U.S. patent application number 11/716051 was filed with the patent office on 2009-03-12 for asthma associated factors as targets for treating atopic allergies including asthma and related disorders.
Invention is credited to Charles Qu Dong, Kenneth J. Holroyd, Roy C. Levitt, Jamila Louahed, W. Lee Maloy, Mike McLane, Nicholas C. Nicolaides, Yuhong Zhou.
Application Number | 20090068185 11/716051 |
Document ID | / |
Family ID | 22134353 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068185 |
Kind Code |
A1 |
Holroyd; Kenneth J. ; et
al. |
March 12, 2009 |
Asthma associated factors as targets for treating atopic allergies
including asthma and related disorders
Abstract
A new gene in the calcium-activated chloride channel family has
been discovered that is induced by IL-9, thereby providing a
therapeutic target in IL-9 mediated development of atopic allergy,
asthma-related disorders and cystic fibrosis. A method for the
identification and use of small molecule inhibitors of this gene
and its products to treat these disorders has also been discovered.
The invention also includes a method for diagnosing susceptibility
to, and assessing treatment of atopic allergy, asthma-related
disorders by measuring the level of gene expression in biologic
samples using antibody specific for this protein.
Inventors: |
Holroyd; Kenneth J.;
(Plymouth Meeting, PA) ; Levitt; Roy C.; (Plymouth
Meeting, PA) ; Maloy; W. Lee; (Plymouth Meeting,
PA) ; Louahed; Jamila; (Plymouth Meeting, PA)
; McLane; Mike; (Plymouth Meeting, PA) ;
Nicolaides; Nicholas C.; (Plymouth Meeting, PA) ;
Zhou; Yuhong; (Plymouth Meeting, PA) ; Dong; Charles
Qu; (Dresher, PA) |
Correspondence
Address: |
MORGAN LEWIS & BOCKIUS LLP
1111 PENNSYLVANIA AVENUE NW
WASHINGTON
DC
20004
US
|
Family ID: |
22134353 |
Appl. No.: |
11/716051 |
Filed: |
March 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10772437 |
Feb 6, 2004 |
7211254 |
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11716051 |
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10270595 |
Oct 16, 2002 |
6716603 |
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10772437 |
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09623624 |
Feb 13, 2001 |
6576434 |
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PCT/US99/04703 |
Mar 3, 1999 |
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10270595 |
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60076815 |
Mar 3, 1998 |
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Current U.S.
Class: |
424/139.1 ;
514/169; 514/44R; 530/350; 536/23.1 |
Current CPC
Class: |
A61P 11/00 20180101;
Y10S 977/914 20130101; G01N 33/6872 20130101; G01N 2500/00
20130101; C07K 14/705 20130101; A61P 37/08 20180101; A61P 1/00
20180101; A61P 43/00 20180101; A61K 38/00 20130101; A61P 11/06
20180101; Y10S 977/927 20130101 |
Class at
Publication: |
424/139.1 ;
536/23.1; 530/350; 514/169; 514/44 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/00 20060101 C07H021/00; A61K 31/56 20060101
A61K031/56; A61P 11/06 20060101 A61P011/06; A61K 31/7052 20060101
A61K031/7052; C07K 14/00 20060101 C07K014/00 |
Claims
1. A purified and isolated nucleic acid molecule selected from the
group consisting of a nucleic acid molecule having a nucleotide
sequence encoding human ICACC-1 (SEQ ID NO: 6), a nucleic acid
molecule which hybridizes to a nucleic acid molecule having SEQ ID
NO: 1, a nucleic acid molecule encoding murine ICACC-1 (SEQ ID NO:
2), a nucleic acid molecule encoding human ICACC-2 (SEQ ID NO: 4)
and a nucleic acid molecule comprising functionally effective
fragments thereof.
2. A purified and isolated DNA molecule of claim 1 having a
nucleotide sequence encoding murine ICACC-1 (SEQ ID NO: 2) or
functionally effective fragments thereof.
3. A purified and isolated DNA molecule of claim 1 having a
nucleotide sequence encoding human ICACC-2 (SEQ ID NO: 4) or
functionally effective fragments thereof.
4. The purified and isolated nucleic acid molecule of claim 1
wherein said molecule is genomic DNA.
5-10. (canceled)
11. A purified and isolated protein molecule selected from the
group consisting of a purified and isolated protein having an amino
acid sequence comprising human ICACC-1 (SEQ ID NO: 6) or
functionally effective fragments thereof, a purified and isolated
protein molecule having an amino acid sequence comprising murine
ICACC-1 (SEQ ID NO: 2) or functionally effective fragments thereof
and a purified and isolated protein molecule having an amino acid
sequence comprising human ICACC-1 (SEQ ID NO: 4) or functionally
effective fragments thereof.
12. A purified and isolated protein molecule of claim 11 having an
amino acid sequence comprising murine ICACC-1 (SEQ ID NO: 2) or
functionally effective fragments thereof.
13. A purified and isolated protein molecule of claim 11 having an
amino acid sequence comprising human ICACC-1 (SEQ ID NO: 4) or
functionally effective fragments thereof.
14. A method of alleviating asthma by administering to patients in
need of such treatment an effective amount of a compound or an
antibody that blocks the function of an ICACC to down-regulate the
function of human ICACC-1.
15. A method according to claim 14 wherein the compound is a
chloride channel inhibitor.
16. A method according to claim 14 wherein the compound is an
aminosterol.
17. (canceled)
18. The method of claim 14 wherein the antibody is produced from
peptides that encompass the functional domains of ICACC-1.
19. A method according to claim 14 wherein the antibody is produced
from peptides taken from the group of SEQ ID NO: 11-15.
20. The method of claim 14 wherein the antibody is monoclonal.
21-29. (canceled)
30. Antisense DNA comprising the antisense sequence of a nucleic
acid molecule of claim 1 or active fragments thereof.
31. A method according to claim 14 wherein the compound is the
antisense DNA of claim 30.
32-36. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 60/076,815 which was filed Mar. 3, 1998
and which is herein incorporated by reference in its entirety. This
invention is also related to the subject matter of U.S. patent
application Ser. Nos. 08/697,419; 08/697,360; 08/697,473;
08/697,472; 08/697,471; 08/702,105; 08/702,110; 08/702,168; and
08/697,440, all of which were filed on Aug. 23, 1996 and are
incorporated herein by reference. This application is also related
to U.S. patent application Ser. No. 08/980,872 which was filed Dec.
1, 1997 and which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to modulating activities associated
with the IL-9 pathway for the treatment of atopic allergies and
related disorders such as asthma.
BACKGROUND OF THE INVENTION
[0003] Inflammation is a complex process in which the body's
defense system combats foreign entities. While the battle against
foreign entities may be necessary for the body's survival, some
defense systems respond to foreign entities, even innocuous ones,
as dangerous and thereby damage surrounding tissue in the ensuing
battle.
[0004] Atopic allergy is an ecogenetic disorder, where genetic
background dictates the response to environmental stimuli. The
disorder is generally characterized by an increased ability of
lymphocytes to produce IgE antibodies in response to ubiquitous
antigens. Activation of the immune system by these antigens leads
to allergic inflammation and may occur after ingestion, penetration
through the skin or after inhalation. When this immune activation
occurs and is accompanied by pulmonary inflammation and bronchial
hyperresponsiveness, this disorder is broadly characterized as
asthma. Certain cells are important in this inflammatory reaction
and they include T cells and antigen-presenting cells, B cells that
produce IgE, basophils that bind IgE and eosinophils. These
inflammatory cells accumulate at the site of allergic inflammation
and the toxic products they release contribute to tissue
destruction related to these disorders.
[0005] While asthma is generally defined as an inflammatory
disorder of the airways, clinical symptoms arise from intermittent
air flow obstruction. It is a chronic, disabling disorder that
appears to be increasing in prevalence and severity (Gergen et al.,
1992). It is estimated that 30-40% of the population suffer with
atopic allergy and 15% of children and 5% of adults in the
population suffer from asthma (Gergen et al., 1992). Thus, an
enormous burden is placed on health-care resources.
[0006] Interestingly, while most individuals experience similar
environmental exposures, only certain individuals develop atopic
allergy and asthma. This hypersensitivity to environmental
allergens known as "atopy" is often indicated by elevated serum IgE
levels or abnormally intense skin test response to allergens in
atopic individuals as compared to nonatopics (Marsh et al., 1982).
Strong evidence for a close relationship between atopic allergy and
asthma is derived from the fact that most asthmatics have clinical
and serologic evidence of atopy (Clifford et al., 1987; Gergen,
1991; Burrows et al., 1992; Johannson et al., 1972; Sears et al.,
1991; Halonen et al., 1992). In particular, younger asthmatics have
a high incidence of atopy (Marsh et al., 1982). In addition,
immunologic factors associated with an increase in total serum IgE
levels are very closely related to impaired pulmonary function
(Burrows et al., 1989).
[0007] Both the diagnosis and treatment of these disorders are
problematic (Gergen et al., 1992). The assessment of inflamed lung
tissue is often difficult and frequently the source of the
inflammation cannot be determined. It is now generally accepted
that failure to control pulmonary inflammation leads to significant
loss of lung function over time.
[0008] Current treatments suffer their own set of disadvantages.
The main therapeutic agents, .beta. agonists, reduce the symptoms
thereby transiently improving pulmonary function, but do not affect
the underlying inflammation so that lung tissue remains in
jeopardy. In addition, constant use of .beta. agonists results in
desensitization which reduces their efficacy and safety (Molinoff
et al., 1995). The agents that can diminish the underlying
inflammation, such as anti-inflammatory steroids, have their own
list of disadvantages that range from immunosuppression to bone
loss (Molinoff et al., 1995).
[0009] Because of the problems associated with conventional
therapies, alternative treatment strategies have been evaluated.
Glycophorin A (Chu et al., 1992), cyclosporin (Alexander et al.,
1992; Morely, 1992) and a nonapeptide fragment of interleukin 2
(IL-2) (Zavyalov et al., 1992) all inhibit potentially critical
immune functions associated with homeostasis. What is needed in the
art is a treatment for asthma that addresses the underlying
pathogenesis. Moreover, these therapies must address the episodic
nature of the disorder and the close association with allergy and
intervene at a point downstream from critical immune functions.
[0010] In the related patent applications mentioned above,
applicants have demonstrated that interleukin 9 (IL-9), its
receptor and activities effected by IL-9 are the appropriate
targets for therapeutic intervention in atopic allergy, asthma and
related disorders.
[0011] Mediator release from mast cells by allergen has long been
considered a critical initiating event in allergy. IL-9 was
originally identified as a mast cell growth factor and it has been
demonstrated that IL-9 up-regulates the expression of mast cell
proteases including MCP-1, MCP-2, MCP-4 (Eklund et al., 1993) and
granzyme B (Louahed et al., 1995). Thus, IL-9 appears to serve a
role in the proliferation and differentiation of mast cells.
Moreover, IL-9 up-regulates the expression of the alpha chain of
the high affinity IgE receptor (Dugas et al., 1993). Elevated IgE
levels are considered to be a hallmark of atopic allergy and a risk
factor for asthma. Furthermore, both in vitro and in vivo studies
have shown IL-9 to potentiate the release of IgE from primed B
cells (Petit-Frere et al., 1993).
[0012] There is substantial support for the role of IL-9 gene in
asthma. First, linkage homology between humans and mice suggests
that the same gene is responsible for producing biologic
variability in response to antigen in both species. Second,
differences in expression of the murine IL-9 candidate gene are
associated with biologic variability in bronchial responsiveness.
In particular, reduced expression of IL-9 is associated with a
lower baseline bronchial response in B6 mice. Third, recent
evidence for linkage disequilibrium in data from humans suggests
IL-9 may be associated with atopy and bronchial hyperresponsiveness
consistent with a role for this gene in both species (Doull et al.,
1992). Moreover, a genetic alteration in the human gene appears to
be associated with loss of cytokine function and lower IgE levels.
Fourth, the pleiotropic functions of this cytokine and its receptor
in the allergic immune response strongly support a role for the
IL-9 pathway in the complex pathogenesis of asthma. Fifth, in
humans, biologic variability in the IL-9 receptor also appears to
be associated with atopic allergy and asthma. Finally, despite the
inherited loss of IL-9 receptor function, these individuals appear
to be otherwise healthy. Thus, nature has demonstrated in atopic
individuals that the therapeutic down-regulation of IL-9 and IL-9
receptor genes or genes activated by IL-9 and its receptor is
likely to be safe.
[0013] While the role of the IL-9 gene, its receptor and their
functions in atopic allergy, asthma and related disorders has been
elucidated, a specific need in the art exists for elucidation of
the role of genes which are regulated by IL-9 in the etiology of
these disorders. Furthermore, most significantly, based on this
knowledge, there is a need for the identification of agents that
are capable of regulating the activity of these genes or their gene
products for treating these disorders.
[0014] Cystic fibrosis is yet another disease which effects the
lung and is associated with thick secretions resulting in airway
obstruction and subsequent colonization and infection by inhaled
pathogenic microorganisms (Eng et al., 1996). Cystic fibrosis
airway epithelia exhibit a spectrum of ion transport properties
that differ from normal, including not only defective cAMP-mediated
chloride secretion, but also increased sodium absorption and
increased calcium-mediated chloride secretion (Johnson et al.,
1995). The increase in calcium-mediated chloride secretion is
presumably an attempt to compensate for the overall decrease in
chloride secretion due to the defect in cAMP-mediated chloride
secretion. It does not adequately compensate for this defect,
however, because normal chloride gradients are not maintained.
Thus, potential therapeutic remedies for cystic fibrosis rely on
mechanisms which increase chloride secretion in airway epithelial
cells to compensate for defective cAMP-mediated chloride secretion.
Such mechanisms are capable of restoring the cellular chloride
gradient thereby alleviating sodium hyperabsorption associated with
decreased chloride secretion. A specific need in the art therefore
exists for identification of agents capable of enhancing
calcium-dependent chloride secretion in cystic fibrosis airway
epithelial cells.
SUMMARY OF THE INVENTION
[0015] The present invention includes new genes from the calcium
activated chloride channel gene family designated ICACC (IL-9
Induced Calcium Activated Chloride Channel), particularly the mouse
(SEQ ID NO:1) and human (SEQ ID NO:3 and SEQ ID NO:5) ICACC genes.
The ICACC-1 genes are selectively up-regulated by IL-9 and
therefore part of the IL-9 signaling pathway. The present invention
also includes the protein products of the ICACC genes,
particularly, the mouse (SEQ ID NO:2) and human (SEQ ID NO:4 and
NO:6) ICACC genes.
[0016] The inventors have satisfied the need for diagnosis and
treatment of atopic allergy, asthma and related disorders by
demonstrating the role of ICACC-1 in the pathogenesis of these
disorders. Therapies for these disorders are derived from the
down-regulation of ICACC-1 as a member of the IL-9 pathway.
[0017] The identification of ICACC-1 has led to the discovery of
compounds that are capable of down-regulating its activity.
Activity is defined here as any alteration in either chloride
channel function or expression of ICACC-1. Molecules that
down-regulate ICACC-1 are therefore part of the invention.
Down-regulation is defined here as a decrease in activation,
function or synthesis of ICACC-1, its ligands or activators. It is
further defined to include an increase in the degradation of
ICACC-1 gene, its protein product, ligands or activators.
Down-regulation is therefore achieved in a number of ways. For
example, administration of molecules that can destabilize the
binding of ICACC-1 with its ligands. Such molecules encompass
polypeptide products, including those encoded by the DNA sequences
of the ICACC-1 gene or DNA sequences containing various mutations.
These mutations may be point mutations, insertions, deletions or
spliced variants of the ICACC-1 gene. This invention also includes
truncated polypeptides encoded by the DNA molecules described
above. These polypeptides being capable of interfering with
interaction of ICACC-1 with its ligand and other proteins.
[0018] A further embodiment of this invention includes the
down-regulation of ICACC-1 function by altering expression of the
ICACC-1 gene, the use of antisense gene therapy being an example.
Down-regulation of ICACC-1 expression is accomplished by
administering an effective amount of antisense oligonucleotides.
These antisense molecules can be fashioned from the DNA sequence of
the ICACC-1 gene or sequences containing various mutations,
deletions, insertions or spliced variants. Another embodiment of
this invention relates to the use of isolated RNA or DNA sequences
derived from the ICACC-1 gene. These sequences containing various
mutations such as point mutations, insertions, deletions or spliced
variant mutations of ICACC-1 gene and can be useful in gene
therapy.
[0019] Molecules that increase the degradation of the ICACC-1
protein may also be used to down-regulate its functions and are
within the scope of the invention. Phosphorylation of ICACC-1 may
alter protein stability, therefore kinase inhibitors may be used to
down-regulate its function. Down-regulation of ICACC-1 may also be
accomplished by the use of polyclonal or monoclonal antibodies or
fragments thereof directed against the ICACC-1 protein. Such
molecules are within the claimed invention. This invention further
includes small molecules with the three-dimensional structure
necessary to bind with sufficient affinity to block ICACC-1
interactions with its ligands or block function of the chloride
channel. ICACC-1 blockade resulting in deregulation of calcium and
chloride flux and other processes of proinflammatory cells where it
is expressed make these small molecules useful as therapeutic
agents in treating inflammation associated with atopic allergy,
asthma and related disorders. In a further embodiment, aminosterol
compounds are assessed for their ability to block ICACC-1 induction
by IL-9 or antigen as a means of determining their usefulness in
treating atopic allergies and related disorders.
[0020] The agents discussed above represent various effective
therapeutic compounds in treating atopic allergies, asthma and
other related disorders. Applicants have thus provided antagonists
and methods of identifying antagonists that are capable of
down-regulating ICACC-1. Applicants also provide methods for
down-regulating the activity of ICACC-1 by administering truncated
protein products, chloride channel blockers, aminosterols and the
like.
[0021] Applicants also provide a method for the diagnosis of
susceptibility to atopic allergy, asthma and related disorders by
describing a method for assaying the induction of ICACC-1, its
functions or downstream activities. In a further embodiment,
Applicants provide methods to monitor the effects of ICACC-1
down-regulation as a means to follow the treatment of atopic
allergy and asthma. Applicants also provide a method for diagnosing
autoimmune type diseases such as inflammatory bowel disease (IBD)
where suppression of TH2-associated responses (such as the biologic
responses associated with IL-9) are a common molecular feature. The
constitutive expression of ICACC-1 in the small intestine and colon
suggest that this is a useful marker for monitoring treatment of
TH1 associated disease states such as IBD, where down regulated
expression of ICACC-1 will be associated with the disease.
[0022] In a further embodiment, Applicants identify a disease
state, which can be treated through the up-regulation of ICACC-1.
Applicants provide a method for treating the defect in
cAMP-mediated chloride secretion in cystic fibrosis airway
epithelia by further increasing calcium-dependent chloride
secretion through up-regulation of ICACC-1. This up-regulation of
ICACC-1 resulting in increased chloride secretion and thus
restoration of the cellular chloride gradient resulting in normal
airway epithelial cell function. Applicants provide a method for
treating inflammatory bowel disease (IBD) with local delivery of
ICACC-1 via gene therapy or up regulation of ICACC-1 to enhance
TH2-associated responses for suppressing the TH1-associated IBD
autoimmune disease.
[0023] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principle of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows a schematic diagram of the suppressive PCR cDNA
subtraction technique.
[0025] FIG. 2 shows the nucleotide (SEQ ID NO:1) and amino acid
(SEQ ID NO:2) sequence of the murine ICACC-1 cDNA.
[0026] FIG. 3 shows an alignment of the murine ICACC-1 protein with
a bovine calcium activated chloride channel.
[0027] FIG. 4A shows the nucleotide (SEQ ID NO:3) and amino acid
(SEQ ID NO:4) sequence of the human ICACC-2 cDNA.
[0028] FIG. 4B shows the nucleotide (SEQ ID NO:5) and amino acid
(SEQ ID NO:6) sequence of the human ICACC-1 cDNA.
[0029] FIG. 5 shows an alignment of the murine ICACC-1 protein with
the human ICACC-1 and ICACC-2 protein.
[0030] FIG. 6 shows ICACC-1 expression in the lung of normal mice
(FVB) compared to transgenic mice overexpressing the IL-9 gene
(Tg5).
[0031] FIG. 7 shows the expression of ICACC-1 in the lungs of DBA
and C57B6 mice.
[0032] FIG. 8 shows the expression of ICACC-1 in the lung of the
C57B6 mouse with and without intratracheal administration of
IL-9.
[0033] FIG. 9 shows the expression of ICACC-1 in tissues from
normal (Balb/C) and IL-9 overexpressing (Tg5) mice.
[0034] FIG. 10 shows Aspergillus fumagatus-antigen induced BHR and
eosinophilia in Balb/C mice.
[0035] FIG. 11 shows the tissue distribution of ICACC-1 in naive
and antigen exposed Balb/C mice.
[0036] FIG. 12 shows the suppression of BHR and lung eosinophilia
by anti-IL-9 in mice exposed to Aspergillus fumagatus.
[0037] FIG. 13 shows suppression of ICACC-1 in antigen exposed
animals treated with anti-IL9.
[0038] FIG. 14 shows ICACC-1 induction by IL-9 in human primary
lung epithelial cells (NHBE).
[0039] FIG. 15 shows ICACC-1 induction by IL-9 in human primary
lung cultures.
