U.S. patent application number 12/762532 was filed with the patent office on 2011-09-15 for asthma associated factors as targets for treating atopic allergies including asthma and related disorders.
Invention is credited to U. Prasad Kari, Roy Clifford Levitt, W. Lee Maloy, Nicholas C. Nicolaides.
Application Number | 20110224153 12/762532 |
Document ID | / |
Family ID | 21702397 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110224153 |
Kind Code |
A1 |
Levitt; Roy Clifford ; et
al. |
September 15, 2011 |
Asthma Associated Factors as Targets for Treating Atopic Allergies
Including Asthma and Related Disorders
Abstract
A C to T DNA variation at position 3365 in exon 5 of the human
Asthma Associated Factor 1 (AAF1) produces the predicted amino acid
substitution of a methionine for a threonine at codon 117 of AAF1.
When this substitution occurs in both alleles in one individual, it
is associated with less evidence of atopic allergy including
asthma, fewer abnormal skin test responses, and a lower serum total
IgE. Thus, applicant has identified the existence of a
non-asthmatic, non-atopic phenotype characterized by methionine at
codon 117 when it occurs in both AAF1 gene products in one
individual.
Inventors: |
Levitt; Roy Clifford;
(Ambler, PA) ; Maloy; W. Lee; (Larsdale, PA)
; Kari; U. Prasad; (Hatfield, PA) ; Nicolaides;
Nicholas C.; (Media, PA) |
Family ID: |
21702397 |
Appl. No.: |
12/762532 |
Filed: |
April 19, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11714899 |
Mar 7, 2007 |
7700088 |
|
|
12762532 |
|
|
|
|
10642149 |
Aug 18, 2003 |
7192578 |
|
|
11714899 |
|
|
|
|
09848585 |
May 4, 2001 |
6645492 |
|
|
10642149 |
|
|
|
|
09325571 |
Jun 4, 1999 |
6261559 |
|
|
09848585 |
|
|
|
|
08874503 |
Jun 13, 1997 |
|
|
|
09325571 |
|
|
|
|
08697419 |
Aug 23, 1996 |
|
|
|
08874503 |
|
|
|
|
60002765 |
Aug 24, 1995 |
|
|
|
Current U.S.
Class: |
514/21.4 ;
530/326 |
Current CPC
Class: |
A61K 31/145 20130101;
A61K 31/00 20130101; A61P 37/08 20180101; C07K 14/5425 20130101;
A61P 11/00 20180101; A61K 38/00 20130101; Y10S 514/885 20130101;
C07K 14/7155 20130101; Y10S 514/826 20130101; A61P 11/06 20180101;
A61K 31/13 20130101 |
Class at
Publication: |
514/21.4 ;
530/326 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/08 20060101 C07K007/08; A61P 11/06 20060101
A61P011/06 |
Claims
1.-109. (canceled)
110. A composition comprising a polypeptide comprising the amino
acid sequence of SEQ ID NO: 13 or 14.
111. The composition of claim 110, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 13.
112. The composition of claim 110, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 14.
113. The composition of claim 110, wherein the polypeptide consists
of the amino acid sequence of SEQ ID NO: 13 or 14.
114. A pharmaceutical composition suitable for human use, the
composition comprising a polypeptide comprising the amino acid
sequence of SEQ ID NO: 13 or 14 and a pharmaceutically acceptable
carrier.
115. The pharmaceutical composition of claim 114, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 13.
116. The pharmaceutical composition of claim 114, wherein the
polypeptide comprises the amino acid sequence of SEQ ID NO: 14.
117. The pharmaceutical composition of claim 114, wherein the
polypeptide consists of the amino acid sequence of SEQ ID NO: 13 or
14.
118. The pharmaceutical composition of claim 115, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 14.
119. The pharmaceutical composition of claim 115, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 15.
120. The pharmaceutical composition of claim 115, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 16.
121. The pharmaceutical composition of claim 116, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 13.
122. The pharmaceutical composition of claim 116, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 15.
123. The pharmaceutical composition of claim 116, wherein the
pharmaceutical composition further comprises a polypeptide
comprising the amino acid sequence of SEQ ID NO: 16.
124. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition comprises from about 0.001 to about 5%
by weight of the polypeptide.
125. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition comprises from about 0.01 to about 1% by
weight of the polypeptide.
126. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition is formulated for systemic or topical
administration.
127. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition is formulated for intravenous,
intraperitoneal or intra-lesional administration.
128. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition is formulated as a powder, pill, tablet,
syrup or elixir.
129. The pharmaceutical composition of claim 114, wherein the
pharmaceutical composition is formulated for administration by
inhalation.
130. The pharmaceutical composition of claim 129, wherein the
pharmaceutical composition is formulated for administration by an
inhaler.
131. The pharmaceutical composition of claim 130, wherein the
pharmaceutical composition is formulated for administration by a
metered-dose inhaler.
132. The pharmaceutical composition of claim 130, wherein the
pharmaceutical composition is formulated for administration by a
dry-powder inhaler.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to U.S. Provisional Application
Ser. No. 60/002,765 which was filed Aug. 24, 1995.
