U.S. patent application number 15/299072 was filed with the patent office on 2017-02-09 for elisa for a naturally-occurring soluble truncated form of il-23 receptor.
This patent application is currently assigned to Medical Diagnostic Laboratories, LLC. The applicant listed for this patent is Medical Diagnostic Laboratories, LLC. Invention is credited to Jonathan Brazaitis, Joyce Eskdale, Grant Gallagher, Raymond Yu.
Application Number | 20170038396 15/299072 |
Document ID | / |
Family ID | 45818280 |
Filed Date | 2017-02-09 |
United States Patent
Application |
20170038396 |
Kind Code |
A1 |
Gallagher; Grant ; et
al. |
February 9, 2017 |
ELISA for a Naturally-Occurring Soluble Truncated Form of IL-23
Receptor
Abstract
A naturally-occurring soluble truncated IL-23R.alpha. protein
(i.e., .DELTA.9 IL-23R.alpha.) is shown to be present in a
biological sample and can serve as a diagnostic tool for autoimmune
diseases. There is provided an enzyme-linked immunosorbent assay
(ELISA) and test kit for the serological detection of the soluble
truncated form of IL-23R.alpha. protein. More particularly,
antibody-sandwich ELISA method and kits for .DELTA.9 IL-23R.alpha.
as an antigen were developed to detect .DELTA.9 IL-23R.alpha.
levels in biological samples from a mammal and a human patient and
are used as a diagnostic index. The present disclosed ELISA has
utility as a diagnostic tool to detect Crohn's disease in patients
using EDTA-plasma.
Inventors: |
Gallagher; Grant; (Milltown,
NJ) ; Yu; Raymond; (East Brunswick, NJ) ;
Eskdale; Joyce; (Milltown, NJ) ; Brazaitis;
Jonathan; (Parlin, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Medical Diagnostic Laboratories, LLC |
Hamilton |
NJ |
US |
|
|
Assignee: |
Medical Diagnostic Laboratories,
LLC
Hamilton
NJ
|
Family ID: |
45818280 |
Appl. No.: |
15/299072 |
Filed: |
October 20, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13065867 |
Mar 31, 2011 |
9523073 |
|
|
15299072 |
|
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|
61341465 |
Mar 31, 2010 |
|
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|
61341457 |
Mar 31, 2010 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 5/0602 20130101; G01N 33/6893 20130101; G01N 2333/705
20130101; G01N 2800/368 20130101; C07K 14/7155 20130101; G01N
33/6863 20130101; G01N 2800/065 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Claims
1. A kit for detecting a .DELTA.9 isoform of IL-23R.alpha.,
comprising: a) a first antibody that binds to carboxyl-terminal
truncated .DELTA.9 isoform of IL-23R.alpha., wherein the
carboxyl-terminal truncated .DELTA.9 isoform of IL-23R.alpha. has
the amino acids 318-348 of IL-23R.alpha.; and; b) instructions for
using the antibody for detecting .DELTA.9 isoform of IL-23R.alpha.,
wherein said .DELTA.9 isoform has an amino acid sequence of SEQ ID
NO: 2.
2. The kit of claim 11, wherein said antibody is immobilized to a
solid support.
3. The kit of claim 11, further comprising a second antibody that
specifically binds to extracellular domain of IL-23R.alpha..
4. The kit of claim 11, wherein said first antibody is a monoclonal
antibody and said second antibody is a polyclonal antibody.
5. The kit of claim 11, wherein said instructions provide guidance
to the use of the kit to detect .DELTA.9 isoform of IL-23R.alpha.
in a biological sample.
6. The kit of claim 16, wherein said biological sample is
EDTA-plasma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional of U.S. application
Ser. No. 13/065,867, filed on Mar. 31, 2011, which claims the
benefit under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application Nos. 61/341,457, 61/341,465 filed Mar. 31, 2010, the
contents of which are incorporated by reference herein in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to an immunoassay
for detecting a naturally-occurring soluble truncated form of IL-23
receptor (IL-23R.alpha.). More specifically, the present invention
provides an ELISA assay for a soluble truncated form (e.g.,
.DELTA.9 protein) of IL-23R.alpha.. The assay is useful as a
diagnostic tool in patients afflicted with immunological diseases
including inflammatory bowel diseases (e.g., Crohn's disease),
asthma, and other pathological diseases such as miscarriage.
BACKGROUND OF THE INVENTION
[0003] IL-23 is a heterodimeric molecule comprising a p19 subunit
and a p40 subunit that are two disulfide-linked. IL-23 is
speculated to play an essential role in chronic inflammation and
autoimmune diseases in humans. Mice lacking p19 exhibit a decreased
pro-inflammatory response to experimental autoimmune
encephalomyelitis, inflammatory bowel disease and collagen-induced
arthritis. While IL-23 per se cannot induce the differentiation of
naive CD4 T-cells into Th-17 cells in vitro, the differentiation of
Th17 cells in vivo may require IL-23. The observed protective
effect in p19-deficient mice may relate to the lack of
differentiation of Th17 cells. This is consistent with recent
report that IL-23 synergies with Th17 cell differentiation
cytokines including IL-1, IL-6 and TGF-.beta. to induce expression
of IL-17.
[0004] IL-23 exerts its biological activities by binding to IL-23
receptor (IL-23R). IL-23R comprises an IL-23R.alpha. subunit and an
IL-12R.beta.1 subunit. When IL-23 binds to IL-23R, it leads to
intracellular signaling including phosphorylation of STAT1, STAT3,
STAT4 and STAT5. IL-23R is expressed on T-cells, NK cells,
monocytes, and dendritic cells and its expression pattern
corresponds with the ability of these cells to respond to
IL-23.
[0005] Human IL-23R.alpha. mRNA is 2.8 kb long and contains 11
exons (NM_144701). The translated full-length IL-23R.alpha. protein
is a type-I transmembrane protein (629 amino acids) and contains at
least three (3) known structural domains: (1) a signal peptide
domain; (2) an extracellular region containing a N-terminal
fibronectin III-like domain; and (3) a 253 amino acid residue
cytoplasmic domain with three (3) potential tyrosine
phosphorylation sites.
[0006] Christi Parham et al. first discovered the genomic and
structural organization of the IL-23R (composed of an IL-23.alpha.
subunit and an IL-12R.beta.1 subunit). While IL-23 is shown to bind
to IL-23R and mediates Jak-STAT cell signaling, Parham explicitly
stated their inability to demonstrate human IL-23R-Ig and soluble
human IL-23R.alpha.-V5-His6 (composed of the entire extracellular
domain--amino acids 1-353) as effective antagonists for human
IL-23R. Daniel J. Cua et al. disclose treatment methods for
multiple sclerosis, neuropathic pain, and inflammatory bowel
disorders using antibodies against IL-23 and its receptor. Contrary
to Parham's statement, Cua proposes using a soluble receptor based
on the extracellular region of a subunit of the IL-23 receptor
(PCT/US2004/003126) as an antagonist. A recombinant human
IL-23R.alpha. Fc chimeric protein is commercially available
(R&D Systems) and claimed to have the ability to inhibit IL-23
induced IL-17 secretion in a mouse splenocytes system. It remains
unclear as to whether any of these proposed soluble IL-23R.alpha. s
may in fact exist in vivo as a naturally-occurring protein, let
alone the possibility that such soluble IL-23Rs may possess ability
to block IL-23R.alpha. mediated cell signaling. To this end, Daniel
J. Cua et al. (PCT/US2004/003126) failed to provide any evidence
that a soluble IL-23 receptor can indeed block IL-23 mediated cell
signaling as well as inhibiting Th17 producing cells.
[0007] Recent evidence suggests that IL-23R.alpha. gene may undergo
extensive alternative splicing. There are at least twenty-four (24)
potential gene transcripts for IL-23R.alpha.. From these
IL-23R.alpha. alternatively spliced mRNA sequences, there appears
at least four (4) deduced putative translated proteins: (1) a short
premature IL-23R.alpha. extracellular peptide; (2) a possible
soluble form of IL-23R.alpha. lacking a transmembrane/intracellular
domain; (3) a full-length IL-23R.alpha. with truncated
extracellular region; and (4) a non-responsive membrane-bound
receptor isoform of IL-23R.alpha. with deletion in intracellular
signaling components.
[0008] Although many gene transcripts for IL-23 R.alpha. (i.e.,
IL-23R splice variants) are suggested, it is important to point out
that their actual existence in vivo is presently unknown. There is
little information regarding whether any of the deduced
IL-23R.alpha. translated products actually exist in vivo, let alone
the function of these IL-23R.alpha. protein variants, if any.
[0009] Accordingly, there is a continuing need for a diagnostic
assay that detects a measurable level of IL-23R.alpha. variants in
a biological sample in a mammal, specifically an accurate ELISA
that measures an isoform of IL-23R.alpha.. The assay would enable
the assessment of a pathological role of IL-23R.alpha. using
biological samples obtained from patients. The present inventors
overcome the prior art deficiency and discovered an ELISA assay for
quantifying a naturally-occurring soluble truncated form of
IL-23R.alpha. (i.e., .DELTA.9 IL-23R.alpha.) in plasma. The present
ELISA reveals that a particular form (i.e., .DELTA.9 IL-23R.alpha.)
of IL-23R.alpha. constitutes a major spliced variant form of
IL-23R.alpha. in plasma and that its level correlates with
inflammatory bowel diseases such as Crohn's disease.
SUMMARY OF THE INVENTION
[0010] In one aspect, the present invention provides a method for
detecting a .DELTA.9 isoform of IL-23 receptor (IL-23R.alpha.) in a
biological sample, comprising the steps of: a) obtaining a
biological sample; b) incubating said biological sample with a
capture reagent immobilized on a solid support to bind a .DELTA.9
isoform of IL-23R.alpha., wherein the capture reagent comprises a
first antibody; and (c) detecting IL-23R.alpha. bound to said
immobilized capture reagent by contacting the bound IL-23R.alpha.
with a second antibody against IL-23R.alpha., coupled with a
detecting agent. Preferably, the first antibody recognizes or binds
to the carboxyl-terminus (C-terminus) of IL-23R.alpha.. More
preferably, the first antibody recognizes the exon 8 of the
IL-23R.alpha. (i.e., at a site between amino acids 318-348). It is
intended that the present invention also cover the first antibody
that may equivalently recognize the proximity of amino acids
318-348 and still function as capture antibody. Such an antibody
would be specific for detecting .DELTA.9.
[0011] The second antibody preferably recognizes the extracellular
domain of the IL-23R.alpha., so as to provide detection for
.DELTA.9. Preferably, the first antibody recognizes an epitope that
is distinct (i.e., does not overlap) with that of the second
antibody.
[0012] The first antibody may be a monoclonal antibody or a
polyclonal antibody. Preferably, the first antibody is a monoclonal
antibody. The second antibody may also be a monoclonal antibody or
a polyclonal antibody. Preferably, the second antibody is a
polyclonal antibody.
[0013] Preferably, the biological sample is selected from the group
consisting of blood and plasma. Preferably, the biological sample
comprising EDTA. More preferably, the biological sample is
EDTA-treated plasma.
[0014] Preferably, the incubating step is performed at a pH is
about 6.0 to about 10.0. More preferably, the incubating step is
performed at pH is about 9.5.
[0015] Preferably, the incubating step is performed at a
temperature of about 0.degree. C. to about 25.degree. C. More
preferably, the incubating step is performed at a temperature of
about 4.degree. C.
[0016] Preferably, the incubating step is performed for about 0.5
to about 16 hours. More preferably, the incubating step is
performed for about 3 hours.
[0017] In another aspect, the present invention provides a kit for
detecting .DELTA.9 isoform of IL-23R.alpha., comprising: a) a first
antibody that binds to carboxyl-terminal truncated .DELTA.9 isoform
of IL-23R.alpha., wherein the carboxyl-terminal truncated .DELTA.9
isoform of IL-23R comprises amino acids 318-348 of IL-23R.alpha.;
and; b) instructions for using the antibody for detecting .DELTA.9
isoform of IL-23R.alpha..
[0018] Preferably, said first antibody is immobilized to a solid
support. Preferably, the kit further comprises a second antibody
that specifically binds to extracellular domain of
IL-23R.alpha..
[0019] Preferably, said first antibody is a monoclonal antibody and
said second antibody is a polyclonal antibody.
[0020] Preferably, said instructions provide guidance to the use of
the kit to detect .DELTA.9 isoform of IL-23R.alpha. in a biological
sample.
[0021] Preferably, the kit detects .DELTA.9 isoform of
IL-23R.alpha. in plasma.
DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 depicts schematically the constructs of wild-type
IL-23R.alpha. and IL-23R.alpha. variants. Partial deletion of exon
11 (p.DELTA.11), deletion of exon 9 (.DELTA.9) and deletion of exon
8 and 9 (.DELTA.8,9) resulted in open reading frame shift, which
generated pre-mature translational termination signal. However,
deletion of exon 8 (.DELTA.8) was in-frame deletion. Therefore,
.DELTA.8 variant used the same translation termination signal at
Exon 11 as wild-type IL-23R.alpha.. These expression constructs
were constructed by PCR amplification and cloned into pcDNA3.3
expression vector using TOPO cloning kit from Invitrogen. All the
constructs were sequence verified.
[0023] FIG. 2 depicts the domain organization of the expressed
IL-23R.alpha. proteins. Wild-type IL-23R.alpha. contains signal
peptide at its N-terminus followed by extracellular domain,
transmembrane region and cytoplasmic domain. Partial deletion of
exon 11 (p.DELTA.11) caused removal of almost entire cytoplasmic
domain. Deletion of exon 8 (.DELTA.8) resulted in internal removal
of C-terminal region of extracellular domain while the
transmembrane region and cytoplasmic domain remained intact.
Deletion of exon 9 (.DELTA.9) and deletion of exon 8 and 9
(.DELTA.8,9) both resulted in complete removal of transmembrane
region and cytoplasmic domain. Therefore, .DELTA.9 and .DELTA.8,9
were predicted to be secreted proteins. .DELTA.9 protein contained
the extracellular domain from amino acids 1 to 348 whereas
.DELTA.8,9 protein contains extracellular domain from amino acids
1-318. All the expressed proteins were tagged with Flag epitope at
C-terminus for detection purpose using Anti-Flag M2 antibody.
[0024] FIG. 3 depicts the transient transfection of the
IL-23R.alpha. expression constructs into 293T cells by Fugene HD.
Cell lysates were prepared after 48 hours post-transfection.
Cultured media were also collected. Expression levels of wild-type
IL-23R.alpha. and its variants were examined by immunoblot using
Anti-Flag M2 antibody. All expression constructs produced similar
level of proteins in the cell lysates. However, only .DELTA.9 and
.DELTA.8,9 were found in the cultured media, which demonstrated
that these two proteins are actively secreted out of the cells.
[0025] FIG. 4 depicts the specificity of the mouse
anti-hIL-23R.alpha. antibody. Cell lysates prepared from the 293T
transient transfection experiment (by Fugene HD) were used in the
immunoblot assay. The mouse anti-hIL-23R.alpha. antibody recognized
wild-type IL-23R.alpha. (weakly), p.DELTA.11 and .DELTA.9. More
importantly, this antibody was highly sensitively to .DELTA.9.
However, this antibody failed to detect .DELTA.8 and .DELTA.8,9
proteins indicating that the antibody recognized the C-terminal
region of extracellular domain (amino acids 319-348) encoded by the
exon 8.
[0026] FIG. 5 depicts an immunoblot experiment using a mouse
anti-hIL-23R.alpha. (anti-human IL-23R.alpha.) antibody to detect
the soluble recombinant .DELTA.9 protein in the culture media
obtained from 293T cells transfected with either control plasmid or
.DELTA.9 expression plasmid.
[0027] FIG. 6 depicts the specificity of biotinylated goat
anti-hIL-23R.alpha. antibody in the immunoblot experiment using
cellular lysates from the 293T cells transfected with five (5)
different expression plasmids hosting wild-type hIL-23R.alpha.,
p.DELTA.11, .DELTA.8, .DELTA.9, and .DELTA.8,9. All the recombinant
proteins were tagged with Flag sequence at the C-terminus. The top
panel was the immunoblot experiment using anti-Flag antibody to
show that all the recombinant proteins were expressed at comparable
level. The bottom panel was the immunoblot experiment using
biotinylated goat anti-IL-23R.alpha. antibody.
[0028] FIG. 7 depicts the ELISA sandwich system. The capture
reagents can be either be anti-hIL-23R.alpha. antibody, IL-23 p19
subunit fused to Fc region of IgG or IL-23R.alpha. binding peptide
fused to Fc region of IgG. The capture reagents were coated on the
solid support (e.g., microtiter plate) to capture the soluble form
of the IL-23R.alpha.. The capture antibody specifically captured
the soluble IL-23R.alpha. on the ELISA plate. The captured soluble
IL-23R.alpha. was then detected by a biotinlyated goat
anti-hIL-23R.alpha. antibody. The antibody-antigen sandwich was
detected by streptavidin conjugated with horseradish peroxidase
(HRP). The peroxidase activity (representing the level of .DELTA.9)
was measured by addition of tetramethylbenzidine (TMB) substrate.
The color intensity was in direct proportion to the amount of the
bound IL-23R.alpha.. Color development was stopped and the
intensity of the color was measured at optical density (OD) 450 nm
on a microtiter plate reader.
[0029] FIG. 8 depicts the ELISA assay result. The ELISA was
performed using the culture media containing .DELTA.9 protein or
the same culture media spiked with either 100 ng IL-23 or 100 ng
IL-12 (negative control). The presence of IL-23 or IL-12 gave same
ELISA signal as that in the absence of the spike. This result
suggested that binding of IL-23 to .DELTA.9 protein or the presence
of IL-12 do not influence the ELISA assay to detect .DELTA.9
protein.
[0030] FIG. 9 depicts the optimization of ELISA. Different
concentrations of capture and detection antibodies were used to
detect 100 ng of antigen, which is a recombinant protein containing
entire extracellular domain of hIL-23R.alpha. fused with Fc region
of human IgG1 obtained from R&D systems. The combination of
capture antibody at 5 .mu.g/ml and detection antibody at 1.6
.mu.g/ml gave the best signal in the ELISA when antigen was
incubated at room temperature for 2 hours.
[0031] FIG. 10 depicts a titration experiment on capture and
detection antibodies with extended antigen incubation (4.degree. C.
for 16 hours).
[0032] FIG. 11 depicts two (2) different incubation conditions for
antigen when capture antibody at 5 .mu.g/ml and detection antibody
at 1.6 .mu.g/ml were used.
[0033] FIG. 12 depicts two (2) different coating buffer conditions
(i.e., PBS and carbonate buffer) when capture antibody at 5
.mu.g/ml and detection antibody at 1.6 .mu.g/ml were used.
[0034] FIG. 13 depicts the purified recombinant .DELTA.9 protein
from the intracellular source (i.e., cell lysates from 293T) on the
SDS-PAGE gel stained with the Coomassie blue to reveal the purity
and estimate the quantity.
[0035] FIG. 14 depicts the purified recombinant .DELTA.9 protein
from the secreted source (i.e., culture medium from 293T) on the
SDS-PAGE gel stained with the Coomassie blue to reveal the purity
and estimate the quantity.
[0036] FIG. 15 depicts the validation of developed ELISA using
purified recombinant .DELTA.9 protein from whole cell lysates
prepared from 293T cell transfected with .DELTA.9 expression
plasmid.
[0037] FIG. 16 depicts the validation of developed ELISA using
recombinant .DELTA.9 protein purified from the secreted source
(i.e., culture medium from 293T).
[0038] FIG. 17 depicts the ELISA experiment to detect the secreted
.DELTA.9 recombinant protein in the culture medium from the 293T
cells transfected with the .DELTA.9 expression plasmid. The result
represents two (2) independent transfection experiments performed
in the 293T cells. FIG. 16 depicts that .DELTA.9 IL-23R.alpha.
protein is secreted. The developed ELISA detects the purified
.DELTA.9 recombinant IL-23R.alpha..
[0039] FIG. 18 depicts the use of ELISA to measure serological
levels of .DELTA.9 IL-23R.alpha. in the sera or plasma from three
(3) human donors. EDTA-plasma, heparin-plasma and serum were
prepared from each donor.
[0040] FIG. 19 depicts the EDTA plasma dilution study for ELISA.
Experiment was performed using plasma samples from two (2)
donors.
[0041] FIG. 20 depicts the spike and recovery experiment performed
on the EDTA-plasma samples obtained from four (4) donors. 100 ng of
IL-23R/Fc fusion protein was spiked into the plasma samples. Around
40% of IL-23R/Fc was recovered in the ELISA assay.
[0042] FIG. 21 depicts the spike and recovery experiment performed
on the synovial fluids obtained from three (3) donors. 100 ng of
IL-23R/Fc fusion protein was spiked into the fluids. Around 50% of
IL-23R/Fc was recovered in the ELISA assay.
[0043] FIG. 22 depicts the spike and recovery experiment performed
on the cerebrospinal fluids obtained from five (5) donors. 100 ng
of IL-23R/Fc fusion protein was spiked into the fluids. More than
80% of IL-23R/Fc was recovered in the ELISA assay.
[0044] FIG. 23 depicts the spike and recovery experiment performed
on the amniotic fluids obtained from four (4) donors. 100 ng of
IL-23R/Fc fusion protein was spiked into the fluids. More than 80%
of IL-23R/Fc was recovered in the ELISA assay.
[0045] FIG. 24 depicts the standard curve generated using
recombinant protein of human IL-23R.alpha., which is composed of
extracellular domain of IL-23R.alpha. and Fc region of IgG1. This
standard curve was used to calculate the amount of
naturally-occurring .DELTA.9 protein in EDTA-plasma from O.D. 450
value.
[0046] FIG. 25 depicts an ELISA experiment performed to detect the
serological level of .DELTA.9 in the plasma samples obtained from
the healthy human donors with no known history of Crohn's disease.
Totally, fifty-two (52) control plasma samples were analyzed. The
mean and median .DELTA.9 values were 41.5 ng/mL and 27.8 ng/mL,
respectively.
[0047] FIG. 26 depicts the ELISA experiment performed to detect the
serological level of .DELTA.9 protein in the EDTA-plasma samples
obtained from the human donors with a medical history of Crohn's
disease. The mean and median values of .DELTA.9 IL-23R.alpha. level
were 155.4 ng/mL and 144.5 ng/mL, respectively. Both mean and
median in Crohn's patients were higher than those in the normal
group. The difference between the two groups was statistically
significant (p=0.0014; Student's t-test, two-tailed).
[0048] FIG. 27 depicts the ELISA experiment performed to detect the
serological levels of .DELTA.9 protein the EDTA-plasma samples
obtained from patients with active Crohn's disease. The mean and
median values of .DELTA.9 protein in the EDTA-plasma were 131.45
ng/mL and 135.87 ng/mL, respectively. Both mean and median values
of .DELTA.9 protein in active Crohn's disease patients were higher
than those in the control group (mean: 90.47 ng/mL; median: 88.67
ng/mL). The difference between the two groups was statistically
significant (p=0.0074; Student's t-test, two-tailed). The mean and
median values of .DELTA.9 protein in the EDTA-plasma of the
inactive Crohn's disease patients were 109.72 ng/mL and 98.63
ng/mL, respectively.
[0049] FIG. 28 depicts the ELISA experiment performed to detect the
serological levels of .DELTA.9 protein in the EDTA-plasma samples
obtained from patients with inactive Crohn's disease with or
without resection of an intestinal tract (i.e., part of the
intestine was removed). The mean and median values of .DELTA.9
protein in the EDTA-plasma of resection patients were 141.57 ng/mL
and 139.49 ng/mL, respectively. The mean and median values of
.DELTA.9 protein in the EDTA-plasma of non-resection patients were
75.40 ng/mL and 77.98 ng/mL, respectively. Note that the .DELTA.9
protein in resection group is significantly higher than those in
non-resection patients. The difference between the two groups was
statistically significant (p=0.009; Student's t-test,
two-tailed).
[0050] FIG. 29 depicts the ELISA experiment performed to detect the
serological levels of .DELTA.9 protein in the EDTA-plasma samples
obtained from non-pregnant women and pregnant women (with not more
than eleven (11) weeks gestation). The mean and median values of
.DELTA.9 protein in the pregnant women were 41.5 ng/mL and 27.8
ng/mL, respectively. The mean and median values of .DELTA.9 protein
in the control non-pregnant women were 100.6 ng/mL and 92.5 ng/mL,
respectively. The .DELTA.9 protein levels in the non-pregnant women
were found to be higher than those in the pregnant women, and the
difference between the two groups was statistically significant
(p<0.0001; Student's t-test, two-tailed).
[0051] FIG. 30 depicts the ELISA experiment performed to detect the
serological level of .DELTA.9 protein in the EDTA-plasma samples
obtained from pregnant women. The serological level of .DELTA.9
protein is plotted according to weeks of gestation. Pregnant women
of greater than twenty (20) weeks gestation (i.e., mean: 3.17
ng/mL, median: 2.68 ng/mL) had lower levels when compared to those
of twenty (20) weeks or less (i.e., mean: 14.76 ng/mL, median: 4.19
ng/mL). The difference between the two groups was statistically
significant (p<0.004; Student's t-test, two-tailed).
[0052] FIG. 31 depicts the ELISA experiment performed to detect the
serological level of .DELTA.9 protein in amniotic fluid samples
obtained from pregnant women. The serological level of .DELTA.9
protein is plotted according to weeks of gestation. Pregnant women
of greater than twenty (20) weeks gestation (i.e., mean: 4.89
ng/mL, median: 4.79 ng/mL) had lower levels than those of twenty
(20) weeks or less (i.e., mean: 15.40 ng/mL, median: 4.18
ng/mL).