[0040] FIG. 16 shows antisera generated against ICACC-1 peptides is
able to recognize native ICACC-1
[0041] FIG. 17 shows IL-9 induces eotaxin production from
epithelial cells in primary lung cultures
[0042] FIG. 18 shows suppression of IL-9 induced eotaxin by
chloride channel blockers
DETAILED DESCRIPTION OF THE INVENTION
[0043] The inventors have resolved a crucial need in the art by
elucidating critical genes in the IL-9 pathway and compositions
affecting that pathway which may be used in the diagnosis,
prevention or treatment of atopic allergy including asthma and
related disorders. Asthma encompasses inflammatory disorders of the
airways with reversible airflow obstruction. Atopic allergy refers
to atopy and related disorders including asthma, bronchial
hyperresponsiveness, rhinitis, urticaria, allergic inflammatory
disorders of the bowel and various forms of eczema. Atopy is a
hypersensitivity to environmental allergens expressed as the
elevation of serum total IgE or abnormal skin test responses to
allergens as compared to controls. Bronchial hyperresponsiveness is
a heightened broncho constrictor response to a variety of
stimuli.
A. ICACC Proteins
[0044] The present invention provides isolated ICACC protein,
allelic variants of the protein, and conservative amino acid
substitutions of the protein. As used herein, the is ICACC protein
or polypeptide includes a protein that has the murine amino acid
sequence of SEQ ID NO: 2 or the human amino acid sequence depicted
in SEQ ID No.4 or SEQ ID No.6. The invention includes naturally
occurring allelic variants and proteins that have a slightly
different amino acid sequence than that specifically recited above.
Allelic variants, though possessing a slightly different amino acid
sequence than those recited above, will still have the same or
similar biological functions associated with the ICACC protein.
[0045] As used herein, the family of proteins related to the ICACC
protein refer to proteins that have been isolated from organisms in
addition to humans or mice. The methods used to identify and
isolate other members of the family of proteins related to the
human and/or murine ICACC proteins are described below.
[0046] The proteins of the present invention are preferably in
isolated form. As used herein, a protein is said to be isolated
when physical, mechanical or chemical methods are employed to
remove the protein from cellular constituents that are normally
associated with the protein. A partially isolated protein, as used
herein, includes ICACC proteins isolated in membrane fragments,
including cellular membrane fragments containing a recombinantly
expressed ICACC protein. A skilled artisan can readily employ
standard purification methods to obtain an isolated protein.
[0047] The proteins of the present invention further include
conservative variants of the proteins herein described. As used
herein, a conservative variant refers to alterations in the amino
acid sequence that do not adversely affect the biological functions
of the protein. A substitution, insertion or deletion is said to
adversely affect the protein when the altered sequence prevents or
disrupts a biological function associated with the protein. For
example, the overall charge, structure or hydrophobic/hydrophilic
properties of the protein may be altered without adversely
affecting a biological activity. Accordingly, the amino acid
sequence can be altered, for example to render the peptide more
hydrophobic or hydrophilic, without adversely affecting the
biological activities of the protein.
[0048] Ordinarily, the allelic variants, the conservative
substitution variants, and the members of the protein family, will
have an amino acid sequence having at least about 55%, at least
about 75% amino acid sequence identity with the murine sequence set
forth in SEQ ID No.2 or the human sequences of SEQ ID NO: 4 or SEQ
ID No.6, more preferably at least 80%, even more preferably at
least 90%, and most preferably at least 95%. Identity or homology
with respect to such sequences is defined herein as the percentage
of amino acid residues in the candidate sequence that are identical
with the known peptides, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as
part of the sequence identity. N-terminal, C-terminal or internal
extensions, deletions, or insertions into the peptide sequence
shall not be construed as affecting homology.
[0049] Thus, the proteins of the present invention include
molecules having the amino acid sequence disclosed in SEQ ID Nos.
2, 4 or 6; fragments thereof having a consecutive sequence of at
least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid
residues of the ICACC protein; amino acid sequence variants of such
sequence wherein an amino acid residue has been inserted N- or
C-terminal to, or within, the disclosed sequence; and amino acid
sequence variants of the disclosed sequence, or their fragments as
defined above, that have been substituted by another residue.
Contemplated variants further include those containing
predetermined mutations by, e.g., homologous recombination,
site-directed or PCR mutagenesis, and the corresponding proteins of
other animal species, including but not limited to rabbit, rat,
porcine, bovine, ovine, equine and non-human primate species, and
the alleles or other naturally occurring variants of the family of
proteins; and derivatives wherein the protein has been covalently
modified by substitution, chemical, enzymatic, or other appropriate
means with a moiety other than a naturally occurring amino acid
(for example a detectable moiety such as an enzyme or
radioisotope).
[0050] As described below, members of the family of proteins can be
used: 1) to identify agents which modulate at least one activity of
the protein; 2) in methods of identifying binding partners for the
protein; and 3) as an antigen to raise polyclonal or monoclonal
antibodies.
B. Nucleic Acid Molecules
[0051] The murine ICACC-1 gene was identified by subtractive cDNA
cloning experiments that were performed in order to identify genes
specifically induced by IL-9. A schematic diagram of the
subtractive cDNA cloning method is provided in FIG. 1. RNA derived
from lungs of transgenic mice overexpressing the murine IL-9
transgene (Tg5) was used to isolate genes expressed in response to
IL-9 as opposed to those which are not expressed in the parental
strain (FVB). FIG. 6 shows a Northern blot with RNA from a lung of
a Tg5 mouse (right lane) and a FVB mouse (left lane) demonstrating
these findings. Expression of ICACC-1 was also observed in the lung
of the DBA murine strain which has been shown to express elevated
baseline IL-9 levels in their lungs (FIG. 7). ICACC-1 expression
was not observed in the lungs of the C57B6 strain where IL-9
expression is below the limits of detection (FIG. 7) (Nicolaides et
al., 1997). The direct effect of IL-9 on inducing ICACC-1
expression was demonstrated when IL-9 was instilled into the
trachea of the C57B6 mouse. The results of this experiment
demonstrated that ICACC-1 was expressed in the lungs of the IL-9
instilled mice but not in naive or vehicle treated mice (FIG. 8),
indicating that this gene is induced by IL-9. The results also show
that ICACC-1 gene expression is induced in the lung of antigen
exposed mice which exhibit asthmatic-like features (BHR, lung
eosinophilia) (FIGS. 10 and 12). The antigen induced BHR and lung
eosinophilia can be suppressed in mice by treatment with anti-IL9
(FIG. 12), which also results in down regulation of ICACC-1 (FIG.
13).
[0052] The murine ICACC-1 gene displayed significant homology
(.about.50%) with a member of the bovine calcium activated chloride
channel family (FIG. 3) (Cunningham et al., 1995). The full length
cDNA was cloned from a murine cDNA library (FIG. 2). Several EST
were identified which displayed partial homology to the murine
ICACC-1. These EST were obtained from the IMAGE consortium
(Lawrence Livermore National Laboratory) and sequenced. A full
length cDNA sequence was isolated for human ICACC-1 and 2 by
library screening and 5'- and 3' RACE cloning (Clonetech). Analysis
of the encoded murine protein sequence identified several conserved
motifs including multiple transmembrane domains and several
phosphorylation and glycosylation sites.
[0053] Expression of murine ICACC-1 was undetectable using standard
commercial tissue blots but elevated expression of ICACC-1 was
observed in lung, lymph node, colon, spleen, stomach, uterus and
ovary derived from IL-9 transgenic mice (FIG. 9). Interestingly,
these tissues all contain various epithelial cell types, suggesting
that this gene may be restricted to IL-9 responsive epithelial
cells. This data is supported by the finding that ICACC-1 gene
expression is induced in antigen exposed mice and this induction
can be suppressed by anti-IL9 treatment (FIGS. 10, 12, and 13).
[0054] To further understand which cell type is capable of
expressing ICACC-1, Applicants tested human lung epithelial cells
for their responsiveness to IL-9 as determined by ICACC-1 induced
gene expression. As shown in FIG. 14, the human lung epithelial
cell line designated NHBE (Clonetics), expressed ICACC-1 mRNA when
grown in the presence, but not in the absence of IL-9. When human
primary lung cultures were grown in the presence of recombinant
IL-9, ICACC-1 expression was induced in contrast to cell cultures
grown in medium alone (FIG. 15).
[0055] The nucleic acid molecules of the present invention include
nucleic acid molecules that encode the proteins having SEQ ID No.2,
SEQ ID No.4, SEQ ID NO: 6 and the related proteins herein
described, preferably in isolated form. As used herein, "nucleic
acid" is defined as RNA or DNA that encodes a protein or peptide as
defined above, or is complementary to nucleic acid sequence
encoding such peptides, or hybridizes to such nucleic acid and
remains stably bound to it under appropriate stringency conditions,
or encodes a polypeptide sharing at least 55% sequence identity,
75% sequence identity, preferably at least 80%, and more preferably
at least 85%, with the peptide sequences. Specifically contemplated
are genomic DNA, cDNA, mRNA and antisense molecules, as well as
nucleic acids based on alternative backbones or including
alternative bases whether derived from natural sources or
synthesized. Such hybridizing or complementary nucleic acids,
however, are defined further as being novel and unobvious over any
prior art nucleic acid including that which encodes, hybridizes
under appropriate stringency conditions, or is complementary to
nucleic acid encoding a protein according to the present
invention.
[0056] Homology or identity is determined by BLAST (Basic Local
Alignment Search Tool) analysis using the algorithm employed by the
programs blastp, blastn, blastx, tblastn and tblastx (Karlin, et
al. Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul,
S. F. J. Mol. Evol. 36: 290-300 (1993), fully incorporated by
reference) which are tailored for sequence similarity searching.
The approach used by the BLAST program is to first consider similar
segments between a query sequence and a database sequence, then to
evaluate the statistical significance of all matches that are
identified and finally to summarize only those matches which
satisfy a preselected threshold of significance. For a discussion
of basic issues in similarity searching of sequence databases, see
Altschul et al. (Nature Genetics 6: 119-129 (1994)) which is fully
incorporated by reference. The search parameters for histogram,
descriptions, alignments, expect (i.e., the statistical
significance threshold for reporting matches against database
sequences), cutoff, matrix and filter are at the default settings.
The default scoring matrix used by blastp, blastx, tblastn, and
tblastx is the BLOSUM62 matrix (Henikoff, et al. Proc. Natl. Acad.
Sci. USA 89: 10915-10919 (1992), fully incorporated by reference).
For blastn, the scoring matrix is set by the ratios of M (i.e., the
reward score for a pair of matching residues) to N (i.e., the
penalty score for mismatching residues), wherein the default values
for M and N are 5 and -4, respectively.
[0057] "Stringent conditions" are those that (1) employ low ionic
strength and high temperature for washing, for example, 0.015M
NaCl/0.0015M sodium titrate/0.1% SDS at 50.degree. C., or (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C. Another example is use of 50% formamide,
5.times.SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's
solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and
10% dextran sulfate at 42.degree. C., with washes at 42.degree. C.
in 0.2.times.SSC and 0.1% SDS. A skilled artisan can readily
determine and vary the stringency conditions appropriately to
obtain a clear and detectable hybridization signal.
[0058] As used herein, a nucleic acid molecule is said to be
"isolated" when the nucleic acid molecule is substantially
separated from contaminant nucleic acid encoding other polypeptides
from the source of nucleic acid.
[0059] The present invention further provides fragments of the
encoding nucleic acid molecule. As used herein, a fragment of an
encoding nucleic acid molecule refers to a small portion of the
entire protein encoding sequence. The size of the fragment will be
determined by the intended use. For example, if the fragment is
chosen so as to encode an active portion of the protein, the
fragment will need to be large enough to encode the functional
region(s) of the protein or may encode regions of homology between
the ICACC proteins in FIG. 5. If the fragment is to be used as a
nucleic acid probe or PCR primer, then the fragment length is
chosen so as to obtain a relatively small number of false positives
during probing/priming.
[0060] Fragments of the encoding nucleic acid molecules of the
present invention (i.e., synthetic oligonucleotides) that are used
as probes or specific primers for the polymerase chain reaction
(PCR), or to synthesize gene sequences encoding proteins of the
invention can easily be synthesized by chemical techniques, for
example, the phosphotriester method of Matteucci, et al., (J. Am.
Chem. Soc. 103:3185-3191, 1981) or using automated synthesis
methods. In addition, larger DNA segments can readily be prepared
by well known methods, such as synthesis of a group of
oligonucleotides that define various modular segments of the gene,
followed by ligation of oligonucleotides to build the complete
modified gene.
[0061] The encoding nucleic acid molecules of the present invention
may further be modified so as to contain a detectable label for
diagnostic and probe purposes. A variety of such labels are known
in the art and can readily be employed with the encoding molecules
herein described. Suitable labels include, but are not limited to,
biotin, radiolabeled nucleotides and the like. A skilled artisan
can employ any of the art known labels to obtain a labeled encoding
nucleic acid molecule.
[0062] Modifications to the primary structure itself by deletion,
addition, or alteration of the amino acids incorporated into the
protein sequence during translation can be made without destroying
the activity of the protein. Such substitutions or other
alterations result in proteins having an amino acid sequence
encoded by a nucleic acid falling within the contemplated scope of
the present invention.
C. Isolation of Other Related Nucleic Acid Molecules
[0063] As described above, the identification of the murine nucleic
acid molecule having SEQ ID NO:1 and the human nucleic acid
molecules having SEQ ID No.3 or SEQ ID No 5 allows a skilled
artisan to isolate nucleic acid molecules that encode other members
of the ICACC protein family in addition to the murine or human
sequences herein described.
[0064] Essentially, a skilled artisan can readily use the amino
acid sequence of SEQ ID NOS: 2, 4 or 6 to generate antibody probes
to screen expression libraries prepared from appropriate cells.
Typically, polyclonal antiserum from mammals such as rabbits
immunized with the purified protein (as described below) or
monoclonal antibodies can be used to probe a mammalian cDNA or
genomic expression library, such as lambda gtII library, to obtain
the appropriate coding sequence for other members of the protein
family. The cloned cDNA sequence can be expressed as a fusion
protein, expressed directly using its own control sequences, or
expressed by constructions using control sequences appropriate to
the particular host used for expression of the enzyme.
[0065] Alternatively, a portion of the coding sequence herein
described can be synthesized and used as a probe to retrieve DNA
encoding a member of the protein family from any mammalian
organism. Oligomers containing approximately 18-20 nucleotides
(encoding about a 6-7 amino acid stretch) are prepared and used to
screen genomic DNA or cDNA libraries to obtain hybridization under
stringent conditions or conditions of sufficient stringency to
eliminate an undue level of false positives.
[0066] Additionally, pairs of oligonucleotide primers can be
prepared for use in a polymerase chain reaction (PCR) to
selectively clone an encoding nucleic acid molecule. A PCR
denature/anneal/extend cycle for using such PCR primers is well
known in the art and can readily be adapted for use in isolating
other encoding nucleic acid molecules.
D. rDNA Molecules Containing a Nucleic Acid Molecule
[0067] The present invention further provides recombinant DNA
molecules (rDNAs) that contain a ICACC coding sequence. As used
herein, a rDNA molecule is a DNA molecule that has been subjected
to molecular manipulation in situ. Methods for generating rDNA
molecules are well known in the art, for example, see Sambrook et
al., Molecular Cloning (1989). In the preferred rDNA molecules, a
coding DNA sequence is operably linked to expression control
sequences and/or vector sequences.
[0068] The choice of vector and/or expression control sequences to
which one of the protein family encoding sequences of the present
invention is operably linked depends directly, as is well known in
the art, on the functional properties desired, e.g., protein
expression, and the host cell to be transformed. A vector
contemplated by the present invention is at least capable of
directing the replication or insertion into the host chromosome,
and preferably also expression, of the structural gene included in
the rDNA molecule.
[0069] Expression control elements that are used for regulating the
expression of an operably linked protein encoding sequence are
known in the art and include, but are not limited to, inducible
promoters, constitutive promoters, secretion signals, and other
regulatory elements. Preferably, the inducible promoter is readily
controlled, such as being responsive to a nutrient in the host
cell's medium.
[0070] In one embodiment, the vector containing a coding nucleic
acid molecule will include a prokaryotic replicon, i.e., a DNA
sequence having the ability to direct autonomous replication and
maintenance of the recombinant DNA molecule extrachromosomally in a
prokaryotic host cell, such as a bacterial host cell, transformed
therewith. Such replicons are well known in the art. In addition,
vectors that include a prokaryotic replicon may also include a gene
whose expression confers a detectable marker such as a drug
resistance. Typical bacterial drug resistance genes are those that
confer resistance to ampicillin or tetracycline.
[0071] Vectors that include a prokaryotic replicon can further
include a prokaryotic or bacteriophage promoter capable of
directing the expression (transcription and translation) of the
coding gene sequences in a bacterial host cell, such as E. coli. A
promoter is an expression control element formed by a DNA sequence
that permits binding of RNA polymerase and transcription to occur.
Promoter sequences compatible with bacterial hosts are typically
provided in plasmid vectors containing convenient restriction sites
for insertion of a DNA segment of the present invention. Typical of
such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available
from Biorad Laboratories, (Richmond, Calif.), pPL and pKK223
available from Pharmacia, Piscataway, N.J.
[0072] Expression vectors compatible with eukaryotic cells,
preferably those compatible with vertebrate cells, can also be used
to form a rDNA molecules that contains a coding sequence.
Eukaryotic cell expression vectors are well known in the art and
are available from several commercial sources. Typically, such
vectors are provided containing convenient restriction sites for
insertion of the desired DNA segment. Typical of such vectors are
pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International
Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the vector pCDM8
described herein, and the like eukaryotic expression vectors.
[0073] Eukaryotic cell expression vectors used to construct the
rDNA molecules of the present invention may further include a
selectable marker that is effective in an eukaryotic cell,
preferably a drug resistance selection marker. A preferred drug
resistance marker is the gene whose expression results in neomycin
resistance, i.e., the neomycin phosphotransferase (neo) gene.
(Southern et al., J. Mol. Anal. Genet. 1:327-341, 1982.)
Alternatively, the selectable marker can be present on a separate
plasmid, and the two vectors are introduced by co-transfection of
the host cell, and selected by culturing in the appropriate drug
for the selectable marker.
E. Host Cells Containing an Exogenously Supplied Coding Nucleic
Acid Molecule
[0074] The present invention further provides host cells
transformed with a nucleic acid molecule that encodes an ICACC
protein, preferably an ICACC-1 protein, of the present invention.
The host cell can be either prokaryotic or eukaryotic. Eukaryotic
cells useful for expression of a protein of the invention are not
limited, so long as the cell line is compatible with cell culture
methods and compatible with the propagation of the expression
vector and expression of the gene product. Preferred eukaryotic
host cells include, but are not limited to, yeast, insect and
mammalian cells, preferably vertebrate cells such as those from a
mouse, rat, monkey or human cell line. Preferred eukaryotic host
cells include Chinese hamster ovary (CHO) cells available from the
ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from
the ATCC as CRL 1658, baby hamster kidney cells (BHK), and the like
eukaryotic tissue culture cell lines.
Any prokaryotic host can be used to express a rDNA molecule
encoding a protein of the invention. The preferred prokaryotic host
is E. coli.
[0075] Transformation of appropriate cell hosts with a rDNA
molecule of the present invention is accomplished by well known
methods that typically depend on the type of vector used and host
system employed. With regard to transformation of prokaryotic host
cells, electroporation and salt treatment methods are typically
employed, see, for example, Cohen et al., Proc. Natl. Acad. Sci.
USA 69:2110, 1972; and Maniatis et al., Molecular Cloning. A
Laboratory Mammal, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1982). With regard to transformation of vertebrate
cells with vectors containing rDNAs, electroporation, cationic
lipid or salt treatment methods are typically employed, see, for
example, Graham et al., Virol. 52:456, 1973; Wigler et al., Proc.
Natl. Acad. Sci. USA 76:1373-76, 1979.
[0076] Successfully transformed cells, i.e., cells that contain a
rDNA molecule of the present invention, can be identified by well
known techniques including the selection for a selectable marker.
For example, cells resulting from the introduction of an rDNA of
the present invention can be cloned to produce single colonies.
Cells from those colonies can be harvested, lysed and their DNA
content examined for the presence of the rDNA using a method such
as that described by Southern, J. Mol. Biol. 98:503, 1975, or
Berent et al., Biotech. 3:208, 1985 or the proteins produced from
the cell assayed via an immunological method.
F. Production of Recombinant Proteins Using a rDNA Molecule
[0077] The present invention further provides methods for producing
an ICACC protein of the invention using nucleic acid molecules
herein described. In general terms, the production of a recombinant
form of a protein typically involves the following steps:
[0078] First, a nucleic acid molecule is obtained that encodes a
protein of the invention, such as the nucleic acid molecule
depicted in SEQ ID NOS.1, 3 or 5 or the open reading frames of
these molecules. If the encoding sequence is uninterrupted by
introns, it is directly suitable for expression in any host.
[0079] The nucleic acid molecule is then preferably placed in
operable linkage with suitable control sequences, as described
above, to form an expression unit containing the protein open
reading frame. The expression unit is used to transform a suitable
host and the transformed host is cultured under conditions that
allow the production of the recombinant protein. Optionally the
recombinant protein is isolated from the medium or from the cells;
recovery and purification of the protein may not be necessary in
some instances where some impurities may be tolerated.
[0080] Each of the foregoing steps can be done in a variety of
ways. For example, the desired coding sequences may be obtained
from genomic fragments and used directly in appropriate hosts. The
construction of expression vectors that are operable in a variety
of hosts is accomplished using appropriate replicons and control
sequences, as set forth above. The control sequences, expression
vectors, and transformation methods are dependent on the type of
host cell used to express the gene and were discussed in detail
earlier. Suitable restriction sites can, if not normally available,
be added to the ends of the coding sequence so as to provide an
excisable gene to insert into these vectors. A skilled artisan can
readily adapt any host/expression system known in the art for use
with the nucleic acid molecules of the invention to produce
recombinant protein.
