Glu Ser
Arg Gly Ser Asp Pro Lys Pro Glu Asn Pro Ala Cys 770 775 780 Pro Trp
Thr Val Leu Pro Ala Gly Asp Leu Pro Thr His Asp Gly Tyr785 790 795
800 Leu Pro Ser Asn Ile Asp Asp Leu Pro Ser His Glu Ala Pro Leu Ala
805 810 815 Asp Ser Leu Glu Glu Leu Glu Pro Gln His Ile Ser Leu Ser
Val Phe 820 825 830 Pro Ser Ser Ser Leu His Pro Leu Thr Phe Ser Cys
Gly Asp Lys Leu 835 840 845 Thr Leu Asp Gln Leu Lys Met Arg Cys Asp
Ser Leu Met Leu 850 855 860
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