[0053] FIG. 32 depicts the nucleotide sequence of .DELTA.9 (SEQ ID
NO: 1) and amino acid sequence of .DELTA.9 (SEQ ID NO: 2).
[0054] FIG. 33 depicts the nucleotide sequence of .DELTA.8,9 (SEQ
ID NO: 3) and amino acid sequence of .DELTA.8,9 (SEQ ID NO: 4).
[0055] FIG. 34 depicts the nucleotide sequence of p.DELTA.11 (SEQ
ID NO: 8) and amino acid sequence of p.DELTA.11 (SEQ ID NO: 9).
[0056] FIG. 35 depicts the nucleotide sequence of .DELTA.8 (SEQ ID
NO: 10) and amino acid sequence of .DELTA.8 (SEQ ID NO: 11).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0057] As used herein, the term "IL-23R" refers to interleukin-23
receptor. IL-23R is composed of two (2) subunits: IL-23R.alpha. and
IL-12R.beta.1. The IL-23R.alpha. gene is located on chromosome
1p31.3. The native form of human IL-23R.alpha. mRNA is 2.8 kb long
and contains 11 exons (NM_144701). The transcribed mRNA is
translated into a full-length protein of 629 amino acids, the
sequence of which is listed in NM_144701. The full-length
translated IL-23R.alpha. protein is a type I cytokine receptor and
forms with human IL-12R.beta.1 to form the heterodimeric IL-23
receptor. Human IL-12R.beta.1 also partners with human
IL-12R.beta.2 to form the cell-surface IL-12 receptor. When bound
to IL-23, this protein triggers a series of cell signaling event
including activation of Janus kinase 2 (JAK2), and transcription
activator STAT3 (i.e., IL-23R mediated cell signaling). IL-23R is
present on many immune system cells, including T cells, natural
killer (NK) cells, monocytes, and dendritic cells.
[0058] As used herein, for purposes of this application, the term
".DELTA.9" refers to the naturally-occurring truncated
IL-23R.alpha. protein resulting from IL-23R mRNA splicing. For
purposes of this application, ".DELTA.9 variant", ".DELTA.9
isoform", and ".DELTA.9 protein" are used interchangeably to refer
to this particular naturally-occurring truncated IL-23R.alpha.
protein. The .DELTA.9 protein has 348 amino acids plus eight (8)
novel amino acid sequences unique to .DELTA.9 protein (a total of
356 amino acids). The signal sequence (i.e., 1-23 amino acids) on
the immature .DELTA.9 protein (located inside the cells) is cleaved
before the mature .DELTA.9 protein is released outside of the
cells. The mature .DELTA.9 protein therefore has a total of 333
amino acids (i.e., 24-356). The present ELISA assay can
specifically detect both of these two (2) .DELTA.9 forms (i.e.,
immature .DELTA.9 and mature .DELTA.9). For purposes of this
application, therefore, the term ".DELTA.9" is intended to include
both of these two (2) forms.
[0059] As used herein, the term ".DELTA.8,9" refers to the
naturally-occurring truncated IL-23R.alpha. resulting from IL-23
gene splicing. The .DELTA.8,9 has 318 amino acids plus eight (8)
novel amino acid sequence unique to .DELTA.8,9 (a total of 326
amino acids). The signal sequence (i.e., 1-23 amino acids) on the
.DELTA.8,9 is cleaved before the mature .DELTA.8,9 protein is
released. Therefore, the mature .DELTA.8,9 has a total of 303 amino
acids (i.e., 24-326). For purposes of this application, the term
".DELTA.8,9" is intended to include both of the two (2) forms.
[0060] As used herein, the term "detecting" refers to quantitative
measurements of IL-23R in a biological sample.
[0061] As used herein, the term "biological sample" refers to a
body sample from a mammal, preferably from a human. Biological
sample may be obtained from patients inflicted with autoimmune
diseases. Biological samples include biological fluids such as
serum, plasma, lymph fluid, synovial fluid, amniotic fluid, urine,
cerebro-spinal fluid, saliva, tissue culture medium, tissue
extracts and the like. The preferred biological sample is serum or
plasma.
[0062] As used herein, the term "capture reagent" refers to a
reagent capable of binding and capturing a target molecule in a
sample such that under suitable condition, the capture
reagent-target molecule complex can be separated from the rest of
the sample. Typically, the capture reagent is immobilized. In a
sandwich immunoassay, the capture reagent is preferably an antibody
or a mixture of different antibodies against a target antigen.
[0063] As used herein, the term "detectable antibody" refers to an
antibody that is capable of being detected either directly through
a label amplified by a detection means, or indirectly through,
e.g., another antibody that is labeled. For direct labeling, the
antibody is typically conjugated to a moiety that is detectable by
some means. The preferred detectable antibody is biotinylated
antibody.
[0064] As used herein, the term "detection means" refers to a
moiety or technique used to detect the presence of the detectable
antibody in the ELISA herein and includes detection agents that
amplify the immobilized label such as label captured onto a
microtiter plate. Preferably, the detection means is a fluorimetric
detection agent such as avidin or streptavidin.
[0065] As used herein, the term "antibody" is used in the broadest
sense and includes monoclonal antibodies, polyclonal antibodies,
multivalent antibodies, multi-specific antibodies, and antibody
fragments so long as they exhibit the desired binding
specificity.
[0066] As used herein, the term "monoclonal antibody" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. 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 et al. Nature 256:495 (1975). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the
techniques described in Clackson et al. Nature 352:624-628 (1991)
and Marks et al. J. Mol. Biol. 222:581-597 (1991).
[0067] The monoclonal antibodies herein may include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or
light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (Morrison et al. Proc. Natl. Acad. Sci. USA
81:6851-6855 (1984)).
[0068] As used herein, the term "mammal" refers to any animal
classified as a mammal, including humans, and animals. Preferably,
the mammal is a human.
[0069] As used herein, the term "autoimmune disease" refers to a
pathological condition in mammals that is typically characterized
by an unregulated immune cell activity. Examples of autoimmune
include but are not limited to, inflammatory bowel disease, Crohn's
disease, asthma and the like. Preferably, the autoimmune diseases
are characterized by an increased Th17 activity.
[0070] As used herein, the term "inflammatory bowel disease" means
an inflammatory disease in bowel that involves Th17 cells. Crohn's
disease represents an exemplary inflammatory bowel disease.
[0071] As used herein, the term "Crohn's disease" is an
inflammatory disease of the intestines. It primarily causes
abdominal pain, diarrhea (which may be bloody if inflammation is at
its worst), vomiting, or weight loss. Crohn's disease is believed
to be an autoimmune disease, in which the body's immune system
attacks the gastrointestinal tract, causing inflammation.
[0072] As used herein, the term "active Crohn's disease" refers to
a clinical state where at the time the patient is displaying
symptoms of Crohn's disease such as cramping, bloody stool,
diarrhea and the like.
[0073] As used herein, the term "inactive Crohn's disease patient"
refers to a clinical state where at the time the patient (although
he/she has been diagnosed as having Crohn's disease) is not
displaying symptoms of Crohn's disease such as cramping, bloody
stool, diarrhea and the like.
[0074] As used herein, the term "intestinal resection" refers to
the surgical removal of a part of intestinal tract such as
colon.
[0075] The present inventors discovered a hitherto unknown soluble
form of a human IL-23R.alpha. receptor (e.g., .DELTA.9). .DELTA.9
mRNAs is a result of alternative splicing of the IL-23R.alpha. gene
that encodes the native IL-23R.alpha. protein. The splice variant
.DELTA.9 is missing the exon 9 and does not contain a transmembrane
domain and an intracellular domain. In .DELTA.9, Exon 8 joins to
Exon 10 and results in the shift of open reading frame and hence
generates the novel eight (8) amino acid sequences (i.e., GLKEGSYC,
SEQ ID NO: 7). .DELTA.9 mRNA represents up to 20% of human
leukocyte IL-23R.alpha. transcript and thus is a major form of
IL-23R.alpha. mRNA. .DELTA.8,9 mRNA also is detectable in the
Fragment Analysis studies.
[0076] Using an ELISA developed by the present inventors, we
detected the .DELTA.9 protein form of IL-23R.alpha. is secreted and
present as a soluble monomer in a biological sample. The .DELTA.9
protein form is found to be associated with inflammatory bowel
diseases such as Crohn's disease. The .DELTA.9 protein is found to
bind to IL-23 in solution. The present inventors further discovered
that this soluble IL-23R.alpha. form is capable of blocking IL-23
induced STAT3 phosphorylation and Th17 maturation.
[0077] It is known that the native form of human IL-23R.alpha. mRNA
is 2.8 kb long, with 11 exons (NM_144701). This mRNA is translated
into a type-I transmembrane protein of 629 amino acids. The native
human IL-23R.alpha. protein comprises an extracellular domain that
contains 354-residue extracellular domain that includes a signal
peptide, an N-terminal fibronectin-III-like domain, as well as a
253-residue cytoplasmic domain with three potential tyrosine
phosphorylation sites. Genetic studies have suggested an
association the IL-23R.alpha. locus with protection/susceptibility
in autoimmune inflammatory disorders, although the exact
mechanistic basis remains elusive.
[0078] The present inventors have unexpectedly discovered a novel
soluble truncated IL-23R.alpha.. The present invention extends our
previous findings that IL-23R.alpha. mRNA undergoes extensive
alternative splicing--resulting in twenty-four (24) different
potential transcripts. (Kan et al.) Four different classes of
putative translation products could be deduced from these
alternatively spliced mRNA sequences: (i) short premature
IL-23R.alpha. extracellular peptides; (ii) soluble forms of
IL-23R.alpha. lacking transmembrane/intracellular domains; (iii)
full-length IL-23R.alpha. with a truncated extracellular region and
(iv) a membrane bound receptor isoform of IL-23R.alpha. that lacked
likely intracellular signaling components.
[0079] Using Fragment Analysis, the present inventors surprisingly
discovered that there are six (6) alternative splice mRNA forms in
human leukocytes. One of the forms (i.e., .DELTA.9) represents the
majority alternative splice mRNA form. .DELTA.9 protein is found to
be soluble and exists as monomer, and it has the ability to bind
p19 and inhibit the generation of functional human Th-17 cells in
vitro. Different from that of the native IL-23R.alpha. protein, the
present soluble truncated IL-23R.alpha. lacks a transmembrane
domain and contains 356 amino acids. Another form (i.e.,
.DELTA.8,9) also share the common features as .DELTA.9 (e.g.,
soluble monomer and ability to block IL-23R mediated cell
signaling).
[0080] According to the present invention, the recombinant
IL-23R.alpha. (which is a soluble truncated form of IL-23R.alpha.
protein) contains a unique eight (8) amino acid sequence (GLKEGSYC)
(SEQ ID NO: 7) at its C-terminus (in the proximity of the
transmembrane domain) due to the alternative translation reading
frame on exon 10. When analyzed under conditions of a reducing gel
electrophoresis, the molecular weight of the protein is
approximately .about.65 kDa. The soluble truncated recombinant
protein corresponds to a N-terminal fragment of IL-23R.alpha.
lacking the transmembrane domain and has 356 amino acids (with 348
amino acids correspond to that of the native IL-23R.alpha.). The
amino acid sequence of the soluble truncated IL-23R.alpha. is set
forth in SEQ ID NO: 2.
[0081] According to the present invention, the soluble truncated
recombinant IL-23R.alpha. form of .DELTA.8,9 also contains a unique
eight (8) amino acid sequence (GLKEGSYC) (SEQ ID NO: 7) at its
C-terminus, in the proximity of the transmembrane domain, due to
the exon 8 and exon 9 skipping. When analyzed under conditions of a
reducing gel electrophoresis, the molecular weight of the protein
is approximately .about.60 kDa. The soluble truncated IL-23R.alpha.
protein (.DELTA.8,9) corresponds to a N-terminal fragment of IL-23R
lacking the transmembrane domain and has 356 amino acids (with 348
amino acids correspond to that of the native IL-23R.alpha.). The
amino acid sequence of the soluble truncated IL-23R.alpha. is set
forth in SEQ ID NO: 4.
[0082] In one embodiment, the present invention provides an
isolated IL-23R.alpha. protein that includes the protein selected
from any of the following protein, an isolated protein of a
truncated human IL-23R.alpha. capable of inhibiting IL-23-mediated
cell signaling; a recombinantly produced truncated human
IL-23R.alpha.; or a purified recombinant human truncated
IL-23R.alpha. having an amino acid sequence set forth in SEQ ID NO:
2 and SEQ ID NO: 4.
[0083] The soluble truncated IL-23R.alpha. exists as a monomer and
contains a unique eight (8) amino acid sequence. In one embodiment,
the soluble IL-23R.alpha. is detected in cultured media and can be
recombinantly produced. The isolated truncated IL-23R.alpha.
protein has therapeutic value to alleviate inflammatory bowel
diseases including Crohn's disease.