G. Methods to Identify Binding Partners
[0081] Another embodiment of the present invention provides methods
for use in isolating and identifying binding partners of proteins
of the invention In detail, a protein of the invention is mixed
with a potential binding partner or an extract or fraction of a
cell under conditions that allow the association of potential
binding partners with the protein of the invention. After mixing,
peptides, polypeptides, proteins or other molecules that have
become associated with a protein of the invention are separated
from the mixture. The binding partner that bound to the protein of
the invention can then be removed and further analyzed. To identify
and isolate a binding partner, the entire protein, for instance a
ICACC protein of SEQ ID No.2, SEQ ID No. 4 or SEQ ID NO: 6 can be
used. Alternatively, a fragment of the protein or a membrane
fragment containing the protein may be used.
[0082] As used herein, a cellular extract refers to a preparation
or fraction which is made from a lysed or disrupted cell. The
preferred source of cellular extracts are cells derived from human
tissues or cells.
[0083] A variety of methods can be used to obtain an extract of a
cell. Cells can be disrupted using either physical or chemical
disruption methods. Examples of physical disruption methods
include, but are not limited to, sonication and mechanical
shearing. Examples of chemical lysis methods include, but are not
limited to, detergent lysis and enzyme lysis. A skilled artisan can
readily adapt methods for preparing cellular extracts in order to
obtain extracts for use in the present methods.
[0084] Once an extract of a cell is prepared, the extract is mixed
with the protein of the invention under conditions in which
association of the protein with the binding partner can occur. A
variety of conditions can be used, the most preferred being
conditions that closely resemble conditions found in the cytoplasm
of a human cell. Features such as osmolarity, pH, temperature, and
the concentration of cellular extract used, can be varied to
optimize the association of the protein with the binding
partner.
[0085] After mixing under appropriate conditions, the bound complex
is separated from the mixture. A variety of techniques can be
utilized to separate the mixture. For example, antibodies specific
to a protein of the invention can be used to immunoprecipitate the
binding partner complex. Alternatively, standard chemical
separation techniques such as chromatography and density/sediment
centrifugation can be used.
[0086] After removal of non-associated cellular constituents found
in the extract, the binding partner can be dissociated from the
complex using conventional methods. For example, dissociation can
be accomplished by altering the salt concentration or pH of the
mixture. To aid in separating associated binding partner pairs from
the mixed extract, the protein of the invention can be immobilized
on a solid support. For example, the protein can be attached to a
nitrocellulose matrix or acrylic beads. Attachment of the protein
to a solid support aids in separating peptide/binding partner pairs
from other constituents found in the extract. The identified
binding partners can be either a single protein or a complex made
up of two or more proteins. Alternatively, binding partners may be
identified using a Far-Western assay according to the procedures of
Takayama et al. (1997) Methods Mol. Biol. 69:171-84 or Sauder et
al. J. Gen. Virol. 77(5):991-6 or identified through the use of
epitope tagged proteins or GST fusion proteins.
[0087] Alternatively, the nucleic acid molecules of the invention
can be used in a yeast two-hybrid system, preferably systems for
screening binding partners of membrane proteins. The yeast
two-hybrid system has been used to identify other protein partner
pairs and can readily be adapted to employ the nucleic acid
molecules herein described.
H. Methods to Identify Agents that Modulate the Expression a
Nucleic Acid Encoding and ICACC Protein.
[0088] Another embodiment of the present invention provides methods
for identifying agents that modulate the expression of a nucleic
acid encoding a protein of the invention such as a protein having
the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6. Such assays
may utilize any available means of monitoring for changes in the
expression level of the nucleic acids of the invention. As used
herein, an agent is said to modulate the expression of a nucleic
acid of the invention, for instance a nucleic acid encoding the
protein having the sequence of SEQ ID NO:2 or SEQ ID NO:6 if it is
capable of up- or down-regulating expression of the nucleic acid in
a cell.
[0089] Agents of the invention may relate to antisense or gene
therapy. It is now known in the art that altered DNA molecules can
be tailored to provide a selected effect, when provided as
antisense or gene therapy. The native DNA segment coding for
ICACC-1 has two strands; a sense strand and an antisense strand
held together by hydrogen bonds. The mRNA coding for the receptor
has a nucleotide sequence identical to the sense strand, with the
expected substitution of thymidine by uridine. Thus, based upon the
knowledge of the receptor sequence, synthetic oligonucleotides can
be synthesized. These oligonucleotides can bind to the DNA and RNA
coding for ICACC-1. The active fragments of the invention, which
are complementary to mRNA and the coding strand of DNA, are usually
at least about 15 nucleotides, more usually at least 20
nucleotides, preferably 30 nucleotides and more preferably may be
50 nucleotides or more. The binding strength between the sense and
antisense stands is dependent upon the total hydrogen bonds.
Therefore, based upon the total number of bases in the mRNA, the
optimal length of the oligonucleotide sequence may be easily
calculated by the skilled artisan.
[0090] The sequence may be complementary to any portion of the
sequence of the mRNA. For example, it may be proximal to the
5'-terminus or capping site or downstream from the capping site,
between the capping site and the initiation codon and may cover all
or only a portion of the non-coding region or the coding region.
The particular site(s) to which the antisense sequence binds will
vary depending upon the degree of inhibition desired, the
uniqueness of the sequence, the stability of the antisense
sequence, etc.
[0091] In the practice of the invention, expression of ICACC-1 is
down-regulated by administering an effective amount of antisense
oligonucleotide sequences described above. The oligonucleotide
compounds of the invention bind to the mRNA coding for human
ICACC-1 thereby inhibiting expression (translation) of these
proteins. The isolated DNA sequences, containing various mutations
such as point mutations, insertions, deletions or spliced mutations
of ICACC-1 are useful in gene therapy as well.
[0092] In one assay format for agents, cell lines that contain
reporter gene fusions between the open reading frame and any
assayable fusion partner may be prepared. Numerous assayable fusion
partners are known and readily available including the firefly
luciferase gene and the gene encoding chloramphenicol
acetyltransferase (Alam et al. (1990) Anal. Biochem. 188:245-254).
Cell lines containing the reporter gene fusions are then exposed to
the agent to be tested under appropriate conditions and time.
Differential expression of the reporter gene between samples
exposed to the agent and control samples identifies agents which
modulate the expression of a nucleic acid encoding an ICACC-1
protein.
[0093] Additional assay formats may be used to monitor the ability
of the agent to modulate the expression of a nucleic acid encoding
a protein of the invention, such as the protein having SEQ ID NO:2
or SEQ ID NO:6. For instance, mRNA expression may be monitored
directly by hybridization to the nucleic acids of the invention.
Cell lines are exposed to the agent to be tested under appropriate
conditions and time and total RNA or mRNA is isolated by standard
procedures such those disclosed in Sambrook et al. (Molecular
Cloning: A Laboratory Manual, 2nd Ed. Clod Spring Harbor Laboratory
Press, 1989).
Probes to detect differences in RNA expression levels between cells
exposed to the agent and control cells may be prepared from the
nucleic acids of the invention. It is preferable, but not
necessary, to design probes which hybridize only with target
nucleic acids under conditions of high stringency. Only highly
complementary nucleic acid hybrids form under conditions of high
stringency. Accordingly, the stringency of the assay conditions
determines the amount of complementarity which should exist between
two nucleic acid strands in order to form a hybrid. Stringency
should be chosen to maximize the difference in stability between
the probe:target hybrid and potential probe:non-target hybrids.
[0094] Probes may be designed from the nucleic acids of the
invention through methods known in the art. For instance, the G+C
content of the probe and the probe length can affect probe binding
to its target sequence. Methods to optimize probe specificity are
commonly available in Sambrook et al. (Molecular Cloning: A
Laboratory Approach, Cold Spring Harbor Press, NY, 1989) or Ausubel
et al. (Current Protocols in Molecular Biology, Greene Publishing
Co., NY, 1995).
[0095] Hybridization conditions are modified using known methods,
such as those described by Sambrook et al. and Ausubel et al. as
required for each probe. Hybridization of total cellular RNA or RNA
enriched for polyA RNA can be accomplished in any available format.
For instance, total cellular RNA or RNA enriched for polyA RNA can
be affixed to a solid support and the solid support exposed to at
least one probe comprising at least one, or part of one of the
sequences of the invention under conditions in which the probe will
specifically hybridize. Alternatively, nucleic acid fragments
comprising at least one, or part of one of the sequences of the
invention can be affixed to a solid support, such as a porous glass
wafer. The glass wafer can then be exposed to total cellular RNA or
polyA RNA from a sample under conditions in which the affixed
sequences will specifically hybridize. Such glass wafers and
hybridization methods are widely available, for example, those
disclosed by Beattie (WO 95/11755). By examining for the ability of
a given probe to specifically hybridize to an RNA sample from an
untreated cell population and from a cell population exposed to the
agent, agents, which up or down regulate the expression of a
nucleic acid encoding an ICACC protein, preferably an ICACC-1
protein, are identified.
[0096] Hybridization for qualitative and quantitative analysis of
mRNAs may also be carried out by using a RNase Protection Assay
(i.e., RPA, see Ma et al. (1996) Methods 10: 273-238). Briefly, an
expression vehicle comprising cDNA encoding the gene product and a
phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3
or SP6 RNA polymerase) is linearized at the 3' end of the cDNA
molecule, downstream from the phage promoter, wherein such a
linearized molecule is subsequently used as a template for
synthesis of a labeled antisense transcript of the cDNA by in vitro
transcription. The labeled transcript is then hybridized to a
mixture of isolated RNA (i.e., total or fractionated mRNA) by
incubation at 45.degree. C. overnight in a buffer comprising 80%
formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The
resulting hybrids are then digested in a buffer comprising 40
.mu.g/ml ribonuclease A and 2 .mu.g/ml ribonuclease. After
deactivation and extraction of extraneous proteins, the samples are
loaded onto urea/polyacrylamide gels for analysis.
[0097] In another assay format for agents which effect the
expression of the instant gene products, cells or cell lines would
first be identified which express said gene products
physiologically (e.g., see the Figures for tissue distribution).
Cell and/or cell lines so identified would be expected to comprise
the necessary cellular machinery such that the fidelity of
modulation of the transcriptional apparatus is maintained with
regard to exogenous contact of agent with appropriate surface
transduction mechanisms and/or the cytosolic cascades. Further,
such cells or cell lines would be transduced or transfected with an
expression vehicle (e.g., a plasmid or viral vector) construct
comprising an operable non-translated 5'-promoter containing end of
the structural gene encoding the instant gene products fused to one
or more antigenic fragments, which are peculiar to the instant gene
products, wherein said fragments are under the transcriptional
control of said promoter and are expressed as polypeptides whose
molecular weight can be distinguished from the naturally occurring
polypeptides or may further comprise an immunologically distinct
tag. Such a process is well known in the art (see Maniatis). Cells
may be exposed to IL-9.
[0098] Cells or cell lines transduced or transfected as outlined
above would then be contacted with agents under appropriate
conditions; for example, the agent comprises a pharmaceutically
acceptable excipient and is contacted with cells comprised in an
aqueous physiological buffer such as phosphate buffered saline
(PBS) at physiological pH, Eagles balanced salt solution (BSS) at
physiological pH, PBS or BSS comprising serum or conditioned media
comprising PBS or BSS and/or serum incubated at 37.degree. C. Said
conditions may be modulated as deemed necessary by one of skill in
the art. Subsequent to contacting the cells with the agent, said
cells will be disrupted and the polypeptides of the disruptate are
fractionated such that a polypeptide fraction is pooled and
contacted with an antibody to be further processed by immunological
assay (e.g., ELISA, immunoprecipitation or Western blot). The pool
of proteins isolated from the "agent contacted" sample will be
compared with a control sample where only the excipient is
contacted with the cells and an increase or decrease in the
immunologically generated signal from the "agent contacted" sample
compared to the control will be used to distinguish the
effectiveness of the agent.
I. Methods to Identify Agents that Modulate at Least One Activity
of an ICACC Protein.
[0099] Another embodiment of the present invention provides methods
for identifying agents that modulate at least one activity of a
protein of the invention, such as a protein having the amino acid
sequence of SEQ ID No.2, SEQ ID NO:4 or SEQ ID No.6 and preferably,
an ICACC-1 protein. Such methods or assays may utilize any means of
monitoring or detecting the desired activity.
[0100] Specific assays may be based on monitoring the cellular
functions of ICACC-1. Antagonists of the invention include those
molecules that interact or bind to ICACC-1 and inactivate this
receptor. To identify other allosteric, inverse or weak antagonists
of the invention, one may test for binding to ICACC-1. The present
invention includes antagonists of ICACC-1 that block activation of
this receptor. Antagonists are compounds that are themselves devoid
of pharmacological activity but cause effects by preventing the
action of an agonist. To identify an antagonist of the invention,
one may test for competitive binding with natural ligands of
ICACC-1. Assays of antagonistic binding and activity can be derived
from monitoring ICACC-1 functions for down-regulation as described
herein and in the cited literature. The binding of the antagonist
may involve all known types of interactions including ionic forces,
hydrogen bonding, hydrophobic interactions, van der Waals forces
and covalent bonds. In many cases, bonds of multiple types are
important in the interaction of an agonist or antagonist with a
molecule like ICACC-1.
[0101] In a further embodiment, these compounds may be analogues of
ICACC-1 or its ligands. ICACC-1 analogues may be produced by point
mutations in the isolated DNA sequence for the gene, nucleotide
substitutions and/or deletions which can be created by methods that
are all well described in the art (Simoncsits et al., 1994). This
invention also includes spliced variants of ICACC-1 and discloses
isolated nucleic acid sequences of ICACC-1, which contain deletions
of one or more of its exons. The term "spliced variants" as used
herein denotes a purified and isolated DNA molecule encoding human
ICACC-1 comprising at least one exon. There is no evidence of
naturally expressed spliced mutants in the art. It must be
understood that these exons may contain various point
mutations.
[0102] Structure-activity relationships may be used to modify the
antagonists of the invention. For example, the techniques of X-ray
crystallography and NMR may be used to make modifications of the
invention. For example, one can create a three-dimensional
structure of human ICACC-1 that can be used as a template for
building structural models of deletion mutants using molecular
graphics. These models can then be used to identify and construct a
ligand for ICACC-1 which alters normal chloride channel function.
In still another embodiment, these compounds may also be used as
dynamic probes for ICACC-1 structure and to develop ICACC-1
antagonists using cell lines or other suitable means of assaying
ICACC-1 activity.
[0103] In addition, this invention also provides compounds that
prevent the synthesis or reduce the biologic stability of ICACC-1.
Biologic stability is a measure of the time between the synthesis
of the molecule and its degradation. For example, the stability of
a protein, peptide or peptide mimetic (Kauvar, 1996) therapeutic
may be prolonged by using D-amino acids or shortened by altering
its sequence to make it more susceptible to enzymatic
degradation.
[0104] In another embodiment, antagonists of the invention are
antibodies to ICACC-1. The antibodies to ICACC-1 may be either
monoclonal or polyclonal, made using standard techniques well known
in the art (See Harlow & Lane's Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1988). They can be
used to block ICACC-1 activation by binding to extracellular
regions of the protein required for ligand binding or activation.
In one embodiment, the antibodies interact with ICACC-1, in another
they interact with the ligands for ICACC-1. The ICACC-1 used to
elicit these antibodies can be the ICACC-1 protein or any of the
ICACC-1 variants or fragments discussed above. Antibodies are also
produced from peptide sequences of ICACC-1 using standard
techniques in the art (see Protocols in Immunology, John Wiley
& Sons, 1994).
[0105] In one assay format, the relative amounts of ICACC-1 protein
of the invention between a cell population that has been exposed to
the agent to be tested compared to an un-exposed control cell
population may be assayed. In this format, probes such as specific
antibodies are used to monitor the differential expression of the
protein in the different cell populations. Cell lines or
populations are exposed to the agent to be tested under appropriate
conditions and time. Cellular lysates may be prepared from the
exposed cell line or population and a control, unexposed cell line
or population. The cellular lysates are then analyzed with the
probe.
[0106] Antibody probes are prepared by immunizing suitable
mammalian hosts in appropriate immunization protocols using the
peptides. polypeptides or proteins of the invention if they are of
sufficient length, or, if desired, or if required to enhance
immunogenicity, conjugated to suitable carriers. Methods for
preparing immunogenic conjugates with carriers such as BSA, KLH, or
other carrier proteins are well known in the art. In some
circumstances, direct conjugation using, for example, carbodiimide
reagents may be effective; in other instances linking reagents such
as those supplied by Pierce Chemical Co., Rockford, Ill., may be
desirable to provide accessibility to the hapten. The hapten
peptides can be extended at either the amino or carboxy terminus
with a Cys residue or interspersed with cysteine residues, for
example, to facilitate linking to a carrier. Administration of the
immunogens is conducted generally by injection over a suitable time
period and with use of suitable adjuvants, as is generally
understood in the art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
[0107] While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein or modifications which effect immortaization of
lymphocytes or spleen cells, as is generally known. The
immortalized cell lines secreting the desired antibodies are
screened by immunoassay in which the antigen is the peptide hapten.
polypeptide or protein. When the appropriate immortalized cell
culture secreting the desired antibody is identified, the cells can
be cultured either in vitro or by production in ascites fluid.
[0108] The desired monoclonal antibodies are then recovered from
the culture supernatant or from the ascites supernatant. Fragments
of the monoclonals or the polyclonal antisera which contain the
immunologically significant portion can be used as antagonists, as
well as the intact antibodies. Use of immunologically reactive
fragments, such as the Fab, Fab', of F(ab').sub.2 fragments is
often preferable, especially in a therapeutic context, as these
fragments are generally less immunogenic than the whole
immunoglobulin.
[0109] The antibodies or fragments may also be produced, using
current technology, by recombinant means. Antibody regions that
bind specifically to the desired regions of the protein can also be
produced in the context of chimeras with multiple species origin.
Agents that are assayed in the above method can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of the a protein of the invention alone or with its
associated substrates, binding partners, etc. An example of
randomly selected agents is the use a chemical library or a peptide
combinatorial library, or a growth broth of an organism.
[0110] As used herein, an agent is said to be rationally selected
or designed when the agent is chosen on a nonrandom basis which
takes into account the sequence of the target site and/or its
conformation in connection with the agent's action.
[0111] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. A skilled artisan can readily recognize that there
is no limit as to the structural nature of the agents of the
present invention.
The peptide agents of the invention can be prepared using standard
solid phase (or solution phase) peptide synthesis methods, as is
known in the art. In addition, the DNA encoding these peptides may
be synthesized using commercially available oligonucleotide
synthesis instrumentation and produced recombinantly using standard
recombinant production systems. The production using solid phase
peptide synthesis is necessitated if non-gene-encoded amino acids
are to be included.
[0112] Another class of agents of the present invention are
antibodies immunoreactive with critical positions of proteins of
the invention. Antibody agents are obtained by immunization of
suitable mammalian subjects with peptides, containing as antigenic
regions, those portions of the protein intended to be targeted by
the antibodies.
J. Uses for Agents that Modulate at Least One Activity of an ICACC
Protein.
[0113] Further evidence defining the role of ICACC-1 in the
pathogenesis of atopic allergy, bronchial hyperresponsiveness,
asthma and related disorders is derived directly from the
Applicants observation that IL-9 selectively induces ICACC-1. Thus,
the pleiotropic role for IL-9, which is important to a number of
antigen induced responses is dependent in part, on the
up-regulation of ICACC-1 in cells critical to atopic allergy. When
the functions of IL-9 are down-regulated by antibody pretreatment
prior to aerosol challenge with antigen, animals can be completely
protected from the antigen induced responses. These responses
include: bronchial hyperresponsiveness, eosinophilia and elevated
cell counts in bronchial lavage, histologic changes in lung
associated with inflammation and elevated serum IgE. The
suppression of IL-9 and asthmatic-like responses is associated with
down regulated expression of ICACC-1 (FIG. 13). Thus, treatment of
such responses, which underlie the pathogenesis of atopic allergy
and characterize allergic inflammation associated with this
disorder, by down-regulating ICACC-1, is within the scope of this
invention.
[0114] The involvement of chloride channels in IL-9 biologic
responses is addressed by in vitro primary lung cultures that
produce secreted eotaxin protein upon IL-9 stimulation (FIG. 17).
The treatment of these cultures with known chloride channel
inhibitors results in suppression of the IL-9 induced eotaxin
response (FIG. 18) and thus provides an assay for screening for
ICACC-1 inhibitors. In another embodiment cell lines in which
ICACC-1 expression vectors are introduced can be used to screen for
specific chloride channel inhibitors.
[0115] Applicants also teach the down-regulation of ICACC-1 by
administering antagonists of ICACC-1. The skilled artisan will
recognize that all molecules containing the requisite
three-dimensional structural conformation critical for activation
of, or ligand binding to ICACC-1 are within the scope of this
invention.
[0116] The demonstration of an IL-9 sequence associated with an
asthma-like phenotype and one associated with the absence of an
asthma-like phenotype, indicates that the inflammatory response to
antigen in the lung is IL-9 dependent. Down-regulating ICACC-1,
which is selectively induced downstream in the IL-9 pathway, will
therefore protect against this antigen induced response.
[0117] In addition to the direct regulation of the ICACC-1 gene,
this invention also encompasses methods of inhibiting the
intracellular signaling by ICACC-1. It is known in the art that
highly exergonic phosphoryl-transfer reactions are catalyzed by
various enzymes known as kinases. In other words, a kinase
transfers phosphoryl groups between ATP and a metabolite. Included
within the scope of this invention are specific inhibitors of
protein kinases. Thus, inhibitors of these kinases are useful in
the down-regulation of ICACC-1 and are therefore useful in the
treatment of atopic allergies and asthma.
[0118] In still another aspect of the invention, surprisingly,
aminosterol compounds were found to be useful in the inhibition of
ICACC-1 induction by IL-9. Aminosterol compounds which are useful
in this invention are described in U.S. patent application Ser. No.
08/290,826 and its related application Ser. Nos. 08/416,883 and
08/478,763 as well as in 08/483,059 and its related application
Ser. Nos. 08/483,057, 08/479,455, 08/479,457, 08/475,572,
08/476,855, 08/474,799 and 08/487,443, which are specifically
incorporated herein by reference in their entirety.
[0119] While a therapeutic potential for ICACC-1 down-regulation
has been identified, Applicants have also recognized a therapeutic
potential for up-regulation of ICACC-1 as well. Patients with
cystic fibrosis are hampered by lung disease characterized by thick
secretions, which cause airway obstruction and subsequent
colonization and infection by inhaled pathogenic microorganisms
(Eng et al., 1996). Airway epithelia from cystic fibrosis patients
exhibit a broad spectrum of ion transport properties that differ
from normal, including not only defective cAMP-mediated chloride
secretion, but also increased sodium absorption and increased
calcium-mediated chloride secretion (Johnson et al., 1995).
Restoration of overall chloride secretion in primary cystic
fibrosis airway epithelial cells leads to correction of sodium
hyperabsorption and normal airway epithelial cell function (Johnson
et al., 1995). Applicants therefore provide a method for treating
cystic fibrosis by further increasing calcium-dependent chloride
secretion in these cells through up-regulation of ICACC-1 activity
in airway epithelia. In this manner, the decrease in chloride
secretion due to the defect in cAMP-mediated chloride secretion is
compensated for through up-regulation of ICACC-1. The result being
a restoration of the cellular chloride gradient and normal airway
epithelial cell function. In another indication, up regulation of
ICACC-1 will be useful for treating autoimmune associated diseases
such as IBD.
[0120] As provided in the Examples, the proteins and nucleic acids
of the invention, such as the ICACC-1 proteins having the amino
acid sequence of SEQ ID NOS: 2 or 6, are induced by IL-9. Agents
that modulate or down-regulate the expression of the protein or
agents such as agonists or antagonists of at least one activity of
the protein may be used to modulate biological and pathologic
processes associated with the protein's function and activity. As
used herein, a subject can be any mammal, so long as the mammal is
in need of modulation of a pathological or biological process
mediated by a protein of the invention.
[0121] The term "mammal" is meant an individual belonging to the
class Mammalia. The invention is particularly useful in the
treatment of human subjects.
[0122] Pathological processes refer to a category of biological
processes which produce a deleterious effect. For example,
expression of a protein of the invention may be associated with
atopic allergy, asthma and/or cystic fibrosis. As used herein, an
agent is said to modulate a pathological process when the agent
reduces the degree or severity of the process. For instance, atopic
allergy, asthma and/or cystic fibrosis may be prevented or disease
progression modulated by the administration of agents which reduce
or modulate in some way the expression or at least one activity of
a protein of the invention.
[0123] The agents of the present invention can be provided alone,
or in combination with other agents that modulate a particular
pathological process. For example, an agent of the present
invention can be administered in combination with anti-asthma
agents. As used herein, two agents are said to be administered in
combination when the two agents are administered simultaneously or
are administered independently in a fashion such that the agents
will act at the same time.
[0124] The agents of the present invention can be administered via
parenteral, subcutaneous, intravenous, intramuscular,
intraperitoneal, transdermal, or buccal routes. Alternatively, or
concurrently, administration may be by the oral route or directly
to the lungs. The dosage administered will be dependent upon the
age, health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of the
effect desired.
[0125] The compounds used in the method of treatment of this
invention may be administered systemically or topically, depending
on such considerations as the condition to be treated, need for
site-specific treatment, quantity of drug to be administered and
similar considerations.
[0126] Topical administration may be used. Any common topical
formation such as a solution, suspension, gel, ointment or salve
and the like may be employed. Preparation of such topical
formulations are well described in the art of pharmaceutical
formulations as exemplified, for example, by Remington's
Pharmaceutical Sciences. For topical application, these compounds
could also be administered as a powder or spray, particularly in
aerosol form. The active ingredient may be administered in
pharmaceutical compositions adapted for systemic administration. As
is known, if a drug is to be administered systemically, it may be
confected as a powder, pill, tablet or the like or as a syrup or
elixir for oral administration. For intravenous, intraperitoneal or
intra-lesional administration, the compound will be prepared as a
solution or suspension capable of being administered by injection.
In certain cases, it may be useful to formulate these compounds in
suppository form or as an extended release formulation for deposit
under the skin or intramuscular injection. In a preferred
embodiment, the compounds of this invention may be administered by
inhalation. For inhalation therapy the compound may be in a
solution useful for administration by metered dose inhalers or in a
form suitable for a dry powder inhaler.
[0127] An effective amount is that amount which will down-regulate
ICACC-1. A given effective amount will vary from condition to
condition and in certain instances may vary with the severity of
the condition being treated and the patient's susceptibility to
treatment. Accordingly, a given effective amount will be best
determined at the time and place through routine experimentation.
However, it is anticipated that in the treatment of atopic allergy
and asthma-related disorders in accordance with the present
invention, a formulation containing between 0.001 and 5 percent by
weight, preferably about 0.01 to 1%, will usually constitute a
therapeutically effective amount. When administered systemically,
an amount between 0.01 and 100 mg per kg body weight per day, but
preferably about 0.1 to 10 mg/kg, will effect a therapeutic result
in most instances.
[0128] The invention also includes pharmaceutical compositions
comprising the compounds of the invention together with a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers are described in Remington's Pharmaceutical Sciences, Mack
Publishing Company, 1995. In addition to the pharmacologically
active agent, the compositions of the present invention may contain
suitable pharmaceutically acceptable carriers comprising excipients
and auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically for delivery
to the site of action. Suitable formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form, for example, water-soluble salts. In addition,
suspensions of the active compounds as appropriate oily injection
suspensions may be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, for example, ethyl oleate or triglycerides.
Aqueous injection suspensions may contain substances which increase
the viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers. Liposomes can also be used
to encapsulate the agent for delivery into the cell.
[0129] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral or topical administration. Indeed, all three types of
formulations may be used simultaneously to achieve systemic
administration of the active ingredient.
[0130] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0131] In practicing the methods of this invention, the compounds
of this invention may be used alone or in combination, or in
combination with other therapeutic or diagnostic agents. In certain
preferred embodiments, the compounds of this invention may be
coadministered along with other compounds typically prescribed for
these conditions according to generally accepted medical practice.
The compounds of this invention can be utilized in vivo, ordinarily
in mammals, preferably in humans.
[0132] In still another embodiment, the compounds of the invention
may be coupled to chemical moieties, including proteins that alter
the functions or regulation of ICACC-1 for therapeutic benefit in
atopic allergy and asthma (Kreitman et al., 1994). These proteins
may include in combination other inhibitors of cytokines and growth
factors including anti-IL-4, anti-IL-5, anti-IL-3, anti-IL-2,
anti-IL-13, anti-IL-11 and anti-IL-10 that may offer additional
therapeutic benefit in atopic allergy and asthma. In addition, the
molecules of the invention may also be conjugated through
phosphorylation to biotinylate, thioate, acetylate, iodinate using
any of the cross-lining reagents well known in the art.
K. Diagnostics
[0133] Also included in the invention are methods of diagnosing
susceptibility to atopic allergy and related disorders and for
treating these disorders based on the relationship between IL-9,
its receptor and ICACC-1.
[0134] These disorders also include the monitoring of ICACC-1 gene
expression for the diagnosis of autoimmune disease of the bowel
such as inflammatory bowel disease (IBD). In the case of IBD the
lack or suppression of ICACC-1 gene expression would be a
diagnostic marker for the disease and the ability to follow ICACC-1
levels would aid in monitoring treatment.
[0135] One diagnostic embodiment involves the recognition of
variations in the DNA sequence of ICACC-1. One method involves the
introduction of a nucleic acid molecule (also known as a probe)
having a sequence complementary to ICACC-1 of the invention under
sufficient hybridizing conditions, as would be understood by those
in the art. In one embodiment, the sequence will bind specifically
to one allele of ICACC-1 or a fragment thereof and in another
embodiment will bind to both alleles. Another method of recognizing
DNA sequence variation associated with these disorders is direct
DNA sequence analysis by multiple methods well known in the art
(Ott, 1991). Another embodiment involves the detection of DNA
sequence variation in the ICACC-1 gene associated with these
disorders (Schwengel et al., 1993; Sheffield et al., 1993; Orita et
al., 1989; Sarkar et al., 1992; Cotton, 1989). These include the
polymerase chain reaction, restriction fragment length polymorphism
analysis and single stranded conformational analysis.
[0136] The practice of the present invention will employ the
conventional terms and techniques of molecular biology,
pharmacology, immunology and biochemistry that are within the
ordinary skill of those in the art. For example, see Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold
Spring Harbor Laboratory Press, 1985.
[0137] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
EXAMPLES
Example 1
cDNA Difference Analysis of IL-9 Expressed Genes
[0138] Lungs extracted from transgenic IL-9 mice (Tg5) were used to
isolate IL-9 induced genes. Tg5 is a FVB mouse overexpressing the
IL-9 gene as previously described (Renauld et al., 1994). This
strain has been shown to overexpress IL-9 in most tissues of the
mouse. In order to identify specific IL-9 induced genes,
suppressive PCR cDNA difference analysis was performed on mRNA from
lungs of Tg5 mice and parental FVB mice using a commercially
available PCR-select cDNA subtraction kit (Clonetech).
[0139] cDNA synthesis. Total RNA was prepared from lungs of FVB and
Tg5 mice using Trizol as described by the manufacturer (Gibco/BRL).
Lungs were removed from euthanized mice and frozen in liquid
nitrogen. Frozen lungs were then placed in Trizol and pulverized
using a tissue grinder. Polyadenylated RNA was purified from total
RNA with oligo(dT) cellulose columns (Pharmacia). Double stranded
cDNA was prepared using Superscript II reverse transcriptase and an
oligo(dT) primer as suggested by the manufacturer (Clonetech). cDNA
was then prepared by phenol-chloroform extraction and ethanol
precipitation. Products were resuspended in nuclease-free water and
analyzed on agarose gels to determine quality of products as
described below.
[0140] cDNA difference analysis protocol. Differential cDNA
analysis of Tg5 and FVB lungs was carried out following the
manufacturers protocol (Clonetech) as depicted in FIG. 1. The
results of the subtraction between the cDNA of these lungs resulted
in the generation of 1200 recombinant clones. Analysis of these
clones revealed multiples of several species, each accounting for
2-5% of the library. The most prominent transcript in the library
was the IL-9 cDNA which served as a control for the efficiency of
subtraction since it was a subtraction between an IL-9
constitutively expressing mouse (Tg5) and its parental control.
Another cDNA which was found in multiple copies (represented 3% of
library) was a novel calcium activated chloride channel which is
described below.
Example 2
Identification of the Murine ICACC-1 cDNA in the Lung of IL-9
Transgenic Mice
[0141] ICACC-1 probes as described in Example 1 were used to probe
a murine lung cDNA library (Clonetech) according to the
manufacturers recommendation. One million recombinant clones were
screened and several overlapping phage were identified. Subsequent
screens enabled identification and isolation of a single plaque
containing a phagemid which was then transformed into a
double-stranded plasmid by phage rescue according to the
manufacturers protocol. Recombinant clones were prepared and
sequenced using primers directed to the plasmid vector as well as
internal sequences identified from the partially subtracted probe.
Clones were then aligned and contiged to generate the full-length
sequence.
[0142] The 2931 bp cDNA isolated contained an open reading frame
encoding a protein of 925 amino acids. FIG. 2 shows the nucleotide
and amino acid sequence of the murine ICACC-1 cDNA. A nucleotide
BLAST (Altschul et al., 1990) database search of GenBank with the
full length cDNA revealed that it was similar to the bovine
chloride channel protein. FIG. 3 shows an alignment to the bovine
calcium activated chloride channel cDNA. Motif analysis of the
encoded polypeptide demonstrated several features such as multiple
transmembrane regions and glycosylation sites. The primary sequence
of murine ICACC-1 was used to perform an EST database search and
several undescribed human ESTs were found to be homologous to small
portions of the novel cDNA. FIGS. 4A and 4B show the sequences of
the human ICACC-1 and ICACC-2 genes. Both full length human ICACC
sequences were obtained by screening a human cDNA library.
Example 3
ICACC-1 is Induced in vivo by IL-9 in Murine Cells
[0143] To confirm that ICACC-1 is induced by IL-9 in the lung, RNA
from the lungs of Tg5 and FVB mice were isolated as described in
Example 1. cDNA was generated using random hexamers (Pharmacia) and
Superscript II (Gibco/BRL) as suggested by the manufacturer.
Message was analyzed by PCR as described (Nicolaides et al., 1995)
and via Northern blot. Primers used to generate murine ICACC-1
message were; sense 5'-CCAGATCCACACCAAAACGAGAAG-3' (SEQ ID NO:7)
(nucleotides 689-712) and antisense 5'-CACTGTCAAAGGTCACCATCCCGA-3'
(SEQ ID NO:8) (nucleotides 1041-1064) which produce a gene product
of 376 bp. DHFR was assayed as an internal control to measure for
cDNA integrity using primers previously described Nicolaides et
al., 1991). Amplification conditions used were 95.degree. C. for 30
seconds, 58.degree. C. for 1.5 minutes and 72.degree. C. for 1.5
minutes for 35 cycles. For Northern blot analysis, total RNA
derived from Tg5 or FVB lungs was electrophoresed on 1.5%
formaldehyde gels, transferred to nylon membranes and probed with a
DNA fragment comprising the murine ICACC-1 cDNA.
[0144] The results of the expression studies demonstrated that
ICACC-1 is specifically expressed in the lung of the IL-9
transgenic mouse but not in the parental strain (FIG. 6). This data
demonstrated a direct effect of IL-9 on ICACC-1 expression in the
lung, where IL-9 responsive cells contained within the lung express
ICACC-1.
Example 4
ICACC-1 Expression can be Induced in the Murine Lung by IL-9
[0145] ICACC-1 gene expression was assessed in vivo using the C57B6
mouse (bronchial hyporesponsive) which does not express detectable
levels of IL-9 and the DBA mouse (bronchial hyperresponsive) which
expresses robust levels of IL-9 (Nicolaides et al., 1997). RT-PCR
and Northern blot analysis of ICACC-1 from these lungs demonstrated
that ICACC-1 was expressed in the lung of mice which naturally
express high levels of IL-9 (DBA) but not in those with low levels
of IL-9 (C57B6) (FIG. 7).
[0146] To confirm that the expression of IL-9 was critically
related to the expression of ICACC-1 and to control for genetic
background specifically, recombinant murine IL-9 was introduced
into the lung of murine strain C57B6. Recombinant IL-9 was
instilled into the trachea of anesthetized mice by addition of 50
.mu.l of a 0.1 mg/ml IL-9 solution or vehicle alone (0.1% bovine
serum albumin) daily for ten days. After ten days, the mice were
euthanized and lungs extracted for either RNA expression analysis
using Trizol as described by the manufacturer (Gibco/BRL) or
Western blot analysis to determine levels of IL-9 instilled. The
Western blot analysis for IL-9 demonstrated that direct addition of
IL-9 to the lung resulted in an increase of overall amount of IL-9
in the lung while none was observed in the mouse instilled with
vehicle alone.
[0147] Expression of ICACC-1 RNA was measured as described in
Example 3. RT-PCR analysis for ICACC-1 RNA expression indicated
that expression increased when recombinant IL-9 was administered to
the lungs of the C57B6 mice, while no expression was observed in
the lungs of mice treated with vehicle only (FIG. 8). This data
demonstrates a direct role of IL-9 on inducing ICACC-1 expression
in the lung.
Example 5
Tissue Distribution of ICACC-1 in Mice
[0148] To address the possibility that ICACC-1 expression occurs
only in the presence of IL-9 expression, various organs were
extracted from Tg5 mice and analyzed for RNA expression via
Northern blot. BALBc mice were used as a control because they
express low levels of IL-9 in the lung when compared to Tg5 mice.
Tissue blots derived from BALBc murine organs were commercially
obtained (Clonetech) while tissue blots for Tg5 murine organs were
prepared by extracting organs followed by freezing in liquid
nitrogen. Total RNA was extracted from each of these organs using
Trizol as described by the manufacturer (Gibco/BRL). RNA was gel
electrophoresed and analyzed as described in Example 4. Lanes were
standardized by probing with .beta.-actin as an internal
control.
[0149] Tissue blots were probed using a DNA fragment comprising the
ICACC-1 cDNA. As shown in FIG. 9 (bottom), no signal was observed
in any of the tissues present on blots from normal mice. Analysis
of ICACC-1 expression in Tg5 organs revealed expression in the
lung, lymph node, colon, spleen, stomach, ovary and uterus (FIG. 9,
top). This data demonstrated that ICACC-1 is expressed in several
tissues in mice overexpressing IL-9 but not in those with low IL-9
levels. This data suggests that ICACC-1 may play a role in the
physiology of these organs in response to IL-9.
Example 5A
Induction of ICACC-1 in the Lung by Exposure to Antigen
[0150] Antigen sensitzation and phenotyping of animals was carried
out as previously described (McLane, M P, et al. Am. J. Respir.
Cell Mol. Biol. 19:713-720, 1998). Briefly, Balb/C mice were
intranasally exposed to Aspergillus fumagatus for 34 weeks. One day
after the final exposure, antigen exposed mice and naive controls
were phenotyped for bronchial hyperresponsiveness (BHR) and
cellularity in the airway. After phenotyping, organs were removed
and total RNA was prepared as described in Example 5 and ICACC-1
expression was assessed in naive and antigen treated tissues. As
shown in FIG. 10, antigen exposed Balb/C mice had a significant
increase in BHR (FIG. 10A) and inflammatory cell influx (the
majority being eosinophils) as compared to controls (FIG. 10B).
These features are very similar to clinical human asthma, and
reinforce the notion that this is a relevant model to study
molecular mechanisms and pharmaceutical target discovery for the
development of asthma drugs. ICACC-1 gene expression was tightly
associated with the asthmatic-like lung where robust expression was
found in the antigen treated lung (bottom panel, FIG. 11), while no
expression was found in the naive "normal" lung (top panel, FIG.
11). These data suggest that: 1) ICACC-1 is a potential therapeutic
target for the treatment of asthma, and 2) inhibiting the
expression or function of ICACC-1 will result in no toxic effects
to the lung.
Example 5B
Inhibition of Antigen Induced induction of ICACC-1 in the Lung with
Anti-IL-9
[0151] IL-9 is a major mediator of the asthmatic response in man
and mouse models of asthma (Nicolaides, et al. Proc. Natl. Acad.
Sci. 94:13175-13180, 1997; McLane, M P, et al. Am. J. Respir. Cell
Mol. Biol. 19:713-720, 1998; Temann et al., J. Exp. Med.
188:1307-1320, 1998: Levitt and Nicolaides, Emerg. Thera. Targets
3:1-11, 1999). The use of IL-9 blocking antibodies in antigen
exposed mice suppresses the asthmatic-like phenotype (bronchial
hyperresponsiveness and influx of inflammatory cells such as
eosinophils). (B6D2)F1 mice were exposed to Aspergillus fumagatus
antigen as described in Example 5A on day 0, 7, 14, 21, and 22. A
subset of mice were also treated with 200 .mu.gs of anti-mIL9
(Pharmingen hamster antimouse IL-9) intra nasally on day 0, 7, 14,
and 21; or an isotype control Ig; or saline alone. All mice and
naive controls were phenotyped for BHR and BAL analysis as
described in Example 5A. As shown in FIG. 12A, anti-IL9 treatment
(Asp+.alpha.-mIL9) was able to significantly suppress BHR to levels
near that of naive, while isotype control Ig (Asp+Ig) had no effect
on reducing BHR. A similar result was found for airway eosinophilia
where a significant eosinophilia resulted upon antigen treatment
(Asp -) that was suppressed by anti-mIL9 treatment
(Asp+.alpha.-mIL9). Northern blot analysis of whole lungs from
these mice showed that anti-mIL9 also suppressed ICACC-1 gene
expression found in lungs of antigen exposed mice (FIG. 13). GADPH
which is a ubiquitously expressed house keeping gene was used as a
control to assure equal loading of RNA and overall gene expression.
Together, these data demonstrate a tight correlation of ICACC-1
gene expression and the asthmatic response. These data suggest that
blocking ICACC-1 expression or function would suppress the
asthmatic response.