[0084] In a preferred embodiment, the present invention provides a
recombinant soluble IL-23R.alpha., which has the amino acid
sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 4.
[0085] The present invention provides an isolated nucleic acid
molecule encoding a truncated human IL-23R.alpha. protein lacking a
transmembrane domain. In one embodiment, the isolated nucleic acid
molecule is a DNA, preferably the isolated DNA is genomic DNA. In
another embodiment, the isolated DNA molecule is a cDNA molecule.
In one embodiment, the isolated nucleic acid molecule is an RNA
molecule. In an embodiment, the isolated nucleic acid molecule
encodes a human IL-23R.alpha. having an amino acid sequence set
forth in SEQ ID NO: 2, wherein the nucleotide sequence of the
isolated DNA molecule is set forth in SEQ ID NO: 1. In an
embodiment, the isolated nucleic acid molecule encodes a human
IL-23R.alpha. having an amino acid sequence set forth in SEQ ID NO:
4, wherein the nucleotide sequence of the isolated DNA molecule is
set forth in SEQ ID NO: 3.
[0086] The present invention provides a recombinantly produced
human IL-23R.alpha. lacking a transmembrane domain. The present
invention provides a purified recombinant human truncated
IL-23R.alpha. having the amino acid sequence set forth in SEQ ID
NO: 2 or SEQ ID NO: 4.
[0087] In one embodiment, the present invention provides an ELISA
to aid detecting the circulating level of the soluble truncated
IL-23R.alpha. protein. One of ordinary skill in the art would
recognize the use of commercially-available antibodies in the
present developed .DELTA.9 ELISA. Using an ELISA, it is
demonstrated that .DELTA.9 protein is present at low levels in the
periphery of healthy individuals. Similarly, the present inventors
believe that .DELTA.8,9 are present in patients suffering from
inflammatory bowel diseases. Soluble cytokine receptors may be
generated by several mechanisms, including proteolytic cleavage of
receptor ectodomains, alternative splicing of mRNA transcripts or
transcription of distinct genes. The present inventors believe that
.DELTA.9 (and .DELTA.8,9) present in the circulation is solely a
result of alternative splicing of the native IL-23R.alpha. mRNA.
Given the human genome project is completed, it is believed that it
is highly unlikely that there is a distinct gene encoded for a
soluble form of IL-23R.alpha. chain.
[0088] The present invention also includes a kit for carrying out
the methods of the invention. The subject kit comprises a first
antibody specific for a carboxyl-truncated region of IL-23R.alpha..
In one embodiment, the first antibody is a monoclonal antibody.
Preferably, the first antibody recognizes and binds to the carboxyl
terminus of .DELTA.9 isoform of IL-23R.alpha.. More preferably, at
or near exon 8 (i.e., amino acid residues 318-348).
[0089] In one embodiment, the kit comprises a second antibody. In
one embodiment, the second antibody is a polyclonal antibody.
Preferably, the second antibody recognizes and binds to the
extracellular domain of the .DELTA.9 isoform of IL-23R.alpha..
Preferably, the binding site of the first antibody does not overlap
with that of the second antibody.
[0090] In other embodiments, the second antibody contains a
labeling component. Such labeling component includes a detection
means. One of ordinary skill in the art would appreciate the
detection means to include streptavidin conjugated with horseradish
peroxidase (HRP), which specifically binds biotin on the detection
antibody. The peroxidase activity (representing the level of
.DELTA.9) was measured by addition of tetramethylbenzidine (TMB)
substrate.
[0091] In one embodiment, the present kit can further include, if
desired, one or more of various conventional components, such as,
for example, containers with one or more buffers, detection
reagents or antibodies. Printed instructions, either as inserts or
as labels, indicating quantities of the components to be used and
guidelines for their use, can also be included in the kit.
[0092] The present kit may further comprises a detection antibody,
which is either directly or indirectly detectable, and which binds
and allows the quantification of the relative carboxyl-terminal
truncated IL-23R.alpha. levels.
[0093] The present kit may also contain a control full-length
recombinant full-length and .DELTA.9 IL-23R.alpha. dilution series,
where the dilution series represents a range of appropriate
standards with which a user of the kit can compare their results
and estimate-the level of .DELTA.9 IL-23R.alpha. in their sample.
Fluorescence or color development results may also be compared to a
standard curve of fluorescence or color density provided by the
kit.
[0094] The present invention will be better understood from the
following experimental studies. One of ordinary skill in the art
would readily appreciate that the specific methods and results
discussed therein are not intended to limit the invention. The
experimental studies merely serve illustrative purposes, and the
invention is more fully described by the claims which follow
thereafter.
EXPERIMENTAL STUDIES
Example 1
Generation of Splice Variant Forms of IL-23R.alpha.
[0095] In this series of study, we generated IL-23R.alpha.
constructs for wild-type (WT) and four (4) IL-23R.alpha. isoforms.
These IL-23R.alpha. constructs include WT, .DELTA.8, .DELTA.9,
.DELTA.8,9, and p.DELTA.11 IL-23 isoforms (FIGS. 1 and 2): [0096]
(i) WT represents wild-type IL-23R.alpha.. It contains 629 amino
acids. 1-353 amino acids are the extracellular domain. 377-629
amino acids are the cytoplasmic domain. The transmembrane domain is
encoded in the amino acids from 354 to 376. The first 23 amino
acids represent the signal sequence (a.a. 1-23). [0097] (ii)
p.DELTA.11 represents a partial deletion of exon 11. This partial
deletion results in frame shift and thus generates the pre-mature
translational termination signal. The p.DELTA.11 protein contains
the amino acids from 1 to 413. Due to the frame shift, p.DELTA.11
protein contains a novel amino acid "R" at the C-terminal end.
[0098] (iii) .DELTA.8 represents the deletion of exon 8 and is an
in-frame deletion. .DELTA.8 variant used the same translation
termination signal at exon 11 as that of the wild-type
IL-23R.alpha.. The .DELTA.8 protein contains amino acids from 1 to
318 fused to amino acids from 350 to 629. Therefore, the .DELTA.8
protein has internal deletion from amino acid 319 to 349, which is
encoded by exon 8. [0099] (iv) .DELTA.9 represents the deletion of
exon 9. This exon 9 deletion results in frame shift and thus
generates the pre-mature translational termination signal at exon
10. The .DELTA.9 protein contains the amino acids from 1 to 348.
Due to the frame shift, the .DELTA.9 protein contains 8 novel amino
acids "GLKEGSYC" at the C-terminal end. [0100] (v) .DELTA.8,9
represents the deletion of exon 8 and exon 9. The exon 8 and 9
deletions result in frame shift and thus generates the pre-mature
translational termination signal at exon 10. The .DELTA.8,9 protein
contains the amino acids from 1 to 318. Due to the frame shift, the
.DELTA.8,9 protein contains 8 novel amino acids "GLKEGSYC" at the
C-terminal end.
[0101] All five (5) IL-23R.alpha. expression constructs were
constructed by PCR amplification. The expression constructs were
cloned into pcDNA3.3 expression vector using TOPO cloning
(Invitrogen). Correct nucleotide sequences of all the expression
constructs were confirmed by DNA sequencing.
[0102] The domain organization of the expressed IL-23R.alpha.
proteins is summarized in FIG. 2. The wild-type IL-23R.alpha.
contains a signal peptide (amino acid 1-23) at its N-terminus
followed by an extracellular domain (amino acid 24-353),
transmembrane region (amino acid 354-376), and cytoplasmic domain
(amino acid 377-629). Deletion of exon 8 (.DELTA.8) results in an
internal removal of C-terminal region of extracellular domain while
the transmembrane region and the cytoplasmic domain remains intact.
Partial deletion of exon 11 (p.DELTA.11) results in the truncation
of cytoplasmic domain but leaves both extracellular domain and
transmembrane region intact. Deletion of the exon 9 (.DELTA.9) and
deletion of the exons 8 and 9 (.DELTA.8,9) both result in the
complete removal of a transmembrane region and cytoplasmic domain.
Thus, .DELTA.9 and .DELTA.8,9 are predicted to be secreted
proteins.
[0103] .DELTA.9 IL-23R.alpha. protein contains the extracellular
domain (amino acid 1-348) whereas .DELTA.8,9 IL-23R.alpha. protein
(amino acid 1-318) has deletion at the C-terminal region of the
extracellular domain.
[0104] The IL-23R.alpha. expression constructs were transfected
into a mammalian cell (i.e., 293T cells). The expressed
IL-23R.alpha. proteins were prepared for subsequently use to study
the specificity of anti-hIL-23R.alpha. antibodies. All the
expressed proteins are tagged with Flag epitope at the C-terminus
for detection purpose using an anti-Flag M2 antibody (Sigma).
Example 2
Expression of IL-23R.alpha. Constructs
[0105] We performed the transient transfection of the IL-23R.alpha.
expression constructs into 293T cells by Fugene HD. Cell lysates
were prepared after .DELTA.8 hours of post-transfection. Cultured
media were also collected.
[0106] Expression levels of the wild-type IL-23R.alpha. and its
four (4) variants were examined by immunoblot using anti-Flag M2
antibody. All expression constructs produced similar level of
proteins in the cell lysates (FIG. 3 Top panel).
[0107] Noted that only .DELTA.9 (amino acid 1-348) and .DELTA.8,9
(amino acid 1-318) were found in the cultured media, indicating
that these two proteins are actively secreted from the cells (FIG.
3 Bottom panel).
Example 3
ELISA Development
[0108] Using the cellular lysates as described in Example 2, we
proceeded to develop a Sandwich ELISA system. The ELISA system
allows detection of the soluble form of human IL-23R.alpha.
(.DELTA.9). In this ELISA system, two anti-hIL-23R.alpha.
antibodies are required, each recognizing different epitopes on
IL-23R.alpha.. The cell lysates obtained from the transient
transfection experiment (Example 2) were used to examine the
antibody specificity and epitope mapping.
[0109] a) Capture Antibody
[0110] Mouse anti-hIL-23R.alpha. antibody (R&D Systems) was
used in the immunoblot assay. This antibody recognizes the
wild-type IL-23R.alpha., p.DELTA.11 and .DELTA.9. More importantly,
this mouse anti-hIL-23R.alpha. antibody is found to be highly
sensitively to .DELTA.9 (FIG. 4 Bottom panel). Therefore, this
antibody was used as a capture antibody in our ELISA system.
[0111] Because this antibody fails to detect .DELTA.8 and
.DELTA.8,9 proteins, it indicates that the antibody recognizes the
C-terminal region of extracellular domain encoded by exon 8 (amino
acid from 319 to 348).
[0112] Since .DELTA.9 is a secreted protein, we also performed an
experiment to show that this mouse antibody is capable to recognize
the secreted .DELTA.9 in the 293T cell culture medium transfected
with .DELTA.9 expression construct (FIG. 5). This result indicates
that this mouse antibody detects not only the intracellular
.DELTA.9 but also secreted .DELTA.9.
[0113] In addition to the commercially available mouse
anti-hIL-23R.alpha. antibody (R&D Systems), we also prepared
different mouse monoclonal antibodies targeted against human
IL-23R.alpha. protein (i.e., amino acid residues 116-129).
Synthetic peptides covering this region were used as antigen and
injected into mice to prepare monoclonal antibodies. Several
hybridoma cells were generated. We selected four (4) hybridoma
cells and obtained purified monoclonal antibodies from these
hybridoma supernatants. All these hybridomas show avid binding
(i.e., high affinity) to the peptide antigen (i.e., amino acid
residues 116-129).
[0114] The four (4) hybridoma clones were identified as 2C8E10,
2C8C4, 3A5C11 and 3A5D11. Monoclonal antibodies secreted by these
hybridoma cells were further purified using Protein A resin
(standard protocol). The purified monoclonal antibodies were tested
in two (2) different validation assays: namely (i)
immunoprecipitation and (ii) ELISA.
[0115] All four (4) monoclonal antibodies were shown to
immunoprecipitate .DELTA.9 protein. Immunoprecipitation was
performed using standard protocol (See "Materials &
Methods).
[0116] Two (2) of the purified monoclonal antibodies from hybridoma
cells (i.e., 3A5C11 and 3A5D11) were tested using our ELISA.
Instead of using the commercially available mouse
anti-hIL-23R.alpha. antibody from R&D as the capture antibody,
we used our purified monoclonal antibodies (i.e., 3A5C11 and
3A5D11) in the ELISA to measure the amount of soluble human
IL-23R.alpha.. We found that both of our monoclonal antibodies are
capable of capturing soluble human IL-23R.alpha. similar to the
commercially available mouse anti-hIL-23R.alpha. antibody (See
Table below).