Example 6
ICACC-1 Inducibility by IL-9 in Human Lung Epithelial Cells
[0152] To assess the ability of ICACC-1 to be induced by IL-9 in
epithelial cells, the human primary lung epithelial cell line NHBE
was assayed for expression levels of ICACC-1 in the presence of
IL-9. 1.times.10.sup.7 cells were harvested and washed three times
with phosphate-buffered saline and plated in medium in the presence
or absence of 50 ng/ml IL-9 for 72 hours. Cells were then harvested
and total RNA was extracted using Trizol as described by the
manufacturer (Gibco/BRL). RNA was processed and reverse transcribed
into cDNA as described in Example 3. Primers used to generate human
ICACC-1 message were; sense 5'-GATTCCAGGAACAGCTAAGC-3' (SEQ ID
NO:9) and antisense 5'-TATTTCATAGCTTGTAGCCTGG-3' (SEQ ID NO:10)
which produce a gene product of 722 bp. .gamma.-actin was assayed
as an internal control to measure for cDNA integrity using primers
previously described (Nicolaides et al., 1991). RT-PCR data derived
from human lung epithelial cells, shows that ICACC-1 is induced in
cells treated with IL-9 while no expression was observed in
untreated cells, indicating that the cells expressing ICACC-1
directly respond to IL-9 (FIG. 14).
[0153] Furthermore, human primary lung cultures that were
established from human lung biopsies were analyzed for IL-9 induced
expression of ICACC-1. Lung tissues were first minced with scissors
and passed through a wire mesh. Tissues were then digested with 175
iU/ml of collagenase (Sigma) for 1 hour at 37.degree. C. Tissue was
passed through 45 .mu.m and 15 .mu.m filters and then resuspended
in Dubelco Iscove's medium, and plated into 10 cm tissue culture
plates. Plates were incubated for 1 hour at 37.degree. C. to allow
macrophages to adhere to the plate and then non-adherent cells were
harvested and resuspended at 2.times.10.sup.5 cell/ml in Dulbelco
Iscoive's medium supplemented with 10% FBS, antibiotics and
cultured at 37.degree. C. with 5% CO.sub.2 for 4-5 days. For
ICACC-1 IL-9 induction studies, cells were incubated for 4-5 days
with or without 20 ng/ml recombinant human IL-9. Cells were then
harvested and total RNA was extracted by trizol as described above.
RNA was reverse transcribed and PCR'd for ICACC-1 using 5' primer
5'-CCCAAAGGAAGCCAACTCTGA-3'' and 3' primer
5'-GTGAATGCCAGGAATGGTGCT-3' which resulted in a 253 bp product.
PMS2 which is a ubiquitously expressed house keeping gene was used
as an internal control as described (Nicolaides et al., Genomics
29: 329-334, 1995). Products were electrophoresed on 2% agarose
gels and visualized by ethidium bromide staining. As shown in FIG.
15, IL-9 induced ICACC-1 expression in human primary lung cultures,
while cultures grown in the absence of IL-9 had no detectable
amounts of ICACC-1.
Example 6A
ICACC-1 Antiserum
[0154] Antisera were prepared to mICACC-1 by immunizing rabbits
with peptides selected from the mICACC-1 sequence using methods
described in Current Protocols in Immunology, Chapter 9, John Wiley
& Sons, Inc. The peptides used for the immunizations were;
residues 309-330, CLVLDKSGSMLNDDRLNRMNQA (SEQ ID NO: 11), residues
357-375, QSELKQLNSGADRDLLIKHC (SEQ ID NO: 12), residues 398422,
KKKYPTDGSEIVLLTDGEDNTISSC (SEQ ID NO: 13), residues 524-546,
TTBPPTIFIWDPSGVEQNGFILDC (SEQ ID NO: 14), residues 590-610,
CPPITVTPVVNKNTGKFPSPVT (SEQ ID NO: 15). The peptides were
synthesized by standard techniques of automated peptide synthesis
as either octavalent multiple antigen peptides (MAP) or as single
peptides. The single peptides were coupled to KLH for immunization
while the MAPs were used uncoupled. Rabbits were immunized with a
mixture of all five peptides either as KLH conjugates or MAPs. Both
immunogens produced useful antisera as indicated by their ability
to immunoprecipitate mICACC-1.
[0155] Immunoprecipitation of in vitro translated ICACC-1 was
performed to analyze the activities of these antibodies. A .sup.35S
labeled ICACC-1 fragment (429 amino acids in length corresponding
to amino acids 289-618 of the full length ICACC-1) was in vitro
translated using TNT Coupled Reticulocyte Lysate Systems (Promega).
Radio labeled ICACC-1 could be immunoprecipitated by 5 .mu.l of
ICACC-1 antisera or 1 .mu.g of protein A purified polyclonal
antibody. To assess the specificity of the ICACC-1 antisera, the
.sup.35S-labeled mIL-9 receptor fragment (60 KD mIL-9R) was used as
a negative control. Under the same precipitation conditions, none
of the mIL-9R protein was precipitated by the ICACC-1 antisera
(FIG. 16). These results indicated that antisera and protein A
purified polyclonal antibodies raised against ICACC-1 could
recognize ICACC-1 and therefore could potentially be used as
pharmaceutical reagents to block ICACC-1 function.
Example 6B
Suppression of IL-9 Induced Eotaxin Expression in the Lung Using
Chloride Channel Blockers
[0156] IL-9 is known to induce eotaxin from lung epithelial cells
(Dong, et. al., submitted for publication, Eur. J. Immunol.). In
situ expression analysis of IL-9 transgenic mice found ICACC-1
expression to be predominant in airway epithelial cells. These
epithelial cells also produce eotaxin, and eotaxin can be induced
by IL-9 in these cells as well as primary lung cultures from a
variety of different mouse strains. Because eotaxin and ICACC-1 are
both induced by IL-9 in lung epithelial cells, it is possible that
inhibiting ICACC-1 can inhibit eotaxin or other cytokines such as
IL-4 or IL-13 (Doucet et al., J. Clin. Invest. 101:2129-2139,
1998.) that induce eotaxin production in lung epithelial cells. To
test this hypothesis, we employed a murine primary lung assays,
where lung cells were harvested from FVB/NJ mice as described in
Example 5A and processed for in vitro analysis as described in
Example 6 for human primary lung culture. Cells were incubated with
or without 20 ng/ml recombinant mIL-9 for 48 hours. After 48 hours,
conditioned supernatant was collected and analyzed for murine
eotaxin production using an eotaxin ELISA kit (R&D Systems).
Recombinant murine eotaxin was used to generate a standard curve.
As shown in FIG. 17, FVB primary cells when cultured with IL-9
produce up to 2 ng/ml of eotaxin in contrast to nearly undetectable
levels in FVB cultures grown in medium alone. A culture derived
from IL-9 transgenic mouse lung (TG5 lane) was used as a positive
control. This assay was used to assess the ability to suppress IL-9
induced eotaxin using chloride channel inhibitors DIDS and SITS.
Cultures were plated with or without mIL-9 in the presence of 0, 30
.mu.M and 100 .mu.M channel blocker. As shown in FIG. 18, eotaxin
production was inhibited 33% and 41% by 100 .mu.M DIDS or SITS
respectively. These data demonstrate the ability to suppress the
biological function of IL-9 on epithelial cells by inhibiting
chloride channel function. These data further indicate that
suppression of a chloride channel such as the asthma associated
ICACC-1 can result in a therapeutic benefit by the suppression of
antigen induced asthmatic responses. This screening assay and
technique can be used to evaluate other IL-9 induced genes whose
products are secreted proteins and is not restricted to using
eotaxin as the only marker. A similar approach will be taken using
the human ICACC-1 and human functional assays to identify
"specific" chloride channel inhibitors that suppress: 1) IL-9
induced effects such as de novo gene expression, and 2) ICACC-1
biologic function(s).
Example 7
Specific Blocking of ICACC-1 Signaling in vivo by Small Molecule
Inhibitors
[0157] To demonstrate the specificity of ICACC-1 signaling which is
induced by IL-9, transfected cells expressing constitutively active
ICACC-1 are treated with chloride channel blocking compounds to
determine if inhibition of ICACC-1 blocks chloride channel
activity. Cells transfected with a constitutively activated ICACC-1
gene are plated at 3000 cells/well in the presence or absence of
IL-9 plus blocking compound and assessed for chloride channel
activity using a fluorescent chloride probe. Wild-type cells do not
exhibit the same amount of chloride channel activity as those
constitutively expressing ICACC-1. The addition of the blocking
compound on chloride channel activity is compared between wild-type
cells and those expressing constitutively activated ICACC-1.
Example 8
Blocking of ICACC-1 Induction by Aminosterols in Murine Lung
[0158] Lungs from the DBA bronchial hyperresponsive mouse are
treated with aminosterol compounds to test for their ability to
block expression of ICACC-1. This group of aminosterols was
identified from the liver of the dogfish shark as a class of
molecules that appear to be antiproliferative. An example of these
compounds are referred to in related U.S. patent application Ser.
No. 08/290,826. This series of aminosterols are assayed for their
ability to inhibit ICACC-1 expression and TH2 activity from the DBA
mouse as described below.
[0159] DBA mice are injected daily intraperitoneally with various
aminosterols at 10 mg/kg for 15 days. At day 15, mice are
phenotyped (see Example 9), euthanized and lungs extracted as
described in Example 1. RNA is isolated and processed for Northern
blot analysis using a ICACC-1 cDNA probe. The level of ICACC-1 RNA
detected by the probe indicates the extent of inhibition by
aminosterols when compared to control. The ability of specific
aminosterols, such as 1459, 1409, 1436 and 1569, to block the
expression of ICACC-1 in vivo is assessed.
Example 9
Role of ICACC-1 in Murine Models of Asthma
Airway Response of Unsensitized Animals
[0160] Certified virus-free male and female mice of the following
strains, DBA, C57B6 and B6D2F1 are purchased from the National
Cancer Institute or Jackson Laboratories (Bar Harbor Me.). IL-9
transgenic mice (Tg5) and their parent strain (FVB), are obtained
from the Ludwig Institute (Brussels, Belgium). Animals are housed
in high-efficiency particulate filtered air laminar flow hoods in a
virus and antigen free facility and allowed free access to food and
water for 3 to 7 days prior to experimental manipulation. The
animal facilities are maintained at 22.degree. C. and the
light:dark cycle is automatically controlled (10:14 hour
light:dark).
[0161] Phenotyping and efficacy of pretreatment. To determine the
bronchoconstrictor response, respiratory system pressure is
measured at the trachea and recorded before and during exposure to
the drug. Mice are anesthetized and instrumented as previously
described. (Levitt et al., 1988; Levitt et al., 1989; Kleeberger et
al., 1990; Levitt et al., 1991; Levitt et al., 1995; Ewart et al.,
1995). Airway responsiveness is measured to one or more of the
following: 5-hydroxytryptamine, acetylcholine, atracurium or a
substance-P analog. A simple and repeatable measure of the change
in peak inspiratory pressure following bronchoconstrictor challenge
is used which has been termed the Airway Pressure Time Index (APTI)
(Levitt et al., 1988; Levitt et al., 1989). The APTI is assessed by
the change in peak respiratory pressure integrated from the time of
injection until the peak pressure returns to baseline or plateau.
The APTI is comparable to airway resistance, however, the APTI
includes an additional component related to the recovery from
bronchoconstriction.
[0162] Prior to sacrifice, whole blood is collected for serum IgE
measurements by needle puncture of the inferior vena cava in
anesthetized animals. Samples are centrifuged to separate cells and
serum is collected and used to measure total IgE levels. Samples
not measured immediately are frozen at -20.degree. C.
[0163] All IgE serum samples are measured using an ELISA
antibody-sandwich assay. Microtiter plates are coated, 50 .mu.l per
well, with rat anti-murine IgE antibody (Southern Biotechnology) at
a concentration of 2.5 .mu.g/ml in a coating buffer of sodium
carbonate-sodium bicarbonate with sodium azide. Plates are covered
with plastic wrap and incubated at 4.degree. C. for 16 hours. The
plates are washed three times with a wash buffer of 0.05% Tween-20
in phosphate-buffered saline, incubating for five minutes for each
wash. Blocking of nonspecific binding sites is accomplished by
adding 200 .mu.l per well 5% bovine serum albumin in
phosphate-buffered saline, covering with plastic wrap and
incubating for 2 hours at 37.degree. C. After washing three times
with wash buffer, duplicate 50 .mu.l test samples are added to each
well. Test samples are assayed after being diluted 1:10, 1:50 and
1:100 with 5% bovine serum albumin in wash buffer. In addition to
the test samples, a set of IgE standards (PharMingen) at
concentrations from 0.8 ng/ml to 200 ng/ml in 5% bovine serum
albumin in wash buffer, are assayed to generate a standard curve. A
blank of no sample or standard is used to zero the plate reader
(background). After adding samples and standards, the plate is
covered with plastic wrap and incubated for 2 hours at room
temperature. After washing three times with wash buffer, 50 .mu.l
of secondary antibody rat anti-murine IgE-horseradish peroxidase
conjugate is added at a concentration of 250 ng/ml in 5% bovine
serum albumin in wash buffer. The plate is covered with plastic
wrap and incubated 2 hours at room temperature. After washing three
times with wash buffer, 100 .mu.l of the substrate 0.5 mg/ml
o-phenylenediamine in 0.1 M citrate buffer is added to every well.
After 5-10 minutes the reaction is stopped with 50 .mu.l of 12.5%
sulfuric acid and absorbance is measured at 490 nm on a MR5000
plate reader (Dynatech). A standard curve is constructed from the
standard IgE concentrations with antigen concentration on the x
axis (log scale) and absorbance on the y axis (linear scale). The
concentration of IgE in the samples is interpolated from the
standard curve.
[0164] Bronchoalveolar lavage and cellular analysis are preformed
as previously described (Kleeberger et al., 1990). Lung histology
is carried out after the lungs are extracted. Since prior
instrumentation may introduce artifact, separate animals are used
for these studies. Thus, a small group of animals is treated in
parallel exactly the same as the cohort undergoing various
pretreatments except these animals are not used for other tests
aside from bronchial responsiveness testing. After bronchial
responsiveness testing, the lungs are removed and submersed in
liquid nitrogen. Cryosectioning and histologic examination is
carried out in a manner obvious to those skilled in the art.
[0165] Polyclonal antibodies which block the murine ICACC-1 pathway
are used therapeutically to down-regulate the functions of, and
assess the importance of this pathway to bronchial responsiveness,
serum IgE and bronchoalveolar lavage in sensitized and unsensitized
mice. After antibody pretreatment, baseline bronchial
hyperresponsiveness, bronchoalveolar lavage and serum IgE levels
relative to Ig matched controls are determined.
Example 10
Role of ICACC-1 in Murine Models of Asthma
Airway Response of Sensitized Animals
[0166] The data of Example 6a demonstrate that antisera is able to
be generated against ICACC-1 that recognizes the native protein
structure as determined by the ability to recognize the protein in
immunoprecipitation studies (FIG. 16). ICACC-1 blocking antibodies
represent potential therapeutic agents in suppressing the function
of ICACC-1. Studies are carried out using antigen sensitized
animals and protocols as described Examples 5A, 5B, and 10. Animals
are given ICACC-1 blocking antibodies via intranasal administration
as described in example 5B and at day 23 animals are phenotyped for
BHR, BAL, and immunoglobulin levels. The effect of pretreatment
with ICACC-1 antibodies is used to assess the effect of
down-regulating ICACC-1 on the asthma phenotype.
[0167] While the invention has been described and illustrated
herein by references to various specific materials, procedures and
examples, it is understood that the invention is not restricted to
the particular combinations of material and procedures selected for
that purpose. Numerous variations of such details can be implied as
will be appreciated by those skilled in the art.