TABLE-US-00001 TABLE 1 Characterization of our prepared monoclonal
antibodies Clone IDs: Immunoprecipitation ELISA as Capture Antibody
2C8E10 Yes N.D. 2C8C4 Yes N.D. 3A5C11 Yes Yes 3A5D11 Yes Yes
[0117] b) Detection Antibody
[0118] Goat anti-human IL-23R.alpha. was used as a detection
antibody. The goat anti-human IL-23R.alpha. is preferably in
biotinylated form.
[0119] We examined the specificity of the biotinlyated goat
anti-human IL-23R.alpha. (FIG. 6 Bottom). We also performed the
immunoblot assay using anti-Flag to show that all the recombinant
proteins expressed at a similar level (FIG. 6 Top). This goat
antibody detects all the expressed proteins. More importantly, the
goat antibody recognizes a different epitope from that of the mouse
antibody.
[0120] As such, the mouse and goat anti-hIL-23R.alpha. antibodies
were used as a "match antibody pair" in the ELISA. The mouse
antibody was used as the capture antibody because of its high
sensitively to .DELTA.9, whereas the biotinlyated goat antibody was
used for detection. Goat anti-hIL-23R.alpha. (R&D Systems) was
used as a detection antibody. The goat anti-hIL-23R.alpha. is
preferably in biotinylated form.
[0121] c) Sandwich ELISA
[0122] FIG. 7 depicts the Sandwich ELISA to detect the soluble form
of IL-23R.alpha. (.DELTA.9). In this particular example, the ELISA
uses 2 different anti-hIL-23R.alpha. antibodies. The mouse
anti-hIL-23R.alpha. antibody was used as capture antibody and
coated on the solid support (e.g., microtiter plate) to capture the
soluble form of the IL-23R.alpha. (.DELTA.9). Because the mouse
antibody only detected the soluble IL-23R.alpha. containing amino
acids from 319 to 348 and .DELTA.9 is the only secreted variant
containing this amino acid sequence among other known sreceted
variants, this antibody specifically captured the .DELTA.9 variant
on the ELISA plate.
[0123] The capture of .DELTA.9 IL-23R.alpha. was then detected by a
biotinlyated goat anti-hIL-23R.alpha. antibody. The goal
anti-hIL-23R.alpha. antibody recognizes a different epitope than
that of mouse anti-human I-23R antibody.
[0124] Alternatively, other capture reagents such as IL-23 p19
subunit fused to Fc region of IgG or IL-23R binding peptide fused
to Fc region of IgG can be used as a substitute for capture
antibody (in this particular example, mouse anti-hIL-23R.alpha.
antibody). This modified ELISA system involves binding of cytokine
(p19-Fc) or peptide (peptide-Fc) to the soluble form of 23R.alpha.
(.DELTA.9).
[0125] In order to prove that the cytokine binding to the soluble
form of the IL-23R.alpha. (.DELTA.9) did not interfere the ELISA
detection by the detection antibody, ELISA was performed using
capture and detection antibodies. Either nothing, IL-23 (100 ng) or
IL-12 (100 ng) was added to the medium containing .DELTA.9 protein.
The result clearly shows that cytokine (IL-23) binding to receptor
(.DELTA.9) does not interfere the ELISA detection. Therefore, this
observation supports our idea that the other capture reagents can
be used as the substitute for capture antibody (FIG. 8).
[0126] d) Detection System
[0127] The antibody-antigen sandwich was detected by streptavidin
conjugated with horseradish peroxidase (HRP), which specifically
binds biotin on the detection antibody. The peroxidase activity
(representing the level of .DELTA.9) was measured by addition of
tetramethylbenzidine (TMB) substrate. The color intensity was in
direct proportion to the amount of the bound IL-23R.alpha.. Color
development was stopped and the intensity of the color was measured
at optical density (OD) 450 nm on a microtiter plate reader.
Example 4
ELISA Optimization
[0128] a) Antibody Concentrations
[0129] In this series of study, we optimized the ELISA system.
Different concentrations of capture and detection antibodies were
used to detect the antigen (100 ng), which represents a recombinant
protein containing entire extracellular domain of human
IL-23R.alpha. fused with Fc region of human IgG1 (obtained from
R&D Systems). The combination of capture antibody at 5 .mu.g/ml
and detection antibody at 1.6 .mu.g/ml yielded an optimal signal in
the ELISA when antigen was incubated at room temperature
(25.degree. C.) for 2 hours (FIG. 9).
[0130] b) Incubation Temperature and Duration
[0131] We conducted a titration experiment on capture and detection
antibodies with extended antigen incubation (FIG. 10). Antigen
incubation time was increased to 16 hours at 4.degree. C. Prolonged
incubation at 4.degree. C. further enhanced the sensitivity of the
ELISA. Capture antibody was used at 5 .mu.g/ml and detection
antibody was used at 1.6 .mu.g/ml. Antigen and sample were
incubated at 4.degree. C. for 16 hours.
[0132] FIG. 11 depicts two different incubation conditions for
antigen when capture antibody at 5 .mu.g/ml and detection antibody
at 1.6 .mu.g/ml were used. Incubation of antigen at 4.degree. C.
for 16 hours increased the signal by .about.67% as compared to that
at room temperature for 2 hours.
[0133] c) Coating Buffer
[0134] FIG. 12 depicts the optimization of the coating buffer.
Because of the hydrophobic nature of the polystyrene surface,
adsorption was allowed to take place at a pH at or slightly above
the pl. At such pH, there is no net charge on the protein so as to
avoid any potential electrostatic repulsion between the protein and
the antibody that is adsorbed on the polystyrene surface. Two
commonly used coating buffers were tested (i.e., 50 mM carbonate pH
9.6 and 10 mM PBS pH 7.2). This step was performed at 4.degree. C.
overnight (i.e., 16-18 hours) to ensure sufficient adsorption and
minimal well-to-well variations. The result indicates that the
carbonate buffer (pH 9.6) used yielded a better signal than that of
the PBS buffer (pH 7.2).
Example 5
Validation of ELISA Using Purified .DELTA.9 Proteins
[0135] a) Purification of Recombinant .DELTA.9 Protein from the
Transient Transfected 293T Cells
[0136] We transfected a mammalian cell (i.e., human embryonic
kidney fibroblast cell; 293T cell) with either the expression
vector alone or the expression vector carrying the .DELTA.9 cDNA.
Cell lysates and culture media were prepared and collected for the
purification purpose (see "Materials & Methods").
[0137] Cells were lysed and cellular lysates were prepared.
.DELTA.9 was then immuno-purified using an anti-Flag M2 affinity
gel (Sigma). The immuno-precipitated .DELTA.9 was eluted by
incubated with excess amount of Flag peptide (see Method). The
purity of .DELTA.9 was assayed by SDS-PAGE gel followed by
Coomassie-blue staining.
[0138] No specific band was detected from 293T cell lysate
transfected with empty vector, whereas .DELTA.9 protein purified
from the cell lysate transfected with .DELTA.9 expression plasmid
showed multiple bands (FIG. 13).
[0139] We also performed the purification experiment using the
culture media from the 293T cells transfected with empty vector or
.DELTA.9 expression plasmid. No protein was detected from 293T cell
lysate transfected with empty vector, whereas the purified .DELTA.9
protein from secreted source (culture media) showed a homogenous
population as a single band of .about.65 kDa in size (FIG. 14).
[0140] FIG. 15 depicts the developed ELISA is validated by using
purified .DELTA.9 protein. .DELTA.9 protein purified from cellular
lysate was used (See Example 5). The ELISA detects the purified
.DELTA.9 protein from the mammalian cells transiently transfected
with .DELTA.9 expression plasmid but not from the cells transiently
transfected with empty expression plasmid.
[0141] FIG. 16 depicts the purified .DELTA.9 protein from secreted
source (culture medium) that was detected by the developed
ELISA.
[0142] In both validation experiments (FIGS. 15 and 16), the
results clearly show that the developed ELISA is capable to detect
purified .DELTA.9 protein.
Example 6
Detection of .DELTA.9 in the 293T Culture Media
[0143] Using the developed and validated ELISA, we proceeded to
examine if the ELISA is capable to detect .DELTA.9 in the cultured
media (FIG. 17). In this series of study, mammalian 293T cells were
transiently transfected with expression vectors carrying either
wild-type IL-23R.alpha. cDNA (WT) or .DELTA.9 cDNA. The culture
media were collected .DELTA.8 hours post-transfection. Two
independent transfection experiments were performed. No signal was
detected in the 293T cell media transfected with the WT expression
construct. However, signal was detected in the 293T cell media
transfected with the .DELTA.9 expression construct. The result
clearly demonstrates that the ELISA is capable to detect the
presence of .DELTA.9 in the cultured media.
Example 7
EDTA-Treatment of Plasma
[0144] a) Heparin-Plasma and Serum
[0145] We examined and compared different types of biological
samples. They included EDTA-plasma, heparin-plasma and serum
prepared from three (3) human subjects. FIG. 18 depicts the use of
ELISA to measure serological levels of .DELTA.9 IL-23R.alpha..
[0146] In the initial experiments using heparin-plasma, we observed
some inconsistency in the detection of .DELTA.9. In particular,
.DELTA.9 was detected in heparin-plasma only in donor #3, but not
in donors #1 and #2 (FIG. 18). We also observed the similar
inconsistency with sera from the three (3) donors (FIG. 18). It
appeared that heparin-plasma and sera were not optimal as a
biological sample.
[0147] b) EDTA-Plasma
[0148] Unlike heparin-plasma and serum, .DELTA.9 IL-23R was
detected consistently in all EDTA-plasma (FIG. 18). The finding was
totally surprisingly. The results indicate that EDTA-plasma can
provide good measurements for serological level of .DELTA.9
IL-23R.
Example 8
Spike-Recovery and Dilution of Biological Samples
[0149] a) Plasma Dilution
[0150] FIG. 19 depicts the plasma dilution study for ELISA. It has
been reported that too much plasma used in the ELISA could result
in inhibition. To optimize the amount of EDTA-plasma used in ELISA,
neat, 2-fold dilution and 10-fold dilution of EDTA-plasma were
prepared. EDTA plasma from two donors was used. In both donors,
neat and 2-fold dilution of EDTA-plasma resulted in similar signal
intensity in the ELISA test, suggesting that 2-fold dilution of
EDTA-plasma is not enough. In addition, the neat EDTA-plasma has
inhibitory effect in the ELISA. 10-fold dilution of EDTA-plasma
resulted in a decrease in signal for around 50-60%, which is in the
detectable range. Therefore, 10-fold dilution of plasma was used in
our serological ELISA test.
[0151] b) Spike-Recovery: EDTA Plasma
[0152] Spike and recovery experiment was performed on the
EDTA-plasma samples obtained from 4 donors. 100 ng of IL-23R/Fc
fusion protein was spiked into the 10-fold diluted plasma samples.
Around 40% of IL-23R/Fc was recovered in the ELISA assay (FIG.
20).
[0153] c) Spike-Recovery: Synovial Fluid
[0154] Spike and recovery experiment was performed on the synovial
fluid samples obtained from 3 donors. 100 ng of IL-23R/Fc fusion
protein was spiked into the 10-fold diluted plasma samples. Around
50% of IL-23R/Fc was recovered in the ELISA assay (FIG. 21).
[0155] d) Spike-Recovery: Cerebrospinal Fluid
[0156] Spike and recovery experiment was performed on the
cerebrospinal fluid samples obtained from 5 donors. 100 ng of
IL-23R/Fc fusion protein was spiked into the 10-fold diluted plasma
samples. More than 80% of IL-23R/Fc was recovered in the ELISA
assay (FIG. 22).
[0157] e) Spike-Recovery: Amniotic Fluid
[0158] Spike and recovery experiment was performed on the amniotic
fluid samples obtained from 4 donors. 100 ng of IL-23R/Fc fusion
protein was spiked into the 10-fold diluted plasma samples. More
than 80% of IL-23R/Fc was recovered in the ELISA assay (FIG.
23).
Example 9
ELISA: Standard Curve
[0159] In order to quantitated the serological levels of .DELTA.9,
standard curve was generated using recombinant protein of human
IL-23R.alpha./Fc, which comprises an extracellular domain of IL-23R
and Fc region of IgG1. This standard curve was used to calculate
the amount of .DELTA.9 IL-23R.alpha. in EDTA-plasma from OD.sub.450
nm value (FIG. 24).
Example 10
Patient Study--Correlation of Increased Level of D9 and Crohn's
Disease
[0160] a) Control Human Subjects
[0161] FIG. 25 depicts the correlation between serological levels
of .DELTA.9 IL-23R.alpha. protein from fifty-two (52) human Healthy
Donors (EDTA-plasma) with no known medical history of inflammatory,
malignant or infectious bowel disease, including Crohn's disease.
The mean and median of .DELTA.9 IL-23R.alpha. levels in this
subject group were 41.5 ng/mL and 27.8 ng/mL, respectively.
[0162] b) Crohn's Disease Patients
[0163] FIG. 26 depicts the serological levels of .DELTA.9
IL-23R.alpha. in ten (10) human subjects with a medical history of
Crohn's disease. The mean and median of .DELTA.9 IL-23R.alpha.
levels in the Crohn's disease patient group were 155.4 ngmL and
144.5 ng/mL, respectively. Note that the serological levels of
.DELTA.9 IL-23R.alpha. in the Crohn's disease patients, when
compared to that Healthy Donor group described in FIG. 25 (Example
10.a) exhibited higher values. The difference between the two
groups was statistically significant (p=0.0014, Student's t-test,
two-tailed).
Example 11
Patient Study--Correlation of Increased Level of .DELTA.9
IL-23R.alpha. with the Presence of Active Crohn's Disease
Patients
[0164] FIG. 27 depicts the correlation between serological levels
of .DELTA.9 IL-23R.alpha. and patients who suffer from an active
Crohn's disease at the time of the study. In this study, we
examined a separate independent group (n=22) of Healthy Donors as
well as a separate independent group of patients who suffered from
active Crohn's disease (n=5). The mean and median of .DELTA.9
IL-23R.alpha. levels in the control Healthy Donors were 90.47 ng/ml
and 88.67 ng/mL, respectively. The mean and median of .DELTA.9
IL-23R.alpha. levels in the active Crohn's disease patient group
were 131.45 ng/mL and 135.87 ng/mL, respectively. Note that the
.DELTA.9 IL-23R.alpha. levels were found to be significantly higher
in the active Crohn's disease patients. The difference between the
two groups was statistically significant (p=0.0074, Student's
t-test, two-tailed).
[0165] We also examined, in a parallel study, the correlation
between serological levels of .DELTA.9 IL-23R.alpha. and patients
who had a prior history of Crohn's disease, but were symptom-free
at the time of the study (which we called inactive Crohn's disease
group; n=22). The mean and median of .DELTA.9 IL-23R.alpha. levels
in the inactive Crohn's disease patient group were 109.72 ng/mL and
98.63 ng/mL, respectively. One possible explanation for this
observation may relate to the possibility that the levels of
.DELTA.9 IL-23R.alpha. subsided incompletely in some patient in
this group.
Example 12
Patient Study--Correlation of Increased Level of .DELTA.9
IL-23R.alpha. with a History of Colonic Resection in Inactive
Crohn's Disease Patients
[0166] In this study, we further examined a different basis for the
observed levels of .DELTA.9 IL-23R.alpha. in the inactive Crohn's
patients. We noted that the entire inactive Crohn's patients could
be further divided into two (2) groups; namely, (i) with an
intestinal resection procedure, or (ii) without an intestinal
resection procedure. Of the 22 inactive Crohn's disease patients,
19 patients had medical history concerning intestinal resection.
Out of these 19 patients, nine (9) patients had previously received
an intestinal resection, while ten (10) patients had never received
such a procedure.
[0167] The mean and median values of .DELTA.9 protein in the
EDTA-plasma of resection patients were 141.57 ng/mL and 139.49
ng/mL, respectively. The mean and median values of .DELTA.9 protein
in the EDTA-plasma of non-resection patients were 75.40 ng/mL and
77.98 ng/mL, respectively. The .DELTA.9 protein in resection
patients was higher than that in the non-resection patients. The
difference between the two groups was statistically significant
(p=0.009; Student's t-test, two-tailed).
Example 13
Pregnancy Study--Correlation of Decreased Levels of .DELTA.9
IL-23R.alpha. with Human Pregnancy
[0168] FIG. 28 depicts the serological detection of .DELTA.9
IL-23R.alpha. levels in the circulation of two groups of women.
Fifteen (15) healthy non-pregnant women between the ages of 18 and
55, and forty-two (42) otherwise healthy, pregnant women of not
more than eleven (11) weeks gestation had .DELTA.9 IL-23R.alpha.
levels determined. The .DELTA.9 IL-23R.alpha. mean and median
levels detected in the pregnant women were 41.5 ng/mL and 27.8
ng/mL, respectively. The .DELTA.9 IL-23R.alpha. mean and median
levels detected in the non-pregnant women were 100.6 ng/mL and 92.5
ng/mL, respectively. The .DELTA.9 IL-23R.alpha. level in
non-pregnant women was higher than that in pregnant women. The
difference between the two groups was statistically significant
(p<0.0001; Student's t-test, two tailed).
Example 14
Pregnancy Study--Correlation of Levels of .DELTA.9 IL-23R.alpha.
with Length of Pregnancy
[0169] a) .DELTA.9 IL-23R.alpha. Levels in Circulating Plasma
[0170] FIG. 29 depicts the serological detection of .DELTA.9
IL-23R.alpha. levels in the circulation of pregnant women.
Seventy-three (73) women with healthy, normal pregnancies had
.DELTA.9 IL-23R.alpha. levels determined at various times between
week seven and week thirty-six. The levels detected up to and
including week 20 (n=47) were higher (mean, 14.76 ng/mL; median
4.19 ng/mL) than those detected after week 20 (n=27; mean 3.17
ng/mL; median 2.68 ng/mL). The difference between the two groups
was statistically significant (p<0.004; Student's t-test, two
tailed). Note that the "week 20" was chosen because miscarriage
(i.e., loss of pregnancy) occurs at or before the 20.sup.th week of
pregnancy (according to the American Congress of Obstetricians and
Gynecologists).
[0171] b) .DELTA.9 IL-23R.alpha. Levels in Amniotic Fluid
[0172] FIG. 30 depicts the serological detection of .DELTA.9
IL-23R.alpha. levels in amniotic fluid during pregnancy. Amniotic
fluid from fourteen (14) women with healthy, normal pregnancies had
.DELTA.9 IL-23R.alpha. levels determined at various times between
week twelve and week thirty-eight. The .DELTA.9 IL-23R.alpha.
levels detected up to and including week 20 (n=8) were higher
(mean, 15.40 ng/mL; median, 4.81 ng/mL) than those detected after
week 20 (n=6; mean, 4.89 ng/mL; median, 4.79 ng/mL). Thus,
circulating levels of .DELTA.9 IL-23R.alpha. (FIG. 29) reflect
those in the amniotic fluid (FIG. 30), allowing monitoring of the
intra-uterine levels of .DELTA.9 IL-23R.alpha. without an invasive
procedure such as amniocentesis. Note that the profile of .DELTA.9
IL-23R.alpha. in amniotic fluid parallels to that of plasma, albeit
with a slight time delay. Accordingly, it is believed that
detecting changes in .DELTA.9 IL-23R.alpha. levels in plasma can
accurately predict changes in the levels of .DELTA.9 IL-23R.alpha.
levels in amniotic fluid. Because the amniotic fluid bathes the
fetus, we obtained direct evidence that .DELTA.9 IL-23R.alpha. is
present in the amniotic fluid (connecting maternal and fetus). We
speculate the .DELTA.9 IL-23R.alpha. may play a role in
establishing such important connection between mother and
fetus.
[0173] Materials and Methods
[0174] Construction of Expression Constructs
[0175] Human wild-type IL-23R.alpha. was amplified from human
peripheral blood mononuclear cells (PBMC)'s cDNA using the
following primer pair by Pfx high fidelity DNA polymerase
(Invitrogen).
TABLE-US-00002 P1 F: (SEQ ID NO: 5) CAGGTTGAAAGAGGGAAACAGTCT C-Flag
R: (SEQ ID NO: 6) CTCGAGCTACTTGTCATCGTCGTCCTTGTAATCCTTTTCCAAGAGTG
AAATCCTAATG
[0176] The amplified PCR products were run on agarose gels and
purified using DNA gel purification kit from Qiagen. The gel
purified PCR products were cloned into pCDNA3.3 using TOPO TA
cloning kit from Invitrogen. The ligated products were transformed
into Top10 competent cell (Invitrogen). The transformed competent
cells were selected using LB plate containing ampicillin for 16
hours at 37.degree. C. The ampicillin resistant clones were
cultured in 2 mL of LB medium with ampicillin for 16 hours at
37.degree. C. DNA was extracted from the bacteria culture using DNA
mini-preparation kit from Qiagene.
[0177] The DNA was then validated by restriction enzyme digestion
and sequencing. The confirmed expression construct was used to
prepare high quality DNA for transfection using DNA
maxi-preparation kit from Qiagene. The purified DNA was quantified
by Nano-drop (Thermo Scientific).
[0178] The expression constructs of p.DELTA.11, .DELTA.8, .DELTA.9
and .DELTA.8,9 were made by the same approach except using
different primer sets.
[0179] Generation of Expression Constructs
[0180] Expression construct of wild-type IL-23R (WT) was generated
by PCR using Pfx DNA polymerase (Invitrogen). Forward primer (5'
ATGAATCAGGTCACATTCAATG 3') (SEQ ID NO: 12) and reverse primer (5'
CTACTTGTCATCGTCGTCCTTGTAATCCTTTTCCAAGAGTGAAATCCTATTG 3') (SEQ ID
NO: 13) were used to amplify wild-type IL-23R from PBMCs cDNA. The
amplified PCR product was treated with Taq polymerase to add 3'-A
overhang to each end of PCR. The gel-purified product was then
subcloning into mammalian expression plasm id using the pcDNA3.3
TOPO TA Cloning kit from Invitrogen. The correct expression
construct was subjected to validation by sequencing.
[0181] Constructions of p.DELTA.11, .DELTA.9 and .DELTA.89
expression plasmids were performed using the same method except
pcDNA3.3 IL-23R WT was used as PCR template. Difference primer sets
were also used as shown in the following:
TABLE-US-00003 p.DELTA.11 Forward primer: (SEQ ID NO: 14) 5'
ATGAATCAGGTCACATTCAATG 3' Reverse primer: (SEQ ID NO: 15) 5'
CTACTTGTCATCGTCGTCCTTGTAATCTCTCTGTAGCATTTTCA CAACATTGCT 3' .DELTA.9
Forward primer: (SEQ ID NO: 16) 5' ATGAATCAGGTCACATTCAATG 3'
Reverse primer: (SEQ ID NO: 17) 5'
CTACTTGTCATCGTCGTCCTTGTAATCACAATAAGATCCTTCTT TTAATCCAGAAGTAAGGTGC
3' .DELTA.8, 9 Forward primer: (SEQ ID NO: 18) 5'
ATGAATCAGGTCACATTCAATG 3' Reverse primer: (SEQ ID NO: 19) 5'
CTACTTGTCATCGTCGTCCTTGTAATCACAATAAGATCCTTCTT TTAATCCTGTTTCAGGTGTT
3'
[0182] Construction of .DELTA.8 expression plasmid was performed by
PCR overlap extension. Two fragments, fragment 1: Translation start
to Exon 7 and fragment 2: Exon 9 to Translation stop, were
amplified using the following primer pairs.
TABLE-US-00004 Fragment 1 Forward primer: (SEQ ID NO: 20) 5'
ATGAATCAGGTCACATTCAATG 3' Reverse primer: (SEQ ID NO: 21) 5'
CTGTTTCAGGTGTT 3' Fragment 2 Forward primer: (SEQ ID NO: 22) 5'
AACACCTGAAACAG 3' Reverse primer: (SEQ ID NO: 23) 5'
CTACTTGTCATCGTCGTCCTTGTAATCCTTTTCCAAGAGTGAAA TCCTATTG 3'
[0183] Two amplified fragments (1 and 2) were then joined together
by overlap extension. The final combined fragment was subcloned
into pcDNA3.3 TOPO expression vector.
[0184] Western Blotting Analysis
[0185] Cells were collected, washed in PBS and lysed in ProteoJET
mammalian cell lysis reagent (Fermentas) with protease and
phosphatase inhibitors (Sigma). Lysates were centrifuged and
supernatants were prepared for SDS-PAGE by addition of sample
loading buffer (Bio-Rad). Lysates were subjected to 4-12% PAGE
(Bio-Rad) and transferred to Immun-Blot PVDF membrane (Bio-Rad) per
manufacturer's recommendations. Membranes were blocked in 5%
milk/TPBT at room temperature for 1 hour. Membranes were first
probed with antibodies against p-STAT1, p-STAT2, p-STAT3 or p-STAT5
(cell signaling technology), and then stripped and reprobed for
STAT1, STAT2, STAT3 or STAT5 (cell signaling technology).
[0186] Transfection of 293T Cells
[0187] One day before the transient transfection experiment, 293T
cells were trypsinized and cultured on the 10-cm culture plate. The
cell density was maintained at around 60-80% confluence at the time
of transfection. 10 .mu.g of DNA was mixed with 500 .mu.l of
OptiMEM (Invirtogen). 40 .mu.l of FuGene HD transfection reagent
(Roche) was diluted in 500 .mu.l of OptiMEM. The diluted
transfection reagent was then added to DNA mix and vortex for two
seconds to mix the contents. The mixture was incubated at room
temperature for 15 minutes before addition to the 293T cells. Both
culture media and cell lysates were prepared after 36-48 hours
post-transfection for purification of .DELTA.9 IL-23R.alpha..