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Sequence CWU 1
1
1812931DNAMus musculusCDS(8)..(2746) 1ctgcagg atg gaa tct ttg aag
agt cct gtc ttc ctc ttg atc ctc cac 49Met Glu Ser Leu Lys Ser Pro
Val Phe Leu Leu Ile Leu His1 5 10ctt ctg gaa gga gtt ctg agt gag
tcc ctc atc caa ctg aac aac aac 97Leu Leu Glu Gly Val Leu Ser Glu
Ser Leu Ile Gln Leu Asn Asn Asn15 20 25 30ggc tat gag ggc atc gtc
atc gcc ata gac cac gac gtg ccg gaa gat 145Gly Tyr Glu Gly Ile Val
Ile Ala Ile Asp His Asp Val Pro Glu Asp35 40 45gaa gcc ctc att caa
cac ata aag gac atg gtg act cag gcc tct cca 193Glu Ala Leu Ile Gln
His Ile Lys Asp Met Val Thr Gln Ala Ser Pro50 55 60tac ctg ttt gaa
gct aca gga aaa aga ttt tac ttc aaa aat gtt gcc 241Tyr Leu Phe Glu
Ala Thr Gly Lys Arg Phe Tyr Phe Lys Asn Val Ala65 70 75att ttg att
ccc gag agc tgg aag gca aag cct gaa tat acg agg cca 289Ile Leu Ile
Pro Glu Ser Trp Lys Ala Lys Pro Glu Tyr Thr Arg Pro80 85 90aaa ctt
gaa acc ttc aaa aac gct gat gtc ctt gta tca aca acc agc 337Lys Leu
Glu Thr Phe Lys Asn Ala Asp Val Leu Val Ser Thr Thr Ser95 100 105
110cct cta ggc aat gat gag ccc tac acc gaa cat ata gga gca tgt gga
385Pro Leu Gly Asn Asp Glu Pro Tyr Thr Glu His Ile Gly Ala Cys
Gly115 120 125gaa aag ggg atc agg att cac ctg act cct gac ttc tta
gca gga aag 433Glu Lys Gly Ile Arg Ile His Leu Thr Pro Asp Phe Leu
Ala Gly Lys130 135 140aag ctg act cag tat ggg cca caa gac agg acc
ttt gtc cat gag tgg 481Lys Leu Thr Gln Tyr Gly Pro Gln Asp Arg Thr
Phe Val His Glu Trp145 150 155gct cac ttc cga tgg gga gtg ttt aat
gaa tac aac aac gac gag aag 529Ala His Phe Arg Trp Gly Val Phe Asn
Glu Tyr Asn Asn Asp Glu Lys160 165 170ttc tac tta tcc aaa gga aaa
ccc caa gca gtg agg tgt tca gca gcc 577Phe Tyr Leu Ser Lys Gly Lys
Pro Gln Ala Val Arg Cys Ser Ala Ala175 180 185 190att acc ggt aaa
aat caa gtt cgt cgg tgc cag gga ggc agt tgt atc 625Ile Thr Gly Lys
Asn Gln Val Arg Arg Cys Gln Gly Gly Ser Cys Ile195 200 205act aac
gga aag tgt gta atc gac aga gta acg gga ctg tat aaa gac 673Thr Asn
Gly Lys Cys Val Ile Asp Arg Val Thr Gly Leu Tyr Lys Asp210 215
220aat tgt gta ttt gta cca gat cca cac caa aac gag aag gct tcc atc
721Asn Cys Val Phe Val Pro Asp Pro His Gln Asn Glu Lys Ala Ser
Ile225 230 235atg ttt aac caa aat atc aat tct gtg gtt gaa ttc tgt
aca gaa aaa 769Met Phe Asn Gln Asn Ile Asn Ser Val Val Glu Phe Cys
Thr Glu Lys240 245 250aat cac aat caa gaa gcc cca aat gac caa aac
caa cga tgc aat ctc 817Asn His Asn Gln Glu Ala Pro Asn Asp Gln Asn
Gln Arg Cys Asn Leu255 260 265 270cga agc acg tgg gaa gtc atc cag
gaa tct gag gac ttc aag caa acc 865Arg Ser Thr Trp Glu Val Ile Gln
Glu Ser Glu Asp Phe Lys Gln Thr275 280 285act ccc atg aca gcc cag
cca cct gca ccc acc ttc tca ctg ctg caa 913Thr Pro Met Thr Ala Gln
Pro Pro Ala Pro Thr Phe Ser Leu Leu Gln290 295 300att gga caa aga
att gtg tgc tta gtt ctt gat aag tcc ggg agc atg 961Ile Gly Gln Arg
Ile Val Cys Leu Val Leu Asp Lys Ser Gly Ser Met305 310 315ctg aac
gat gat cgt ctt aac cga atg aat cag gca agc cgg ctt ttc 1009Leu Asn
Asp Asp Arg Leu Asn Arg Met Asn Gln Ala Ser Arg Leu Phe320 325
330ctg ctg cag act gtg gag cag gga tcc tgg gtc ggg atg gtg acc ttt
1057Leu Leu Gln Thr Val Glu Gln Gly Ser Trp Val Gly Met Val Thr
Phe335 340 345 350gac agt gct gcc tat gta caa agc gaa ctc aaa cag
tta aac agt ggt 1105Asp Ser Ala Ala Tyr Val Gln Ser Glu Leu Lys Gln
Leu Asn Ser Gly355 360 365gct gac aga gat ctg ctg atc aag cac tta
ccc aca gta tct gca gga 1153Ala Asp Arg Asp Leu Leu Ile Lys His Leu
Pro Thr Val Ser Ala Gly370 375 380ggg aca tct ata tgc tct ggc ctt
cgg aca gca ttt aca gtg ata aag 1201Gly Thr Ser Ile Cys Ser Gly Leu
Arg Thr Ala Phe Thr Val Ile Lys385 390 395aag aag tat cca act gat
gga tct gaa att gtg ctg ctg acc gat ggg 1249Lys Lys Tyr Pro Thr Asp
Gly Ser Glu Ile Val Leu Leu Thr Asp Gly400 405 410gag gac aac acc
att agc agc tgc ttt gac ctg gtg aag cag agc ggg 1297Glu Asp Asn Thr
Ile Ser Ser Cys Phe Asp Leu Val Lys Gln Ser Gly415 420 425 430gcc
atc atc cat aca gtg gcc ctg gga ccg gct gcc gct aaa gag ctt 1345Ala
Ile Ile His Thr Val Ala Leu Gly Pro Ala Ala Ala Lys Glu Leu435 440
445gag cag ctg tcc aaa atg aca gga ggc ctg cag aca tac tct tcg gat
1393Glu Gln Leu Ser Lys Met Thr Gly Gly Leu Gln Thr Tyr Ser Ser
Asp450 455 460cag gtt cag aac aat ggt ctt gtt gat gct ttc gca gca
ctc tcc tca 1441Gln Val Gln Asn Asn Gly Leu Val Asp Ala Phe Ala Ala
Leu Ser Ser465 470 475gga aat gcg gcg atc gct cag cac tcc atc cag
ctg gag agc agg gga 1489Gly Asn Ala Ala Ile Ala Gln His Ser Ile Gln
Leu Glu Ser Arg Gly480 485 490gtt aat ctc cag aat aac caa tgg atg
aat ggc tca gtg atc gtg gac 1537Val Asn Leu Gln Asn Asn Gln Trp Met
Asn Gly Ser Val Ile Val Asp495 500 505 510agc tcg gtg ggc aag gac
acc ttg ttt ctt atc acc tgg aca acg cat 1585Ser Ser Val Gly Lys Asp
Thr Leu Phe Leu Ile Thr Trp Thr Thr His515 520 525cct cct aca ata
ttt atc tgg gat ccc agc gga gtg gaa caa aat ggt 1633Pro Pro Thr Ile
Phe Ile Trp Asp Pro Ser Gly Val Glu Gln Asn Gly530 535 540ttt ata
cta gac aca acc act aag gtg gcc tac ctc caa gtc cca ggc 1681Phe Ile
Leu Asp Thr Thr Thr Lys Val Ala Tyr Leu Gln Val Pro Gly545 550
555acg gct aag gtt ggc ttt tgg aaa tac agc att caa gcg agc tca cag
1729Thr Ala Lys Val Gly Phe Trp Lys Tyr Ser Ile Gln Ala Ser Ser
Gln560 565 570act ctc acc ttg act gtc acc tcc cgt gca gca agt gct
aca ctg cct 1777Thr Leu Thr Leu Thr Val Thr Ser Arg Ala Ala Ser Ala
Thr Leu Pro575 580 585 590cct att aca gtg acc ccg gta gtg aat aag
aac aca ggg aaa ttc ccc 1825Pro Ile Thr Val Thr Pro Val Val Asn Lys
Asn Thr Gly Lys Phe Pro595 600 605agc cct gta aca gtg tat gca agc
att cgc caa gga gcc tcg cct att 1873Ser Pro Val Thr Val Tyr Ala Ser
Ile Arg Gln Gly Ala Ser Pro Ile610 615 620ctc agg gcc agc gtc aca
gcc ttg att gaa tct gtg aat gga aaa aca 1921Leu Arg Ala Ser Val Thr
Ala Leu Ile Glu Ser Val Asn Gly Lys Thr625 630 635gta acc ctg gaa
tta ctg gat aac gga gca ggt gcc gat gcc acc aag 1969Val Thr Leu Glu
Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr Lys640 645 650aat gat
ggt gtc tac tca agg ttt ttt aca gct ttt gat gca aat ggt 2017Asn Asp
Gly Val Tyr Ser Arg Phe Phe Thr Ala Phe Asp Ala Asn Gly655 660 665
670aga tac agc gtt aaa ata tgg gct ctg gga gga gtc act tca gac aga
2065Arg Tyr Ser Val Lys Ile Trp Ala Leu Gly Gly Val Thr Ser Asp
Arg675 680 685cag aga gca gca cct ccg aag aac aga gcc atg tac ata
gat ggc tgg 2113Gln Arg Ala Ala Pro Pro Lys Asn Arg Ala Met Tyr Ile
Asp Gly Trp690 695 700att gag gat ggt gaa gta aga atg aac cca cca
cgt cct gaa act agt 2161Ile Glu Asp Gly Glu Val Arg Met Asn Pro Pro
Arg Pro Glu Thr Ser705 710 715tat gtt caa gac aag cag ctg tgc ttc
agc agg aca tct tca ggg gga 2209Tyr Val Gln Asp Lys Gln Leu Cys Phe
Ser Arg Thr Ser Ser Gly Gly720 725 730tcg ttt gtg gcc acc aat gtc
ccc gca gca gct ccc att cct gac ctc 2257Ser Phe Val Ala Thr Asn Val
Pro Ala Ala Ala Pro Ile Pro Asp Leu735 740 745 750ttt cca ccc tgt
caa atc act gac ctg aag gcc agc atc caa ggg cag 2305Phe Pro Pro Cys
Gln Ile Thr Asp Leu Lys Ala Ser Ile Gln Gly Gln755 760 765aac ctg
gtg aat ctg acg tgg acg gct cct ggg gat gac tac gac cac 2353Asn Leu
Val Asn Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp His770 775
780ggg aga gct tcc aac tac atc atc cga atg agc acc agt atc gtt gat
2401Gly Arg Ala Ser Asn Tyr Ile Ile Arg Met Ser Thr Ser Ile Val
Asp785 790 795ctc agg gac cac ttc aac acc tca ctc caa gtg aac act
acc ggt ctt 2449Leu Arg Asp His Phe Asn Thr Ser Leu Gln Val Asn Thr
Thr Gly Leu800 805 810atc ccc aaa gag gcc agc tct gag gaa atc ttt
gag ttt gaa ctg gga 2497Ile Pro Lys Glu Ala Ser Ser Glu Glu Ile Phe
Glu Phe Glu Leu Gly815 820 825 830ggc aac act ttt gga aat ggc aca
gat atc ttc att gct atc cag gct 2545Gly Asn Thr Phe Gly Asn Gly Thr
Asp Ile Phe Ile Ala Ile Gln Ala835 840 845gtg gat aag tcc aat ctg
aaa tca gaa atc tcc aac att gca cgg gtg 2593Val Asp Lys Ser Asn Leu
Lys Ser Glu Ile Ser Asn Ile Ala Arg Val850 855 860tct gtg ttc atc
ccc gct cag gag ccg ccc att ccc gaa gac tca act 2641Ser Val Phe Ile
Pro Ala Gln Glu Pro Pro Ile Pro Glu Asp Ser Thr865 870 875ccc cct
tgt cct gac atc agc atc aac agc acc att cct ggc atc cac 2689Pro Pro
Cys Pro Asp Ile Ser Ile Asn Ser Thr Ile Pro Gly Ile His880 885
890gtg ctg aag ata atg tgg aag tgg cta ggg gaa atg cag gtg aca cta
2737Val Leu Lys Ile Met Trp Lys Trp Leu Gly Glu Met Gln Val Thr
Leu895 900 905 910ggt ttg cac tgaattttca ggcaagaaat caaccagtca
ttcctttcac 2786Gly Leu Histggagaattt tctaaaaatg tactttagac
ttcctgtagg gggcggtata gtaacactcg 2846aagctgtaaa actgggtctg
ggtgcattaa aaattatctg ttcaaataca aaaaaaaaaa 2906aaaaaaaaaa
aaaaaaaaaa aaaaa 29312913PRTMus musculus 2Met Glu Ser Leu Lys Ser
Pro Val Phe Leu Leu Ile Leu His Leu Leu1 5 10 15Glu Gly Val Leu Ser
Glu Ser Leu Ile Gln Leu Asn Asn Asn Gly Tyr20 25 30Glu Gly Ile Val
Ile Ala Ile Asp His Asp Val Pro Glu Asp Glu Ala35 40 45Leu Ile Gln
His Ile Lys Asp Met Val Thr Gln Ala Ser Pro Tyr Leu50 55 60Phe Glu
Ala Thr Gly Lys Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu65 70 75
80Ile Pro Glu Ser Trp Lys Ala Lys Pro Glu Tyr Thr Arg Pro Lys Leu85
90 95Glu Thr Phe Lys Asn Ala Asp Val Leu Val Ser Thr Thr Ser Pro
Leu100 105 110Gly Asn Asp Glu Pro Tyr Thr Glu His Ile Gly Ala Cys
Gly Glu Lys115 120 125Gly Ile Arg Ile His Leu Thr Pro Asp Phe Leu
Ala Gly Lys Lys Leu130 135 140Thr Gln Tyr Gly Pro Gln Asp Arg Thr
Phe Val His Glu Trp Ala His145 150 155 160Phe Arg Trp Gly Val Phe
Asn Glu Tyr Asn Asn Asp Glu Lys Phe Tyr165 170 175Leu Ser Lys Gly
Lys Pro Gln Ala Val Arg Cys Ser Ala Ala Ile Thr180 185 190Gly Lys
Asn Gln Val Arg Arg Cys Gln Gly Gly Ser Cys Ile Thr Asn195 200
205Gly Lys Cys Val Ile Asp Arg Val Thr Gly Leu Tyr Lys Asp Asn
Cys210 215 220Val Phe Val Pro Asp Pro His Gln Asn Glu Lys Ala Ser
Ile Met Phe225 230 235 240Asn Gln Asn Ile Asn Ser Val Val Glu Phe
Cys Thr Glu Lys Asn His245 250 255Asn Gln Glu Ala Pro Asn Asp Gln
Asn Gln Arg Cys Asn Leu Arg Ser260 265 270Thr Trp Glu Val Ile Gln
Glu Ser Glu Asp Phe Lys Gln Thr Thr Pro275 280 285Met Thr Ala Gln
Pro Pro Ala Pro Thr Phe Ser Leu Leu Gln Ile Gly290 295 300Gln Arg
Ile Val Cys Leu Val Leu Asp Lys Ser Gly Ser Met Leu Asn305 310 315
320Asp Asp Arg Leu Asn Arg Met Asn Gln Ala Ser Arg Leu Phe Leu
Leu325 330 335Gln Thr Val Glu Gln Gly Ser Trp Val Gly Met Val Thr
Phe Asp Ser340 345 350Ala Ala Tyr Val Gln Ser Glu Leu Lys Gln Leu
Asn Ser Gly Ala Asp355 360 365Arg Asp Leu Leu Ile Lys His Leu Pro
Thr Val Ser Ala Gly Gly Thr370 375 380Ser Ile Cys Ser Gly Leu Arg
Thr Ala Phe Thr Val Ile Lys Lys Lys385 390 395 400Tyr Pro Thr Asp
Gly Ser Glu Ile Val Leu Leu Thr Asp Gly Glu Asp405 410 415Asn Thr
Ile Ser Ser Cys Phe Asp Leu Val Lys Gln Ser Gly Ala Ile420 425
430Ile His Thr Val Ala Leu Gly Pro Ala Ala Ala Lys Glu Leu Glu
Gln435 440 445Leu Ser Lys Met Thr Gly Gly Leu Gln Thr Tyr Ser Ser
Asp Gln Val450 455 460Gln Asn Asn Gly Leu Val Asp Ala Phe Ala Ala
Leu Ser Ser Gly Asn465 470 475 480Ala Ala Ile Ala Gln His Ser Ile
Gln Leu Glu Ser Arg Gly Val Asn485 490 495Leu Gln Asn Asn Gln Trp
Met Asn Gly Ser Val Ile Val Asp Ser Ser500 505 510Val Gly Lys Asp
Thr Leu Phe Leu Ile Thr Trp Thr Thr His Pro Pro515 520 525Thr Ile
Phe Ile Trp Asp Pro Ser Gly Val Glu Gln Asn Gly Phe Ile530 535
540Leu Asp Thr Thr Thr Lys Val Ala Tyr Leu Gln Val Pro Gly Thr
Ala545 550 555 560Lys Val Gly Phe Trp Lys Tyr Ser Ile Gln Ala Ser
Ser Gln Thr Leu565 570 575Thr Leu Thr Val Thr Ser Arg Ala Ala Ser
Ala Thr Leu Pro Pro Ile580 585 590Thr Val Thr Pro Val Val Asn Lys
Asn Thr Gly Lys Phe Pro Ser Pro595 600 605Val Thr Val Tyr Ala Ser
Ile Arg Gln Gly Ala Ser Pro Ile Leu Arg610 615 620Ala Ser Val Thr
Ala Leu Ile Glu Ser Val Asn Gly Lys Thr Val Thr625 630 635 640Leu
Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr Lys Asn Asp645 650
655Gly Val Tyr Ser Arg Phe Phe Thr Ala Phe Asp Ala Asn Gly Arg
Tyr660 665 670Ser Val Lys Ile Trp Ala Leu Gly Gly Val Thr Ser Asp
Arg Gln Arg675 680 685Ala Ala Pro Pro Lys Asn Arg Ala Met Tyr Ile
Asp Gly Trp Ile Glu690 695 700Asp Gly Glu Val Arg Met Asn Pro Pro
Arg Pro Glu Thr Ser Tyr Val705 710 715 720Gln Asp Lys Gln Leu Cys
Phe Ser Arg Thr Ser Ser Gly Gly Ser Phe725 730 735Val Ala Thr Asn
Val Pro Ala Ala Ala Pro Ile Pro Asp Leu Phe Pro740 745 750Pro Cys
Gln Ile Thr Asp Leu Lys Ala Ser Ile Gln Gly Gln Asn Leu755 760
765Val Asn Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp His Gly
Arg770 775 780Ala Ser Asn Tyr Ile Ile Arg Met Ser Thr Ser Ile Val
Asp Leu Arg785 790 795 800Asp His Phe Asn Thr Ser Leu Gln Val Asn
Thr Thr Gly Leu Ile Pro805 810 815Lys Glu Ala Ser Ser Glu Glu Ile
Phe Glu Phe Glu Leu Gly Gly Asn820 825 830Thr Phe Gly Asn Gly Thr
Asp Ile Phe Ile Ala Ile Gln Ala Val Asp835 840 845Lys Ser Asn Leu
Lys Ser Glu Ile Ser Asn Ile Ala Arg Val Ser Val850 855 860Phe Ile
Pro Ala Gln Glu Pro Pro Ile Pro Glu Asp Ser Thr Pro Pro865 870 875
880Cys Pro Asp Ile Ser Ile Asn Ser Thr Ile Pro Gly Ile His Val
Leu885 890 895Lys Ile Met Trp Lys Trp Leu Gly Glu Met Gln Val Thr
Leu Gly Leu900 905 910His33190DNAHomo sapiensCDS(120)..(2948)
3cttcttgtgt tcttaaaccc ttgcaagttc agraagaaac ccatctgcat ccatattgaa
60aacctgacac aatgtatgca gcaggctcag tgtgagtgaa ctggaggctt ctctacaac
119atg acc caa agg agc att gca ggt cct att tgc aac ctg aag ttt gtg
167Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cys Asn Leu Lys Phe Val1
5 10 15act ctc ctg gtt gcc tta agt tca gaa ctc cca ttc ctg gga gct
gga 215Thr Leu Leu Val Ala Leu Ser Ser Glu Leu Pro Phe Leu Gly Ala
Gly20 25 30gta cag ctt caa gac aat ggg tat aat gga ttg ctc att gca
att aat 263Val Gln Leu Gln Asp Asn Gly Tyr Asn Gly Leu Leu Ile Ala
Ile Asn35 40 45cct cag gta cct gag aat cag aac ctc atc tca aac att
aag gaa atg 311Pro Gln Val Pro Glu Asn Gln Asn Leu Ile Ser Asn Ile
Lys Glu Met50 55 60ata act gaa gct tca ttt tac cta ttt aat gct acc
aag aga aga gta 359Ile Thr Glu Ala Ser Phe Tyr Leu Phe Asn Ala Thr
Lys Arg Arg Val65 70 75 80ttt ttc aga aat ata aag att tta ata cct
gcc aca tgg aaa gct aat 407Phe Phe Arg Asn Ile Lys Ile Leu Ile Pro
Ala Thr Trp Lys Ala Asn85
90 95aat aac agc aaa ata aaa caa gaa tca tat gaa aag gca aat gtc
ata 455Asn Asn Ser Lys Ile Lys Gln Glu Ser Tyr Glu Lys Ala Asn Val
Ile100 105 110gtg act gac tgg tat agg gca cat gga gat gat cca tac
acc cta caa 503Val Thr Asp Trp Tyr Arg Ala His Gly Asp Asp Pro Tyr
Thr Leu Gln115 120 125tac aga ggg tgt gga aaa gag gga aaa tac att
cat ttc aca cct aat 551Tyr Arg Gly Cys Gly Lys Glu Gly Lys Tyr Ile
His Phe Thr Pro Asn130 135 140ttc cta ctg aat gat aac tta aca gct
ggc tac gga tca cga ggc cga 599Phe Leu Leu Asn Asp Asn Leu Thr Ala
Gly Tyr Gly Ser Arg Gly Arg145 150 155 160gtg ttt gtc cat gaa tgg
gcc cac ctc cgt tgg ggt gtg ttc gat gag 647Val Phe Val His Glu Trp
Ala His Leu Arg Trp Gly Val Phe Asp Glu165 170 175tat aac aat gac
aaa cct ttc tac ata aat ggg caa aat caa att aaa 695Tyr Asn Asn Asp
Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Ile Lys180 185 190gtg aca
agg tgt tca tct gac atc aca ggc att ttt gtg tgt gaa aaa 743Val Thr
Arg Cys Ser Ser Asp Ile Thr Gly Ile Phe Val Cys Glu Lys195 200
205ggt cct tgc ccc caa gaa aac tgt att att agt aag ctt ttt aaa gaa
791Gly Pro Cys Pro Gln Glu Asn Cys Ile Ile Ser Lys Leu Phe Lys
Glu210 215 220gga tgc acc ttt atc tac aat agc acc caa agt gca act
gca tca ata 839Gly Cys Thr Phe Ile Tyr Asn Ser Thr Gln Ser Ala Thr
Ala Ser Ile225 230 235 240atg ttc atg cga agt tta tct tct gtg gtt
gaa ttt tgt aat gca agt 887Met Phe Met Arg Ser Leu Ser Ser Val Val
Glu Phe Cys Asn Ala Ser245 250 255acc cac aac caa gaa gca cca aac
cta cag aac cag atg tgc agc ctc 935Thr His Asn Gln Glu Ala Pro Asn
Leu Gln Asn Gln Met Cys Ser Leu260 265 270aga agt gca tgg gat gta
atc aca gac tct gct gac ttt cac cac agc 983Arg Ser Ala Trp Asp Val
Ile Thr Asp Ser Ala Asp Phe His His Ser275 280 285ttt ccc atg aac
ggg act gag ctt cca cct cct ccc aca ttc tcg ctt 1031Phe Pro Met Asn
Gly Thr Glu Leu Pro Pro Pro Pro Thr Phe Ser Leu290 295 300gta gag
gct ggt gac aaa gtg gtc tgt tta gtg ctg gat gcg tcc agc 1079Val Glu
Ala Gly Asp Lys Val Val Cys Leu Val Leu Asp Ala Ser Ser305 310 315
320aag atg gca gag gct gac aga ctc ctt caa cta caa caa gcc gca gaa
1127Lys Met Ala Glu Ala Asp Arg Leu Leu Gln Leu Gln Gln Ala Ala
Glu325 330 335ttt tat ttg atg cag att gtt gaa att cat acc ttc gtg
ggc att gcc 1175Phe Tyr Leu Met Gln Ile Val Glu Ile His Thr Phe Val
Gly Ile Ala340 345 350agt ttc gac agc aaa gga gag atc aga gcc cag
cta cac caa att aac 1223Ser Phe Asp Ser Lys Gly Glu Ile Arg Ala Gln
Leu His Gln Ile Asn355 360 365agc aat gat gat cga aag ttg ctg gtt
tca tat ctg ccc acc act gta 1271Ser Asn Asp Asp Arg Lys Leu Leu Val
Ser Tyr Leu Pro Thr Thr Val370 375 380tca gct aaa aca gac atc agc
att tgt tca ggg ctt aag aaa gga ttt 1319Ser Ala Lys Thr Asp Ile Ser
Ile Cys Ser Gly Leu Lys Lys Gly Phe385 390 395 400gag gtg gtt gaa
aaa ctg aat gga aaa gct tat ggc tct gtg atg ata 1367Glu Val Val Glu
Lys Leu Asn Gly Lys Ala Tyr Gly Ser Val Met Ile405 410 415tta gtg
acc agc gga gat gat aag ctt ctt ggc aat tgc tta ccc act 1415Leu Val
Thr Ser Gly Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr420 425
430gtg ctc agc agt ggt tca aca att cac tcc att gcc ctg ggt tca tct
1463Val Leu Ser Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser
Ser435 440 445gca gcc cca aat ctg gag gaa tta tca cgt ctt aca gga
ggt tta aag 1511Ala Ala Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr Gly
Gly Leu Lys450 455 460ttc ttt gtt cca gat ata tca aac tcc aat agc
atg att gat gct ttc 1559Phe Phe Val Pro Asp Ile Ser Asn Ser Asn Ser
Met Ile Asp Ala Phe465 470 475 480agt aga att tcc tct gga act gga
gac att ttc cag caa cat att cag 1607Ser Arg Ile Ser Ser Gly Thr Gly
Asp Ile Phe Gln Gln His Ile Gln485 490 495ctt gaa agt aca ggt gaa
aat gtc aaa cct cac cat caa ttg aaa aac 1655Leu Glu Ser Thr Gly Glu
Asn Val Lys Pro His His Gln Leu Lys Asn500 505 510aca gtg act gtg
gat aat act gtg ggc aac gac act atg ttt cta gtt 1703Thr Val Thr Val
Asp Asn Thr Val Gly Asn Asp Thr Met Phe Leu Val515 520 525acg tgg
cag gcc agt ggt cct cct gag att ata tta ttt gat cct gat 1751Thr Trp
Gln Ala Ser Gly Pro Pro Glu Ile Ile Leu Phe Asp Pro Asp530 535
540gga cga aaa tac tac aca aat aat ttt atc acc aat cta act ttt cgg
1799Gly Arg Lys Tyr Tyr Thr Asn Asn Phe Ile Thr Asn Leu Thr Phe
Arg545 550 555 560aca gct agt ctt tgg att cca gga aca gct aag cct
ggg cac tgg act 1847Thr Ala Ser Leu Trp Ile Pro Gly Thr Ala Lys Pro
Gly His Trp Thr565 570 575tac acc ctg aac aat acc cat cat tct ctg
caa gcc ctg aaa gtg aca 1895Tyr Thr Leu Asn Asn Thr His His Ser Leu
Gln Ala Leu Lys Val Thr580 585 590gtg acc tct cgt gcc tcc aac tca
gct gtg ccc cca gcc act gtg gaa 1943Val Thr Ser Arg Ala Ser Asn Ser
Ala Val Pro Pro Ala Thr Val Glu595 600 605gcc ttt gtg gaa aga gac
agc ctc cat ttt cct cat cct gtg atg att 1991Ala Phe Val Glu Arg Asp
Ser Leu His Phe Pro His Pro Val Met Ile610 615 620tat gcc aat gtg
aaa cag gga ttt tat ccc att ctt aat gcc act gtc 2039Tyr Ala Asn Val
Lys Gln Gly Phe Tyr Pro Ile Leu Asn Ala Thr Val625 630 635 640act
gcc aca gtt gag cca gag act gga gat cct gtt acg ctg aga ctc 2087Thr
Ala Thr Val Glu Pro Glu Thr Gly Asp Pro Val Thr Leu Arg Leu645 650
655ctt gat gat gga gca ggt gct gat gtt ata aaa aat gat gga att tac
2135Leu Asp Asp Gly Ala Gly Ala Asp Val Ile Lys Asn Asp Gly Ile
Tyr660 665 670tcg agg tat ttt ttc tcc ttt gct gca aat ggt aga tat
agc ttg aaa 2183Ser Arg Tyr Phe Phe Ser Phe Ala Ala Asn Gly Arg Tyr
Ser Leu Lys675 680 685gtg cat gtc aat cac tct ccc agc ata agc acc
cca gcc cac tct att 2231Val His Val Asn His Ser Pro Ser Ile Ser Thr
Pro Ala His Ser Ile690 695 700cca ggg agt cat gct atg tat gta cca
ggt tac aca gca aac ggt aat 2279Pro Gly Ser His Ala Met Tyr Val Pro
Gly Tyr Thr Ala Asn Gly Asn705 710 715 720att cag atg aat gct cca
agg aaa tca gta ggc aga aat gag gag gag 2327Ile Gln Met Asn Ala Pro
Arg Lys Ser Val Gly Arg Asn Glu Glu Glu725 730 735cga aag tgg ggc
ttt agc cga gtc agc tca gga ggc tcc ttt tca gtg 2375Arg Lys Trp Gly
Phe Ser Arg Val Ser Ser Gly Gly Ser Phe Ser Val740 745 750ctg gga
gtt cca gct ggc ccc cac cct gat gtg ttt cca cca tgc aaa 2423Leu Gly
Val Pro Ala Gly Pro His Pro Asp Val Phe Pro Pro Cys Lys755 760
765att att gac ctg gaa gct gta aaa gta gaa gag gaa ttg acc cta tct
2471Ile Ile Asp Leu Glu Ala Val Lys Val Glu Glu Glu Leu Thr Leu
Ser770 775 780tgg aca gca cct gga gaa gac ttt gat cag ggc cag gct
aca agc tat 2519Trp Thr Ala Pro Gly Glu Asp Phe Asp Gln Gly Gln Ala
Thr Ser Tyr785 790 795 800gaa ata aga atg agt aaa agt cta cag aat
atc caa gat gac ttt aac 2567Glu Ile Arg Met Ser Lys Ser Leu Gln Asn
Ile Gln Asp Asp Phe Asn805 810 815aat gct att tta gta aat aca tca
aag cga aat cct cag caa gct ggc 2615Asn Ala Ile Leu Val Asn Thr Ser
Lys Arg Asn Pro Gln Gln Ala Gly820 825 830atc agg gag ata ttt acg
ttc tca ccc cag att tcc acg aat gga cct 2663Ile Arg Glu Ile Phe Thr
Phe Ser Pro Gln Ile Ser Thr Asn Gly Pro835 840 845gaa cat cag cca
aat gga gaa aca cat gaa agc cac aga att tat gtt 2711Glu His Gln Pro
Asn Gly Glu Thr His Glu Ser His Arg Ile Tyr Val850 855 860gca ata
cga gca atg gat agg aac tcc tta cag tct gct gta tct aac 2759Ala Ile
Arg Ala Met Asp Arg Asn Ser Leu Gln Ser Ala Val Ser Asn865 870 875
880att gcc cag gcg cct ctg ttt att ccc ccc aat tct gat cct gta cct
2807Ile Ala Gln Ala Pro Leu Phe Ile Pro Pro Asn Ser Asp Pro Val
Pro885 890 895gcc aga gat tat ctt ata ttg aaa gga gtt tta aca gca
atg ggt ttg 2855Ala Arg Asp Tyr Leu Ile Leu Lys Gly Val Leu Thr Ala
Met Gly Leu900 905 910ata gga atc att tgc ctt att ata gtt gtg aca
cat cat act tta agc 2903Ile Gly Ile Ile Cys Leu Ile Ile Val Val Thr
His His Thr Leu Ser915 920 925agg aaa aag aga gca gac aag aaa gag
aat gga aca aaa tta tta 2948Arg Lys Lys Arg Ala Asp Lys Lys Glu Asn
Gly Thr Lys Leu Leu930 935 940taaataaata tccaaagtgt cttccttctt
agatataaga cccatggcct tcgactacaa 3008aaacatacta acaaagtcaa
attaacatca aaactgtatt aaaatgcatt gagttttgta 3068caatacagat
aagattttta catggtagat caacaaattc tttttggggg tagattagaa
3128aaccttacac tttggctatg aacaaataat aaaaattatt ctttaaaaaa
aaaaaaaaaa 3188aa 31904943PRTHomo sapiens 4Met Thr Gln Arg Ser Ile
Ala Gly Pro Ile Cys Asn Leu Lys Phe Val1 5 10 15Thr Leu Leu Val Ala
Leu Ser Ser Glu Leu Pro Phe Leu Gly Ala Gly20 25 30Val Gln Leu Gln
Asp Asn Gly Tyr Asn Gly Leu Leu Ile Ala Ile Asn35 40 45Pro Gln Val
Pro Glu Asn Gln Asn Leu Ile Ser Asn Ile Lys Glu Met50 55 60Ile Thr
Glu Ala Ser Phe Tyr Leu Phe Asn Ala Thr Lys Arg Arg Val65 70 75
80Phe Phe Arg Asn Ile Lys Ile Leu Ile Pro Ala Thr Trp Lys Ala Asn85
90 95Asn Asn Ser Lys Ile Lys Gln Glu Ser Tyr Glu Lys Ala Asn Val
Ile100 105 110Val Thr Asp Trp Tyr Arg Ala His Gly Asp Asp Pro Tyr
Thr Leu Gln115 120 125Tyr Arg Gly Cys Gly Lys Glu Gly Lys Tyr Ile
His Phe Thr Pro Asn130 135 140Phe Leu Leu Asn Asp Asn Leu Thr Ala
Gly Tyr Gly Ser Arg Gly Arg145 150 155 160Val Phe Val His Glu Trp
Ala His Leu Arg Trp Gly Val Phe Asp Glu165 170 175Tyr Asn Asn Asp
Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Ile Lys180 185 190Val Thr
Arg Cys Ser Ser Asp Ile Thr Gly Ile Phe Val Cys Glu Lys195 200
205Gly Pro Cys Pro Gln Glu Asn Cys Ile Ile Ser Lys Leu Phe Lys
Glu210 215 220Gly Cys Thr Phe Ile Tyr Asn Ser Thr Gln Ser Ala Thr
Ala Ser Ile225 230 235 240Met Phe Met Arg Ser Leu Ser Ser Val Val
Glu Phe Cys Asn Ala Ser245 250 255Thr His Asn Gln Glu Ala Pro Asn
Leu Gln Asn Gln Met Cys Ser Leu260 265 270Arg Ser Ala Trp Asp Val
Ile Thr Asp Ser Ala Asp Phe His His Ser275 280 285Phe Pro Met Asn
Gly Thr Glu Leu Pro Pro Pro Pro Thr Phe Ser Leu290 295 300Val Glu
Ala Gly Asp Lys Val Val Cys Leu Val Leu Asp Ala Ser Ser305 310 315
320Lys Met Ala Glu Ala Asp Arg Leu Leu Gln Leu Gln Gln Ala Ala
Glu325 330 335Phe Tyr Leu Met Gln Ile Val Glu Ile His Thr Phe Val
Gly Ile Ala340 345 350Ser Phe Asp Ser Lys Gly Glu Ile Arg Ala Gln
Leu His Gln Ile Asn355 360 365Ser Asn Asp Asp Arg Lys Leu Leu Val
Ser Tyr Leu Pro Thr Thr Val370 375 380Ser Ala Lys Thr Asp Ile Ser
Ile Cys Ser Gly Leu Lys Lys Gly Phe385 390 395 400Glu Val Val Glu
Lys Leu Asn Gly Lys Ala Tyr Gly Ser Val Met Ile405 410 415Leu Val
Thr Ser Gly Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr420 425
430Val Leu Ser Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser
Ser435 440 445Ala Ala Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr Gly
Gly Leu Lys450 455 460Phe Phe Val Pro Asp Ile Ser Asn Ser Asn Ser
Met Ile Asp Ala Phe465 470 475 480Ser Arg Ile Ser Ser Gly Thr Gly
Asp Ile Phe Gln Gln His Ile Gln485 490 495Leu Glu Ser Thr Gly Glu
Asn Val Lys Pro His His Gln Leu Lys Asn500 505 510Thr Val Thr Val
Asp Asn Thr Val Gly Asn Asp Thr Met Phe Leu Val515 520 525Thr Trp
Gln Ala Ser Gly Pro Pro Glu Ile Ile Leu Phe Asp Pro Asp530 535
540Gly Arg Lys Tyr Tyr Thr Asn Asn Phe Ile Thr Asn Leu Thr Phe
Arg545 550 555 560Thr Ala Ser Leu Trp Ile Pro Gly Thr Ala Lys Pro
Gly His Trp Thr565 570 575Tyr Thr Leu Asn Asn Thr His His Ser Leu
Gln Ala Leu Lys Val Thr580 585 590Val Thr Ser Arg Ala Ser Asn Ser
Ala Val Pro Pro Ala Thr Val Glu595 600 605Ala Phe Val Glu Arg Asp
Ser Leu His Phe Pro His Pro Val Met Ile610 615 620Tyr Ala Asn Val
Lys Gln Gly Phe Tyr Pro Ile Leu Asn Ala Thr Val625 630 635 640Thr
Ala Thr Val Glu Pro Glu Thr Gly Asp Pro Val Thr Leu Arg Leu645 650
655Leu Asp Asp Gly Ala Gly Ala Asp Val Ile Lys Asn Asp Gly Ile
Tyr660 665 670Ser Arg Tyr Phe Phe Ser Phe Ala Ala Asn Gly Arg Tyr
Ser Leu Lys675 680 685Val His Val Asn His Ser Pro Ser Ile Ser Thr
Pro Ala His Ser Ile690 695 700Pro Gly Ser His Ala Met Tyr Val Pro
Gly Tyr Thr Ala Asn Gly Asn705 710 715 720Ile Gln Met Asn Ala Pro
Arg Lys Ser Val Gly Arg Asn Glu Glu Glu725 730 735Arg Lys Trp Gly
Phe Ser Arg Val Ser Ser Gly Gly Ser Phe Ser Val740 745 750Leu Gly
Val Pro Ala Gly Pro His Pro Asp Val Phe Pro Pro Cys Lys755 760
765Ile Ile Asp Leu Glu Ala Val Lys Val Glu Glu Glu Leu Thr Leu
Ser770 775 780Trp Thr Ala Pro Gly Glu Asp Phe Asp Gln Gly Gln Ala
Thr Ser Tyr785 790 795 800Glu Ile Arg Met Ser Lys Ser Leu Gln Asn
Ile Gln Asp Asp Phe Asn805 810 815Asn Ala Ile Leu Val Asn Thr Ser
Lys Arg Asn Pro Gln Gln Ala Gly820 825 830Ile Arg Glu Ile Phe Thr
Phe Ser Pro Gln Ile Ser Thr Asn Gly Pro835 840 845Glu His Gln Pro
Asn Gly Glu Thr His Glu Ser His Arg Ile Tyr Val850 855 860Ala Ile
Arg Ala Met Asp Arg Asn Ser Leu Gln Ser Ala Val Ser Asn865 870 875
880Ile Ala Gln Ala Pro Leu Phe Ile Pro Pro Asn Ser Asp Pro Val
Pro885 890 895Ala Arg Asp Tyr Leu Ile Leu Lys Gly Val Leu Thr Ala
Met Gly Leu900 905 910Ile Gly Ile Ile Cys Leu Ile Ile Val Val Thr
His His Thr Leu Ser915 920 925Arg Lys Lys Arg Ala Asp Lys Lys Glu
Asn Gly Thr Lys Leu Leu930 935 94052745DNAHomo
sapiensCDS(1)..(2742) 5atg ggg cca ttt aag agt tct gtg ttc atc ttg
att ctt cac ctt cta 48Met Gly Pro Phe Lys Ser Ser Val Phe Ile Leu
Ile Leu His Leu Leu1 5 10 15gaa ggg gcc ctg agt aat tca ctc att cag
ctg aac aac aat ggc tat 96Glu Gly Ala Leu Ser Asn Ser Leu Ile Gln
Leu Asn Asn Asn Gly Tyr20 25 30gaa ggc att gtc gtt gca atc gac ccc
aat gtg cca gaa gat gaa aca 144Glu Gly Ile Val Val Ala Ile Asp Pro
Asn Val Pro Glu Asp Glu Thr35 40 45ctc att caa caa ata aag gac atg
gtg acc cag gca tct ctg tat ctg 192Leu Ile Gln Gln Ile Lys Asp Met
Val Thr Gln Ala Ser Leu Tyr Leu50 55 60ttt gaa gct aca gga aag cga
ttt tat ttc aaa aat gtt gcc att ttg 240Phe Glu Ala Thr Gly Lys Arg
Phe Tyr Phe Lys Asn Val Ala Ile Leu65 70 75 80att cct gaa aca tgg
aag aca aag gct gac tat gtg aga cca aaa ctt 288Ile Pro Glu Thr Trp
Lys Thr Lys Ala Asp Tyr Val Arg Pro Lys Leu85 90 95gag acc tac aaa
aat gct gat gtt ctg gtt gct gag tct act cct cca 336Glu Thr Tyr Lys
Asn Ala Asp Val Leu Val Ala Glu Ser Thr Pro Pro100 105 110ggt aat
gat gaa ccc tac act gag cag atg ggc aac tgt gga gag aag 384Gly Asn
Asp Glu Pro Tyr Thr Glu Gln Met Gly Asn Cys Gly Glu Lys115 120
125ggt gaa agg atc cac ctc act cct gat ttc att gca gga aaa aag tta
432Gly Glu Arg Ile His Leu Thr Pro Asp Phe Ile Ala Gly Lys Lys
Leu130 135 140gct gaa tat gga cca caa ggt agg gca ttt gtc cat gag
tgg gct cat 480Ala
Glu Tyr Gly Pro Gln Gly Arg Ala Phe Val His Glu Trp Ala His145 150
155 160cta cga tgg gga gta ttt gac gag tac aat aat gat gag aaa ttc
tac 528Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Asn Asp Glu Lys Phe
Tyr165 170 175tta tcc aat gga aga ata caa gca gta aga tgt tca gca
ggt att act 576Leu Ser Asn Gly Arg Ile Gln Ala Val Arg Cys Ser Ala
Gly Ile Thr180 185 190ggt aca aat gta gta aag aag tgt cag gga ggc
agc tgt tac acc aaa 624Gly Thr Asn Val Val Lys Lys Cys Gln Gly Gly
Ser Cys Tyr Thr Lys195 200 205aga tgc aca ttc aat aaa gtw aca gga
ctc tat gaa aaa gga tgt gag 672Arg Cys Thr Phe Asn Lys Xaa Thr Gly
Leu Tyr Glu Lys Gly Cys Glu210 215 220ttt gtt ctc caa tcc cgc cag
acg gag aag gct tct ata atg ttt gca 720Phe Val Leu Gln Ser Arg Gln
Thr Glu Lys Ala Ser Ile Met Phe Ala225 230 235 240caa cat gtt gat
tct ata gtt gaa ttc tgt aca gaa caa aac cac aac 768Gln His Val Asp
Ser Ile Val Glu Phe Cys Thr Glu Gln Asn His Asn245 250 255aaa gaa
gct cca aac aag caa aat caa aaa tgc aat ctc cga agc aca 816Lys Glu
Ala Pro Asn Lys Gln Asn Gln Lys Cys Asn Leu Arg Ser Thr260 265
270tgg gaa gtg atc cgt gat tct gag gac ttt aag aaa acc act cct atg
864Trp Glu Val Ile Arg Asp Ser Glu Asp Phe Lys Lys Thr Thr Pro
Met275 280 285aca aca cag cca cca aat ccc acc ttc tca ttg ctg cag
att gga caa 912Thr Thr Gln Pro Pro Asn Pro Thr Phe Ser Leu Leu Gln
Ile Gly Gln290 295 300aga att gtg tgt tta gtc ctt gac aaa tct gga
agc atg gcg act ggt 960Arg Ile Val Cys Leu Val Leu Asp Lys Ser Gly
Ser Met Ala Thr Gly305 310 315 320aac cgc ctc aat cga ctg aat caa
gca ggc cag ctt ttc ctg ctg cag 1008Asn Arg Leu Asn Arg Leu Asn Gln
Ala Gly Gln Leu Phe Leu Leu Gln325 330 335aca gtt gag ctg ggg tcc
tgg gtt ggg atg gtg aca ttt gac agt gct 1056Thr Val Glu Leu Gly Ser
Trp Val Gly Met Val Thr Phe Asp Ser Ala340 345 350gcc cat gta caa
agt gaa ctc ata cag ata aac agt ggc agt gac agg 1104Ala His Val Gln
Ser Glu Leu Ile Gln Ile Asn Ser Gly Ser Asp Arg355 360 365gac aca
ctc gcc aaa aga tta cct gca gca gct tca gga ggg acg tcc 1152Asp Thr
Leu Ala Lys Arg Leu Pro Ala Ala Ala Ser Gly Gly Thr Ser370 375
380atc tgc agc ggg ctt cga tcg gca ttt act gtg att agg aag aaa tat
1200Ile Cys Ser Gly Leu Arg Ser Ala Phe Thr Val Ile Arg Lys Lys
Tyr385 390 395 400cca act gat gga tct gaa att gtg ctg ctg acg gat