[0188] Purification of Intracellular .DELTA.9 IL-23R.alpha.
[0189] Cells were collected, washed in PBS and lysed in ProteoJET
mammalian cell lysis reagent (Fermentas) with protease and
phosphatase inhibitors (Sigma). Lysates were centrifuged and
supernatants were prepared for purification. C-terminal flag-tagged
.DELTA.9 was immuno-precipitated from cellular lysates using
anti-flag M2 affinity gel (Sigma) according to the manufacturer's
instructions. The precipitated .DELTA.9 was eluted by excessive
Flag peptide (Sigma). The quality and quantity of the purified
.DELTA.9 IL-23R.alpha. were measured by PAGE gels (Bio-Rad) and
stained with Coomassie Blue (Bio-Rad).
[0190] Purification of .DELTA.9 IL-23R.alpha. from Culture
Medium
[0191] The purification of .DELTA.9 from culture medium was the
same as that of the intracellular .DELTA.9, except the concentrated
cultured media were used. Expression construct of .DELTA.9 was
transiently transfected into 293T cells by Fugene HD transfection
reagent (Roche applied science). The culture medium from the
transfected cells was collected and then concentrated using Amicon
ultra centrifugal filter 30K (Millipore). C-terminal flag-tagged
.DELTA.9 was immuno-precipitated from the concentrated medium using
anti-flag M2 affinity gel (Sigma) according to the manufacturer's
instructions. The precipitated .DELTA.9 was eluted by excessive
Flag peptide (Sigma). The quality and quantity of purified .DELTA.9
were measured by PAGE gels (Bio-rad) stained with Coomassie Blue
(Bio-rad). The purification steps of .DELTA.9 include: [0192] (1)
Transfecting .DELTA.9 expression construct into 293T cells; [0193]
(2) Collecting culture media after .DELTA.8 hours, 72 hours or 96
hours post-transfection; [0194] (3) Centrifugating to remove dead
cells and unattached cells; [0195] (4) Concentrating culture media
using Amicon Ultra-15 Centrifugal Filter with 30 kDa cutoff; [0196]
(5) Immuno-precipitating .DELTA.9 by anti-Flag M2 Affinity Gel;
[0197] (6) Eluting .DELTA.9 by excessive Flag peptides; and [0198]
(7) Measuring the quantity and purity of purified .DELTA.9 by gel
electrophoresis and Commassie Blue staining.
[0199] Immunoblot Assay (Western Blotting)
[0200] Cells were collected, washed in PBS and lysed in ProteoJET
mammalian cell lysis reagent (Fermentas) with protease and
phosphatase inhibitors (Sigma). Lysates were centrifuged and
supernatants were prepared for SDS-PAGE by addition of sample
loading buffer (Bio-Rad). Lysates were subjected to 4-12% PAGE
(Bio-Rad) and transferred to Immun-Blot PVDF membrane (Bio-Rad) per
manufacturer's recommendations. Membranes were blocked in 5%
milk/TPBT at room temperature for 1 hour. Membranes were probed
with anti-flag (sigma), mouse anti-hIL-23R.alpha. (R&D) and
biotinlyated goat anti-hIL-23R.alpha. (R&D).
[0201] Enzyme-Linked Immunosorbent Assay (ELISA)
[0202] Sandwich ELISA was developed using 5 .mu.g/ml of mouse
anti-hIL-23R.alpha. (R&D) as capture antibody and 1.6 .mu.g/ml
of Goat biotinlyated anti-hIL-23R.alpha. (R&D) as detection
antibody. Capture antibody was first coated on the microtiter plate
using 50 mM of bicarbonate buffer (pH=9.6) at 4.degree. C.
overnight. The plate was then blocked with 10% FBS/TBST at room
temperature for 2 hours. Samples were added to the well and
incubated at 4.degree. C. overnight. Detection antibody in TBST was
added to the wells and incubated at room temperature for 2 hours.
The plate was extensively washed with TBST during each change. The
immuno-complex was detected by addition of Streptavidin-HRP
(R&D) and TMB substrate (eBioscience). The plate was read at
OD.sub.450 nm.
[0203] While the present invention has been described in connection
with specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations of the invention thereof. One of skill in
the art will recognize that various modifications may be made to
the embodiments described herein without departing from the spirit
and scope of the invention, which is defined by the appended
claims. All the references and patents cited in this application
are incorporated by reference in their entirety.
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Sequence CWU 1
1
2311071DNAHomo sapiens 1atgaatcagg tcactattca atgggatgca gtaatagccc
tttacatact cttcagctgg 60tgtcatggag gaattacaaa tataaactgc tctggccaca
tctgggtaga accagccaca 120atttttaaga tgggtatgaa tatctctata
tattgccaag cagcaattaa gaactgccaa 180ccaaggaaac ttcattttta
taaaaatggc atcaaagaaa gatttcaaat cacaaggatt 240aataaaacaa
cagctcggct ttggtataaa aactttctgg aaccacatgc ttctatgtac
300tgcactgctg aatgtcccaa acattttcaa gagacactga tatgtggaaa
agacatttct 360tctggatatc cgccagatat tcctgatgaa gtaacctgtg
tcatttatga atattcaggc 420aacatgactt gcacctggaa tgctgggaag
ctcacctaca tagacacaaa atacgtggta 480catgtgaaga gtttagagac
agaagaagag caacagtatc tcacctcaag ctatattaac 540atctccactg
attcattaca aggtggcaag aagtacttgg tttgggtcca agcagcaaac
600gcactaggca tggaagagtc aaaacaactg caaattcacc tggatgatat
agtgatacct 660tctgcagccg tcatttccag ggctgagact ataaatgcta
cagtgcccaa gaccataatt 720tattgggata gtcaaacaac aattgaaaag
gtttcctgtg aaatgagata caaggctaca 780acaaaccaaa cttggaatgt
taaagaattt gacaccaatt ttacatatgt gcaacagtca 840gaattctact
tggagccaaa cattaagtac gtatttcaag tgagatgtca agaaacaggc
900aaaaggtact ggcagccttg gagttcactg ttttttcata aaacacctga
aacagttccc 960caggtcacat caaaagcatt ccaacatgac acatggaatt
ctgggctaac agttgcttcc 1020atctctacag ggcaccttac ttctggatta
aaagaaggat cttattgtta a 10712356PRTHomo sapiens 2Met Asn Gln Val
Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15 Leu Phe
Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30
His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35
40 45 Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys
Leu 50 55 60 His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile
Thr Arg Ile 65 70 75 80 Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn
Phe Leu Glu Pro His 85 90 95 Ala Ser Met Tyr Cys Thr Ala Glu Cys
Pro Lys His Phe Gln Glu Thr 100 105 110 Leu Ile Cys Gly Lys Asp Ile
Ser Ser Gly Tyr Pro Pro Asp Ile Pro 115 120 125 Asp Glu Val Thr Cys
Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140 Thr Trp Asn
Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val 145 150 155 160
His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165
170 175 Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys
Tyr 180 185 190 Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu
Glu Ser Lys 195 200 205 Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile
Pro Ser Ala Ala Val 210 215 220 Ile Ser Arg Ala Glu Thr Ile Asn Ala
Thr Val Pro Lys Thr Ile Ile 225 230 235 240 Tyr Trp Asp Ser Gln Thr
Thr Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255 Tyr Lys Ala Thr
Thr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270 Asn Phe
Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285
Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290
295 300 Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Val
Pro 305 310 315 320 Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr Trp
Asn Ser Gly Leu 325 330 335 Thr Val Ala Ser Ile Ser Thr Gly His Leu
Thr Ser Gly Leu Lys Glu 340 345 350 Gly Ser Tyr Cys 355 3981DNAHomo
sapiens 3atgaatcagg tcactattca atgggatgca gtaatagccc tttacatact
cttcagctgg 60tgtcatggag gaattacaaa tataaactgc tctggccaca tctgggtaga
accagccaca 120atttttaaga tgggtatgaa tatctctata tattgccaag
cagcaattaa gaactgccaa 180ccaaggaaac ttcattttta taaaaatggc
atcaaagaaa gatttcaaat cacaaggatt 240aataaaacaa cagctcggct
ttggtataaa aactttctgg aaccacatgc ttctatgtac 300tgcactgctg
aatgtcccaa acattttcaa gagacactga tatgtggaaa agacatttct
360tctggatatc cgccagatat tcctgatgaa gtaacctgtg tcatttatga
atattcaggc 420aacatgactt gcacctggaa tgctgggaag ctcacctaca
tagacacaaa atacgtggta 480catgtgaaga gtttagagac agaagaagag
caacagtatc tcacctcaag ctatattaac 540atctccactg attcattaca
aggtggcaag aagtacttgg tttgggtcca agcagcaaac 600gcactaggca
tggaagagtc aaaacaactg caaattcacc tggatgatat agtgatacct
660tctgcagccg tcatttccag ggctgagact ataaatgcta cagtgcccaa
gaccataatt 720tattgggata gtcaaacaac aattgaaaag gtttcctgtg
aaatgagata caaggctaca 780acaaaccaaa cttggaatgt taaagaattt
gacaccaatt ttacatatgt gcaacagtca 840gaattctact tggagccaaa
cattaagtac gtatttcaag tgagatgtca agaaacaggc 900aaaaggtact
ggcagccttg gagttcactg ttttttcata aaacacctga aacaggatta
960aaagaaggat cttattgtta a 9814326PRTHomo sapiens 4Met Asn Gln Val
Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15 Leu Phe
Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30
His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35
40 45 Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys
Leu 50 55 60 His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile
Thr Arg Ile 65 70 75 80 Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn
Phe Leu Glu Pro His 85 90 95 Ala Ser Met Tyr Cys Thr Ala Glu Cys
Pro Lys His Phe Gln Glu Thr 100 105 110 Leu Ile Cys Gly Lys Asp Ile
Ser Ser Gly Tyr Pro Pro Asp Ile Pro 115 120 125 Asp Glu Val Thr Cys
Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140 Thr Trp Asn
Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val 145 150 155 160
His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165
170 175 Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys
Tyr 180 185 190 Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu
Glu Ser Lys 195 200 205 Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile
Pro Ser Ala Ala Val 210 215 220 Ile Ser Arg Ala Glu Thr Ile Asn Ala
Thr Val Pro Lys Thr Ile Ile 225 230 235 240 Tyr Trp Asp Ser Gln Thr
Thr Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255 Tyr Lys Ala Thr
Thr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270 Asn Phe
Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285
Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290
295 300 Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr Gly
Leu 305 310 315 320 Lys Glu Gly Ser Tyr Cys 325 524DNAHomo sapiens
5caggttgaaa gagggaaaca gtct 24658DNAHomo sapiens 6ctcgagctac
ttgtcatcgt cgtccttgta atccttttcc aagagtgaaa tcctaatg 5878PRTHomo
sapiens 7Gly Leu Lys Glu Gly Ser Tyr Cys 1 5 81245DNAHomo sapiens
8atgaatcagg tcactattca atgggatgca gtaatagccc tttacatact cttcagctgg
60tgtcatggag gaattacaaa tataaactgc tctggccaca tctgggtaga accagccaca
120atttttaaga tgggtatgaa tatctctata tattgccaag cagcaattaa
gaactgccaa 180ccaaggaaac ttcattttta taaaaatggc atcaaagaaa
gatttcaaat cacaaggatt 240aataaaacaa cagctcggct ttggtataaa
aactttctgg aaccacatgc ttctatgtac 300tgcactgctg aatgtcccaa
acattttcaa gagacactga tatgtggaaa agacatttct 360tctggatatc
cgccagatat tcctgatgaa gtaacctgtg tcatttatga atattcaggc
420aacatgactt gcacctggaa tgctgggaag ctcacctaca tagacacaaa
atacgtggta 480catgtgaaga gtttagagac agaagaagag caacagtatc
tcacctcaag ctatattaac 540atctccactg attcattaca aggtggcaag
aagtacttgg tttgggtcca agcagcaaac 600gcactaggca tggaagagtc
aaaacaactg caaattcacc tggatgatat agtgatacct 660tctgcagccg
tcatttccag ggctgagact ataaatgcta cagtgcccaa gaccataatt
720tattgggata gtcaaacaac aattgaaaag gtttcctgtg aaatgagata
caaggctaca 780acaaaccaaa cttggaatgt taaagaattt gacaccaatt
ttacatatgt gcaacagtca 840gaattctact tggagccaaa cattaagtac
gtatttcaag tgagatgtca agaaacaggc 900aaaaggtact ggcagccttg
gagttcactg ttttttcata aaacacctga aacagttccc 960caggtcacat
caaaagcatt ccaacatgac acatggaatt ctgggctaac agttgcttcc
1020atctctacag ggcaccttac ttctgacaac agaggagaca ttggactttt
attgggaatg 1080atcgtctttg ctgttatgtt gtcaattctt tctttgattg
ggatatttaa cagatcattc 1140cgaactggga ttaaaagaag gatcttattg
ttaataccaa agtggcttta tgaagatatt 1200cctaatatga aaaacagcaa
tgttgtgaaa atgctacaga gataa 12459414PRTHomo sapiens 9Met Asn Gln
Val Thr Ile Gln Trp Asp Ala Val Ile Ala Leu Tyr Ile 1 5 10 15 Leu
Phe Ser Trp Cys His Gly Gly Ile Thr Asn Ile Asn Cys Ser Gly 20 25
30 His Ile Trp Val Glu Pro Ala Thr Ile Phe Lys Met Gly Met Asn Ile
35 40 45 Ser Ile Tyr Cys Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg
Lys Leu 50 55 60 His Phe Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln
Ile Thr Arg Ile 65 70 75 80 Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys
Asn Phe Leu Glu Pro His 85 90 95 Ala Ser Met Tyr Cys Thr Ala Glu
Cys Pro Lys His Phe Gln Glu Thr 100 105 110 Leu Ile Cys Gly Lys Asp
Ile Ser Ser Gly Tyr Pro Pro Asp Ile Pro 115 120 125 Asp Glu Val Thr
Cys Val Ile Tyr Glu Tyr Ser Gly Asn Met Thr Cys 130 135 140 Thr Trp
Asn Ala Gly Lys Leu Thr Tyr Ile Asp Thr Lys Tyr Val Val 145 150 155
160 His Val Lys Ser Leu Glu Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser
165 170 175 Ser Tyr Ile Asn Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys
Lys Tyr 180 185 190 Leu Val Trp Val Gln Ala Ala Asn Ala Leu Gly Met
Glu Glu Ser Lys 195 200 205 Gln Leu Gln Ile His Leu Asp Asp Ile Val
Ile Pro Ser Ala Ala Val 210 215 220 Ile Ser Arg Ala Glu Thr Ile Asn
Ala Thr Val Pro Lys Thr Ile Ile 225 230 235 240 Tyr Trp Asp Ser Gln
Thr Thr Ile Glu Lys Val Ser Cys Glu Met Arg 245 250 255 Tyr Lys Ala
Thr Thr Asn Gln Thr Trp Asn Val Lys Glu Phe Asp Thr 260 265 270 Asn
Phe Thr Tyr Val Gln Gln Ser Glu Phe Tyr Leu Glu Pro Asn Ile 275 280
285 Lys Tyr Val Phe Gln Val Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp
290 295 300 Gln Pro Trp Ser Ser Leu Phe Phe His Lys Thr Pro Glu Thr
Val Pro 305 310 315 320 Gln Val Thr Ser Lys Ala Phe Gln His Asp Thr
Trp Asn Ser Gly Leu 325 330 335 Thr Val Ala Ser Ile Ser Thr Gly His
Leu Thr Ser Asp Asn Arg Gly 340 345 350 Asp Ile Gly Leu Leu Leu Gly
Met Ile Val Phe Ala Val Met Leu Ser 355 360 365 Ile Leu Ser Leu Ile
Gly Ile Phe Asn Arg Ser Phe Arg Thr Gly Ile 370 375 380 Lys Arg Arg
Ile Leu Leu Leu Ile Pro Lys Trp Leu Tyr Glu Asp Ile 385 390 395 400
Pro Asn Met Lys Asn Ser Asn Val Val Lys Met Leu Gln Arg 405 410
101800DNAHomo sapiens 10atgaatcagg tcactattca atgggatgca gtaatagccc
tttacatact cttcagctgg 60tgtcatggag gaattacaaa tataaactgc tctggccaca
tctgggtaga accagccaca 120atttttaaga tgggtatgaa tatctctata
tattgccaag cagcaattaa gaactgccaa 180ccaaggaaac ttcattttta
taaaaatggc atcaaagaaa gatttcaaat cacaaggatt 240aataaaacaa
cagctcggct ttggtataaa aactttctgg aaccacatgc ttctatgtac
300tgcactgctg aatgtcccaa acattttcaa gagacactga tatgtggaaa
agacatttct 360tctggatatc cgccagatat tcctgatgaa gtaacctgtg
tcatttatga atattcaggc 420aacatgactt gcacctggaa tgctgggaag
ctcacctaca tagacacaaa atacgtggta 480catgtgaaga gtttagagac
agaagaagag caacagtatc tcacctcaag ctatattaac 540atctccactg
attcattaca aggtggcaag aagtacttgg tttgggtcca agcagcaaac
600gcactaggca tggaagagtc aaaacaactg caaattcacc tggatgatat
agtgatacct 660tctgcagccg tcatttccag ggctgagact ataaatgcta
cagtgcccaa gaccataatt 720tattgggata gtcaaacaac aattgaaaag
gtttcctgtg aaatgagata caaggctaca 780acaaaccaaa cttggaatgt
taaagaattt gacaccaatt ttacatatgt gcaacagtca 840gaattctact
tggagccaaa cattaagtac gtatttcaag tgagatgtca agaaacaggc
900aaaaggtact ggcagccttg gagttcactg ttttttcata aaacacctga
aacagacaac 960agaggagaca ttggactttt attgggaatg atcgtctttg
ctgttatgtt gtcaattctt 1020tctttgattg ggatatttaa cagatcattc
cgaactggga ttaaaagaag gatcttattg 1080ttaataccaa agtggcttta
tgaagatatt cctaatatga aaaacagcaa tgttgtgaaa 1140atgctacagg
aaaatagtga acttatgaat aataattcca gtgagcaggt cctatatgtt
1200gatcccatga ttacagagat aaaagaaatc ttcatcccag aacacaagcc
tacagactac 1260aagaaggaga atacaggacc cctggagaca agagactacc
cgcaaaactc gctattcgac 1320aatactacag ttgtatatat tcctgatctc
aacactggat ataaacccca aatttcaaat 1380tttctgcctg agggaagcca
tctcagcaat aataatgaaa ttacttcctt aacacttaaa 1440ccaccagttg
attccttaga ctcaggaaat aatcccaggt tacaaaagca tcctaatttt
1500gctttttctg tttcaagtgt gaattcacta agcaacacaa tatttcttgg
agaattaagc 1560ctcatattaa atcaaggaga atgcagttct cctgacatac
aaaactcagt agaggaggaa 1620accaccatgc ttttggaaaa tgattcaccc
agtgaaacta ttccagaaca gaccctgctt 1680cctgatgaat ttgtctcctg
tttggggatc gtgaatgagg agttgccatc tattaatact 1740tattttccac
aaaatatttt ggaaagccac ttcaatagga tttcactctt ggaaaagtag
180011599PRTHomo sapiens 11Met Asn Gln Val Thr Ile Gln Trp Asp Ala
Val Ile Ala Leu Tyr Ile 1 5 10 15 Leu Phe Ser Trp Cys His Gly Gly
Ile Thr Asn Ile Asn Cys Ser Gly 20 25 30 His Ile Trp Val Glu Pro
Ala Thr Ile Phe Lys Met Gly Met Asn Ile 35 40 45 Ser Ile Tyr Cys
Gln Ala Ala Ile Lys Asn Cys Gln Pro Arg Lys Leu 50 55 60 His Phe
Tyr Lys Asn Gly Ile Lys Glu Arg Phe Gln Ile Thr Arg Ile 65 70 75 80
Asn Lys Thr Thr Ala Arg Leu Trp Tyr Lys Asn Phe Leu Glu Pro His 85
90 95 Ala Ser Met Tyr Cys Thr Ala Glu Cys Pro Lys His Phe Gln Glu
Thr 100 105 110 Leu Ile Cys Gly Lys Asp Ile Ser Ser Gly Tyr Pro Pro
Asp Ile Pro 115 120 125 Asp Glu Val Thr Cys Val Ile Tyr Glu Tyr Ser
Gly Asn Met Thr Cys 130 135 140 Thr Trp Asn Ala Gly Lys Leu Thr Tyr
Ile Asp Thr Lys Tyr Val Val 145 150 155 160 His Val Lys Ser Leu Glu
Thr Glu Glu Glu Gln Gln Tyr Leu Thr Ser 165 170 175 Ser Tyr Ile Asn
Ile Ser Thr Asp Ser Leu Gln Gly Gly Lys Lys Tyr 180 185 190 Leu Val
Trp Val Gln Ala Ala Asn Ala Leu Gly Met Glu Glu Ser Lys 195 200 205
Gln Leu Gln Ile His Leu Asp Asp Ile Val Ile Pro Ser Ala Ala Val 210
215 220 Ile Ser Arg Ala Glu Thr Ile Asn Ala Thr Val Pro Lys Thr Ile
Ile 225 230 235 240 Tyr Trp Asp Ser Gln Thr Thr Ile Glu Lys Val Ser
Cys Glu Met Arg 245 250 255 Tyr Lys Ala Thr Thr Asn Gln Thr Trp Asn
Val Lys Glu Phe Asp Thr 260 265 270 Asn Phe Thr Tyr Val Gln Gln Ser
Glu Phe Tyr Leu Glu Pro Asn Ile 275 280 285 Lys Tyr Val Phe Gln Val
Arg Cys Gln Glu Thr Gly Lys Arg Tyr Trp 290 295 300 Gln Pro Trp Ser
Ser Leu Phe Phe His Lys Thr Pro Glu Thr Asp Asn 305 310 315 320 Arg
Gly Asp Ile Gly Leu Leu Leu Gly Met Ile Val Phe Ala Val Met 325 330
335 Leu Ser Ile Leu Ser Leu Ile Gly Ile Phe Asn Arg Ser Phe Arg Thr
340 345 350 Gly Ile Lys Arg Arg Ile Leu Leu Leu Ile Pro Lys Trp Leu
Tyr Glu 355
360 365 Asp Ile Pro Asn Met Lys Asn Ser Asn Val Val Lys Met Leu Gln
Glu 370 375 380 Asn Ser Glu Leu Met Asn Asn Asn Ser Ser Glu Gln Val
Leu Tyr Val 385 390 395 400 Asp Pro Met Ile Thr Glu Ile Lys Glu Ile
Phe Ile Pro Glu His Lys 405 410 415 Pro Thr Asp Tyr Lys Lys Glu Asn
Thr Gly Pro Leu Glu Thr Arg Asp 420 425 430 Tyr Pro Gln Asn Ser Leu
Phe Asp Asn Thr Thr Val Val Tyr Ile Pro 435 440 445 Asp Leu Asn Thr
Gly Tyr Lys Pro Gln Ile Ser Asn Phe Leu Pro Glu 450 455 460 Gly Ser
His Leu Ser Asn Asn Asn Glu Ile Thr Ser Leu Thr Leu Lys 465 470 475
480 Pro Pro Val Asp Ser Leu Asp Ser Gly Asn Asn Pro Arg Leu Gln Lys
485 490 495 His Pro Asn Phe Ala Phe Ser Val Ser Ser Val Asn Ser Leu
Ser Asn 500 505 510 Thr Ile Phe Leu Gly Glu Leu Ser Leu Ile Leu Asn
Gln Gly Glu Cys 515 520 525 Ser Ser Pro Asp Ile Gln Asn Ser Val Glu
Glu Glu Thr Thr Met Leu 530 535 540 Leu Glu Asn Asp Ser Pro Ser Glu
Thr Ile Pro Glu Gln Thr Leu Leu 545 550 555 560 Pro Asp Glu Phe Val
Ser Cys Leu Gly Ile Val Asn Glu Glu Leu Pro 565 570 575 Ser Ile Asn
Thr Tyr Phe Pro Gln Asn Ile Leu Glu Ser His Phe Asn 580 585 590 Arg
Ile Ser Leu Leu Glu Lys 595 1222DNAHomo sapiens 12atgaatcagg
tcacattcaa tg 221352DNAHomo sapiens 13ctacttgtca tcgtcgtcct
tgtaatcctt ttccaagagt gaaatcctat tg 521422DNAHomo sapiens
14atgaatcagg tcacattcaa tg 221554DNAHomo sapiens 15ctacttgtca
tcgtcgtcct tgtaatctct ctgtagcatt ttcacaacat tgct 541622DNAHomo
sapiens 16atgaatcagg tcacattcaa tg 221764DNAHomo sapiens
17ctacttgtca tcgtcgtcct tgtaatcaca ataagatcct tcttttaatc cagaagtaag
60gtgc 641822DNAHomo sapiens 18atgaatcagg tcacattcaa tg
221964DNAHomo sapiens 19ctacttgtca tcgtcgtcct tgtaatcaca ataagatcct
tcttttaatc ctgtttcagg 60tgtt 642022DNAHomo sapiens 20atgaatcagg
tcacattcaa tg 222114DNAHomo sapiens 21ctgtttcagg tgtt 142214DNAHomo
sapiens 22aacacctgaa acag 142352DNAHomo sapiens 23ctacttgtca
tcgtcgtcct tgtaatcctt ttccaagagt gaaatcctat tg 52
* * * * *