ggg gaa gac aac 1248Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr Asp
Gly Glu Asp Asn405 410 415act ata agt ggg tgc ttt aac gag gtc aaa
caa agt ggt gcc atc atc 1296Thr Ile Ser Gly Cys Phe Asn Glu Val Lys
Gln Ser Gly Ala Ile Ile420 425 430cac aca gtc gct ttg ggg ccc tct
gca gct caa gaa cta gag gag ctg 1344His Thr Val Ala Leu Gly Pro Ser
Ala Ala Gln Glu Leu Glu Glu Leu435 440 445tcc aaa atg aca gga ggt
tta cag aca tat gct tca gat caa gtt cag 1392Ser Lys Met Thr Gly Gly
Leu Gln Thr Tyr Ala Ser Asp Gln Val Gln450 455 460aac aat ggc ctc
att gat gct ttt ggg gcc ctt tca tca gga aat gga 1440Asn Asn Gly Leu
Ile Asp Ala Phe Gly Ala Leu Ser Ser Gly Asn Gly465 470 475 480gct
gtc tct cag cgc tcc atc cag ctt gag agt aag gga tta acc ctc 1488Ala
Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly Leu Thr Leu485 490
495cag aac agc cag tgg atg aat ggc aca gtg atc gtg gac agc acc gtg
1536Gln Asn Ser Gln Trp Met Asn Gly Thr Val Ile Val Asp Ser Thr
Val500 505 510gga aag gac act ttg ttt ctt atc acc tgg aca acg cag
cct ccc caa 1584Gly Lys Asp Thr Leu Phe Leu Ile Thr Trp Thr Thr Gln
Pro Pro Gln515 520 525atc ctt ctc tgg gat ccc agt gga cag aag caa
ggt ggc ttt gta gtg 1632Ile Leu Leu Trp Asp Pro Ser Gly Gln Lys Gln
Gly Gly Phe Val Val530 535 540gac aaa aac acc aaa atg gcc tac ctc
caa atc cca ggc att gct aag 1680Asp Lys Asn Thr Lys Met Ala Tyr Leu
Gln Ile Pro Gly Ile Ala Lys545 550 555 560gtt ggc act tgg aaa tac
agt ctg caa gca agc tca caa acc ttg acc 1728Val Gly Thr Trp Lys Tyr
Ser Leu Gln Ala Ser Ser Gln Thr Leu Thr565 570 575ctg act gtc acg
tcc cgt gcg tcc aat gct acc ctg cct cca att aca 1776Leu Thr Val Thr
Ser Arg Ala Ser Asn Ala Thr Leu Pro Pro Ile Thr580 585 590gtg act
tcc aaa acg aac aag gac acc agc aaa ttc ccc agc cct ctg 1824Val Thr
Ser Lys Thr Asn Lys Asp Thr Ser Lys Phe Pro Ser Pro Leu595 600
605gta gtt tat gca aat att cgc caa gga gcc tcc cca att ctc agg gcc
1872Val Val Tyr Ala Asn Ile Arg Gln Gly Ala Ser Pro Ile Leu Arg
Ala610 615 620agt gtc aca gcc ctg att gaa tca gtg aat gga aaa aca
gtt acc ttg 1920Ser Val Thr Ala Leu Ile Glu Ser Val Asn Gly Lys Thr
Val Thr Leu625 630 635 640gaa cta ctg gat aat gga gca ggt gct gat
gct act aag gat gac ggt 1968Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp
Ala Thr Lys Asp Asp Gly645 650 655gtc tac tca agg tat ttc aca act
tat gac acg aat ggt aga tac agt 2016Val Tyr Ser Arg Tyr Phe Thr Thr
Tyr Asp Thr Asn Gly Arg Tyr Ser660 665 670gta aaa gtg cgg gct ctg
gga gga gtt aac gca gcc aga cgg aga gtg 2064Val Lys Val Arg Ala Leu
Gly Gly Val Asn Ala Ala Arg Arg Arg Val675 680 685ata ccc cag cag
agt gga gca ctg tac ata cct ggc tgg att gag aat 2112Ile Pro Gln Gln
Ser Gly Ala Leu Tyr Ile Pro Gly Trp Ile Glu Asn690 695 700gat gaa
atc caa tgg aat cca cca aga cct gaa att aat aag gat gat 2160Asp Glu
Ile Gln Trp Asn Pro Pro Arg Pro Glu Ile Asn Lys Asp Asp705 710 715
720gtt caa cac aag caa gtg tgt ttc agc aga aca tcc tcg gga ggc tca
2208Val Gln His Lys Gln Val Cys Phe Ser Arg Thr Ser Ser Gly Gly
Ser725 730 735ttt gtg gct tct gat gtc cca aat gct ccc ata cct gat
ctc ttc cca 2256Phe Val Ala Ser Asp Val Pro Asn Ala Pro Ile Pro Asp
Leu Phe Pro740 745 750cct ggc caa atc acc gac ctg aag gcg gaa att
cac ggg ggc agt ctc 2304Pro Gly Gln Ile Thr Asp Leu Lys Ala Glu Ile
His Gly Gly Ser Leu755 760 765att aat ctg act tgg aca gct cct ggg
gat gat tat gac cat gga aca 2352Ile Asn Leu Thr Trp Thr Ala Pro Gly
Asp Asp Tyr Asp His Gly Thr770 775 780gct cac aag tat atc att cga
ata agt aca agt att ctt gat ctc aga 2400Ala His Lys Tyr Ile Ile Arg
Ile Ser Thr Ser Ile Leu Asp Leu Arg785 790 795 800gac aag ttc aat
gaa tct ctt caa gtg aat act act gct ctc atc cca 2448Asp Lys Phe Asn
Glu Ser Leu Gln Val Asn Thr Thr Ala Leu Ile Pro805 810 815aag gaa
gcc aac tct gag gaa gtc ttt ttg ttt aaa cca gaa aac att 2496Lys Glu
Ala Asn Ser Glu Glu Val Phe Leu Phe Lys Pro Glu Asn Ile820 825
830act ttt gaa aat ggc aca gat ctt ttc att gct att cag gct gtt gat
2544Thr Phe Glu Asn Gly Thr Asp Leu Phe Ile Ala Ile Gln Ala Val
Asp835 840 845aag gtc gat ctg aaa tca gaa ata tcc aac att gca cga
gta tct ttg 2592Lys Val Asp Leu Lys Ser Glu Ile Ser Asn Ile Ala Arg
Val Ser Leu850 855 860ttt att cct cca cag act ccg cca gag aca cct
agt cct gat gaa acg 2640Phe Ile Pro Pro Gln Thr Pro Pro Glu Thr Pro
Ser Pro Asp Glu Thr865 870 875 880tct gct cct tgt cct aat att cat
atc aac agc acc att cct ggc att 2688Ser Ala Pro Cys Pro Asn Ile His
Ile Asn Ser Thr Ile Pro Gly Ile885 890 895cac att tta aaa att atg
tgg aag tgg ata gga gaa ctg cag ctg tca 2736His Ile Leu Lys Ile Met
Trp Lys Trp Ile Gly Glu Leu Gln Leu Ser900 905 910ata gcc tag
2745Ile Ala6914PRTHomo sapiens 6Met Gly Pro Phe Lys Ser Ser Val Phe
Ile Leu Ile Leu His Leu Leu1 5 10 15Glu Gly Ala Leu Ser Asn Ser Leu
Ile Gln Leu Asn Asn Asn Gly Tyr20 25 30Glu Gly Ile Val Val Ala Ile
Asp Pro Asn Val Pro Glu Asp Glu Thr35 40 45Leu Ile Gln Gln Ile Lys
Asp Met Val Thr Gln Ala Ser Leu Tyr Leu50 55 60Phe Glu Ala Thr Gly
Lys Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu65 70 75 80Ile Pro Glu
Thr Trp Lys Thr Lys Ala Asp Tyr Val Arg Pro Lys Leu85 90 95Glu Thr
Tyr Lys Asn Ala Asp Val Leu Val Ala Glu Ser Thr Pro Pro100 105
110Gly Asn Asp Glu Pro Tyr Thr Glu Gln Met Gly Asn Cys Gly Glu
Lys115 120 125Gly Glu Arg Ile His Leu Thr Pro Asp Phe Ile Ala Gly
Lys Lys Leu130 135 140Ala Glu Tyr Gly Pro Gln Gly Arg Ala Phe Val
His Glu Trp Ala His145 150 155 160Leu Arg Trp Gly Val Phe Asp Glu
Tyr Asn Asn Asp Glu Lys Phe Tyr165 170 175Leu Ser Asn Gly Arg Ile
Gln Ala Val Arg Cys Ser Ala Gly Ile Thr180 185 190Gly Thr Asn Val
Val Lys Lys Cys Gln Gly Gly Ser Cys Tyr Thr Lys195 200 205Arg Cys
Thr Phe Asn Lys Xaa Thr Gly Leu Tyr Glu Lys Gly Cys Glu210 215
220Phe Val Leu Gln Ser Arg Gln Thr Glu Lys Ala Ser Ile Met Phe
Ala225 230 235 240Gln His Val Asp Ser Ile Val Glu Phe Cys Thr Glu
Gln Asn His Asn245 250 255Lys Glu Ala Pro Asn Lys Gln Asn Gln Lys
Cys Asn Leu Arg Ser Thr260 265 270Trp Glu Val Ile Arg Asp Ser Glu
Asp Phe Lys Lys Thr Thr Pro Met275 280 285Thr Thr Gln Pro Pro Asn
Pro Thr Phe Ser Leu Leu Gln Ile Gly Gln290 295 300Arg Ile Val Cys
Leu Val Leu Asp Lys Ser Gly Ser Met Ala Thr Gly305 310 315 320Asn
Arg Leu Asn Arg Leu Asn Gln Ala Gly Gln Leu Phe Leu Leu Gln325 330
335Thr Val Glu Leu Gly Ser Trp Val Gly Met Val Thr Phe Asp Ser
Ala340 345 350Ala His Val Gln Ser Glu Leu Ile Gln Ile Asn Ser Gly
Ser Asp Arg355 360 365Asp Thr Leu Ala Lys Arg Leu Pro Ala Ala Ala
Ser Gly Gly Thr Ser370 375 380Ile Cys Ser Gly Leu Arg Ser Ala Phe
Thr Val Ile Arg Lys Lys Tyr385 390 395 400Pro Thr Asp Gly Ser Glu
Ile Val Leu Leu Thr Asp Gly Glu Asp Asn405 410 415Thr Ile Ser Gly
Cys Phe Asn Glu Val Lys Gln Ser Gly Ala Ile Ile420 425 430His Thr
Val Ala Leu Gly Pro Ser Ala Ala Gln Glu Leu Glu Glu Leu435 440
445Ser Lys Met Thr Gly Gly Leu Gln Thr Tyr Ala Ser Asp Gln Val
Gln450 455 460Asn Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Ser Ser
Gly Asn Gly465 470 475 480Ala Val Ser Gln Arg Ser Ile Gln Leu Glu
Ser Lys Gly Leu Thr Leu485 490 495Gln Asn Ser Gln Trp Met Asn Gly
Thr Val Ile Val Asp Ser Thr Val500 505 510Gly Lys Asp Thr Leu Phe
Leu Ile Thr Trp Thr Thr Gln Pro Pro Gln515 520 525Ile Leu Leu Trp
Asp Pro Ser Gly Gln Lys Gln Gly Gly Phe Val Val530 535 540Asp Lys
Asn Thr Lys Met Ala Tyr Leu Gln Ile Pro Gly Ile Ala Lys545 550 555
560Val Gly Thr Trp Lys Tyr Ser Leu Gln Ala Ser Ser Gln Thr Leu
Thr565 570 575Leu Thr Val Thr Ser Arg Ala Ser Asn Ala Thr Leu Pro
Pro Ile Thr580 585 590Val Thr Ser Lys Thr Asn Lys Asp Thr Ser Lys
Phe Pro Ser Pro Leu595 600 605Val Val Tyr Ala Asn Ile Arg Gln Gly
Ala Ser Pro Ile Leu Arg Ala610 615 620Ser Val Thr Ala Leu Ile Glu
Ser Val Asn Gly Lys Thr Val Thr Leu625 630 635 640Glu Leu Leu Asp
Asn Gly Ala Gly Ala Asp Ala Thr Lys Asp Asp Gly645 650 655Val Tyr
Ser Arg Tyr Phe Thr Thr Tyr Asp Thr Asn Gly Arg Tyr Ser660 665
670Val Lys Val Arg Ala Leu Gly Gly Val Asn Ala Ala Arg Arg Arg
Val675 680 685Ile Pro Gln Gln Ser Gly Ala Leu Tyr Ile Pro Gly Trp
Ile Glu Asn690 695 700Asp Glu Ile Gln Trp Asn Pro Pro Arg Pro Glu
Ile Asn Lys Asp Asp705 710 715 720Val Gln His Lys Gln Val Cys Phe
Ser Arg Thr Ser Ser Gly Gly Ser725 730 735Phe Val Ala Ser Asp Val
Pro Asn Ala Pro Ile Pro Asp Leu Phe Pro740 745 750Pro Gly Gln Ile
Thr Asp Leu Lys Ala Glu Ile His Gly Gly Ser Leu755 760 765Ile Asn
Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr Asp His Gly Thr770 775
780Ala His Lys Tyr Ile Ile Arg Ile Ser Thr Ser Ile Leu Asp Leu
Arg785 790 795 800Asp Lys Phe Asn Glu Ser Leu Gln Val Asn Thr Thr
Ala Leu Ile Pro805 810 815Lys Glu Ala Asn Ser Glu Glu Val Phe Leu
Phe Lys Pro Glu Asn Ile820 825 830Thr Phe Glu Asn Gly Thr Asp Leu
Phe Ile Ala Ile Gln Ala Val Asp835 840 845Lys Val Asp Leu Lys Ser
Glu Ile Ser Asn Ile Ala Arg Val Ser Leu850 855 860Phe Ile Pro Pro
Gln Thr Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr865 870 875 880Ser
Ala Pro Cys Pro Asn Ile His Ile Asn Ser Thr Ile Pro Gly Ile885 890
895His Ile Leu Lys Ile Met Trp Lys Trp Ile Gly Glu Leu Gln Leu
Ser900 905 910Ile Ala724DNAArtificial SequenceDescription of
Artificial Sequence sense primer for mouse ICACC-1 RNA 7ccagatccac
accaaaacga gaag 24824DNAArtificial SequenceDescription of
Artificial Sequence anti-sense primer for mouse ICACC-1 RNA
8cactgtcaaa ggtcaccatc ccga 24920DNAArtificial SequenceDescription
of Artificial Sequence sense primer for human ICACC-1 RNA
9gattccagga acagctaagc 201022DNAArtificial SequenceDescription of
Artificial Sequence anti-sense primer for human ICACC-1 RNA
10tatttcatag cttgtagcct gg 221121DNAArtificial SequenceDescription
of Artificial Sequence PCR 5' primer for ICACC-1 11cccaaaggaa
gccaactctg a 211221DNAArtificial SequenceDescription of Artificial
Sequence PCR 3' primer for ICACC-1 12gtgaatgcca ggaatggtgc t
211322PRTArtificial SequenceDescription of Artificial Sequence
peptide for immunization to mICACC-1 13Cys Leu Val Leu Asp Lys Ser
Gly Ser Met Leu Asn Asp Asp Arg Leu1 5 10 15Asn Arg Met Asn Gln
Ala201420PRTArtificial SequenceDescription of Artificial Sequence
peptide for immunization to mICACC-1 14Gln Ser Glu Leu Lys Gln Leu
Asn Ser Gly Ala Asp Arg Asp Leu Leu1 5 10 15Ile Lys His
Cys201525PRTArtificial SequenceDescription of Artificial Sequence
peptide for immunization to mICACC-1 15Lys Lys Lys Tyr Pro Thr Asp
Gly Ser Glu Ile Val Leu Leu Thr Asp1 5 10 15Gly Glu Asp Asn Thr Ile
Ser Ser Cys20 251624PRTArtificial SequenceDescription of Artificial
Sequence peptide for immunization to mICACC-1 16Thr Thr His Pro Pro
Thr Ile Phe Ile Trp Asp Pro Ser Gly Val Glu1 5 10 15Gln Asn Gly Phe
Ile Leu Asp Cys201722PRTArtificial SequenceDescription of
Artificial Sequence peptide for immunization to mICACC-1 17Cys Pro
Pro Ile Thr Val Thr Pro Val Val Asn Lys Asn Thr Gly Lys1 5 10 15Phe
Pro Ser Pro Val Thr2018903PRTBos taurus 18Met Val Pro Arg Leu Thr
Val Ile Leu Phe Leu Thr Leu His Leu Leu1 5 10 15Pro Gly Met Lys Ser
Ser Met Val Asn Leu Ile Asn Asn Gly Tyr Asp20 25 30Gly Ile Val Ile
Ala Ile Asn Pro Ser Val Pro Glu Asp Glu Lys Leu35 40 45Ile Gln Asn
Ile Lys Glu Met Val Thr Glu Ala Ser Thr Tyr Leu Phe50 55 60His Ala
Thr Lys Arg Arg Val Tyr Phe Arg Asn Val Ser Ile Leu Ile65 70 75
80Pro Met Thr Trp Lys Ser Lys Ser Glu Tyr Leu Met Pro Lys Gln Glu85
90 95Ser Tyr Asp Gln Ala Glu Val Ile Val Ala Asn Pro Tyr Leu Lys
His100 105 110Gly Asp Asp Pro Tyr Thr Leu Gln Tyr Gly
Arg Cys Gly Glu Lys Gly115 120 125Gln Tyr Ile His Phe Thr Pro Asn
Phe Leu Leu Thr Asn Asn Leu Pro130 135 140Ile Tyr Gly Ser Arg Gly
Arg Ala Phe Val His Glu Trp Ala His Leu145 150 155 160Arg Trp Gly
Ile Phe Asp Glu Tyr Asn Gly Asp Gln Pro Phe Tyr Ile165 170 175Ser
Arg Arg Asn Thr Ile Glu Ala Thr Arg Cys Ser Thr His Ile Thr180 185
190Gly Thr Asn Val Ile Val Lys Cys Gln Gly Gly Ser Cys Ile Thr
Arg195 200 205Pro Cys Arg Arg Asp Ser Gln Thr Gly Leu Tyr Glu Ala
Lys Cys Thr210 215 220Phe Ile Pro Glu Lys Ser Gln Thr Ala Arg Glu
Ser Ile Met Phe Met225 230 235 240Gln Ser Leu His Ser Val Thr Glu
Phe Cys Thr Glu Lys Thr His Asn245 250 255Val Glu Ala Pro Asn Leu
Gln Asn Lys Met Cys Asn Gly Lys Ser Thr260 265 270Trp Asp Val Ile
Met Asn Ser Thr Asp Phe Gln Asn Thr Ser Pro Met275 280 285Thr Glu
Met Asn Pro Pro Thr Gln Pro Thr Phe Ser Leu Leu Lys Ser290 295
300Lys Gln Arg Val Val Cys Leu Val Leu Asp Lys Ser Gly Ser Met
Ser305 310 315 320Ser Glu Asp Arg Leu Phe Arg Met Asn Gln Ala Ala
Glu Leu Phe Leu325 330 335Ile Gln Ile Ile Glu Lys Gly Ser Leu Val
Gly Met Val Thr Phe Asp340 345 350Ser Val Ala Glu Ile Arg Asn Asn
Leu Thr Lys Ile Thr Asp Asp Asn355 360 365Val Tyr Glu Asn Ile Thr
Ala Asn Leu Pro Gln Glu Ala Asn Gly Gly370 375 380Thr Ser Ile Cys
Arg Gly Leu Lys Ala Gly Phe Gln Ala Ile Ile Gln385 390 395 400Ser
Gln Gln Ser Thr Ser Gly Ser Glu Ile Ile Leu Leu Thr Asp Gly405 410
415Glu Asp Asn Glu Ile His Ser Cys Ile Glu Glu Val Lys Gln Ser
Gly420 425 430Val Ile Ile His Thr Ile Ala Leu Gly Pro Ser Ala Ala
Lys Glu Leu435 440 445Glu Thr Leu Ser Asp Met Thr Gly Gly His Arg
Phe Tyr Ala Asn Lys450 455 460Asp Ile Asn Gly Leu Thr Asn Ala Phe
Ser Arg Ile Ser Ser Arg Ser465 470 475 480Gly Ser Ile Thr Gln Gln
Thr Ile Gln Leu Glu Ser Lys Ala Leu Ala485 490 495Ile Thr Glu Lys
Lys Trp Val Asn Gly Thr Val Pro Val Asp Ser Thr500 505 510Ile Gly
Asn Asp Thr Phe Phe Val Val Thr Trp Thr Ile Lys Lys Pro515 520
525Glu Ile Leu Leu Gln Asp Pro Lys Gly Lys Lys Tyr Lys Thr Ser
Asp530 535 540Phe Lys Glu Asp Lys Leu Asn Ile His Ser Ala Arg Leu
Arg Ile Pro545 550 555 560Gly Ile Ala Glu Thr Gly Thr Trp Thr Tyr
Ser Leu Leu Asn Asn His565 570 575Ala Ser Pro Gln Ile Leu Thr Val
Thr Val Thr Thr Arg Ala Arg Ser580 585 590Pro Thr Thr Pro Pro Val
Thr Ala Thr Ala His Met Ser Gln Asn Thr595 600 605Ala His Tyr Pro
Ser Pro Val Ile Val Tyr Ala Gln Val Ser Gln Gly610 615 620Phe Leu
Pro Val Leu Gly Ile Asn Val Thr Ala Ile Ile Glu Thr Glu625 630 635
640Asp Gly His Gln Val Thr Leu Glu Leu Trp Asp Asn Gly Ala Gly
Ala645 650 655Asp Thr Val Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe
Thr Asp Tyr660 665 670Arg Gly Asn Gly Arg Tyr Ser Leu Lys Val His
Ala Glu Ala Arg Asn675 680 685Asn Thr Ala Arg Leu Ser Leu Arg Gln
Pro Gln Asn Lys Ala Leu Tyr690 695 700Ile Pro Gly Tyr Ile Glu Asn
Gly Lys Ile Ile Leu Asn Pro Pro Arg705 710 715 720Pro Glu Val Lys
Asp Asp Leu Ala Lys Ala Glu Ile Glu Asp Phe Ser725 730 735Arg Leu
Thr Ser Gly Gly Ser Phe Thr Val Ser Gly Ala Pro Pro Gly740 745
750Asn His Pro Ser Val Leu Pro Pro Asn Lys Ile Ile Asp Leu Glu
Ala755 760 765Lys Phe Lys Glu Asp His Ile Gln Leu Ser Trp Thr Ala
Pro Ala Asn770 775 780Val Leu Asp Lys Gly Lys Ala Asn Ser Tyr Ile
Ile Arg Ile Ser Lys785 790 795 800Ser Phe Leu Asp Leu Gln Lys Asp
Phe Asp Asn Ala Thr Leu Val Asn805 810 815Thr Ser Ser Leu Lys Pro
Lys Glu Ala Gly Ser Asp Glu Asn Phe Glu820 825 830Phe Lys Pro Glu
Pro Phe Arg Ile Glu Asn Gly Thr Asn Phe Tyr Ile835 840 845Ala Val
Gln Ala Ile Asn Glu Ala Asn Leu Thr Ser Glu Val Ser Asn850 855
860Ile Ala Gln Ala Ile Lys Phe Ile Pro Met Pro Glu Asp Ser Val
Pro865 870 875 880Ala Leu Gly Thr Lys Ile Ser Ala Ile Asn Leu Ala
Ile Phe Ala Leu885 890 895Ala Met Ile Leu Ser Ile Val900
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