FIELD OF THE INVENTION
[0002] This invention relates to regulating IL-9 activity and
treating atopic allergies and related disorders like asthma, based
upon the relationship between IL-9 and its receptor.
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 improperly 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 pulmonary inflammation ensues this disorder is broadly
characterized as asthma. Certain cells are critical to this
inflammatory reaction and they include T cells and antigen
presenting cells, B cells that produce IgE, and mast
cells/basophils and eosinophils that bind IgE. These inflammatory
cells accumulate at the site of allergic inflammation and the toxic
products they release contribute to the tissue destruction related
to the disorder.
[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.sup.1. 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..sup.1 Thus, an enormous burden is placed on our health care
resources.
[0006] The mechanism of susceptibility to atopy and asthma remains
unknown. 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 great skin test response to allergens in
atopic individuals as compared to nonatopics..sup.10 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..sup.4-9 In particular, younger
asthmatics have a high incidence of atopy..sup.10 In addition,
immunologic factors associated with an increase in serum total IgE
levels are very closely related to impaired pulmonary
function..sup.3
[0007] Both the diagnosis and treatment of these disorders are
problematic..sup.1 The assessment of inflamed lung tissue is often
difficult, and frequently the source of the inflammation cannot be
determined. Without knowledge of the source of the airway
inflammation and protection from the inciting foreign environmental
agent or agents, the inflammatory process cannot be interrupted. It
is now generally accepted that failure to control the 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,
i.e., transiently improve pulmonary functions, 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..sup.2 The
agents that can diminish the underlying inflammation, the
anti-inflammatory steroids, have their own known list of
disadvantages that range from immunosuppression to bone
loss..sup.2
[0009] Because of the problems associated with conventional
therapies, alternative treatment strategies have been
evaluated..sup.65-66 Glycophorin A,.sup.64 cyclosporin,.sup.65 and
a nonapeptide fragment of IL-2,.sup.63 all inhibit interleukin-2
dependent T lymphocyte proliferation and therefore, IL-9
production,.sup.51 however, they are known to have many other
effects..sup.2 For example, cyclosporin is used as a
immunosuppressant after organ transplantation. While these agents
may represent alternatives to steroids in the treatment of
asthmatics,.sup.63-66 they inhibit interleukin-2 dependent T
lymphocyte proliferation and potentially critical immune functions
associated with homeostasis. What is needed in the art is the
identification of a pathway critical to the development of asthma
that explains the episodic nature of the disorder and the close
association with allergy that is downstream of these critical
immune functions. Nature demonstrated that this pathway is the
appropriate target for therapy since biologic variability normally
exists at this pathway and these individuals are otherwise
generally not immunocompromised or ill except for their symptoms of
atopy.
[0010] Because of the difficulties related to the diagnosis and
treatment of asthma, the complex pathophysiology of this disorder
is under intensive study. Although this disorder is heterogeneous
and may be difficult to define because it can take many forms,
certain features are found in common among asthmatics. Examples of
such traits include elevated serum IgE levels, abnormal skin test
response to allergen challenge, bronchial hyperresponsiveness
[BHR], bronchodilator reversibility, and airflow
obstruction..sup.3-10 These expressions of these asthma related
phenotypes may be studied as quantitative or qualitative
measures.
[0011] Elevated IgE levels are also closely correlated with BHR, a
heightened bronchoconstrictor response to a variety of
stimuli..sup.4,6,8,9 BHR is believed to reflect the presence of
airway inflammation,.sup.6,8 and is considered a risk factor for
asthma..sup.11-12 BHR is accompanied by bronchial inflammation and
an allergic diathesis in asthmatic individuals..sup.13-21 Even in
children with no symptoms of atopy and asthma, BHR is strongly
associated with elevated IgE levels..sup.19
[0012] A number of studies document a heritable component to atopy
and asthma..sup.4,10,21 However, family studies have been difficult
to interpret since these disorders are significantly influenced by
age and gender, as well as many environmental factors such as
allergens, viral infections, and pollutants..sup.22-24 Moreover,
because there is no known biochemical defect associated with
susceptibility to these disorders, the mutant genes and their
abnormal gene products can only be recognized by the anomalous
phenotypes they produce. Thus, an important first step in isolating
and characterizing a heritable component is identifying the
chromosomal locations of the genes.
[0013] Cookson et al. provided the first description of a genetic
localization for inherited atopy..sup.25 These investigators
described evidence for genetic linkage between atopy and a single
marker on a specific chromosomal region designated 11q13.1. Later,
they suggested evidence of maternal inheritance for atopy at this
locus..sup.26 Although maternal inheritance [genetic imprinting]
had been observed for atopy, it had never been explained
previously. However, efforts to confirm this linkage have not been
generally successful..sup.27-31
[0014] Recently, the .beta. subunit of the high-affinity IgE
receptor was mapped to chromosome 11q, and a putative mutation
associated with atopy has been described in this gene..sup.32,33
However, because of the difficulties by others of replicating this
linkage, the significance of this gene and polymorphism remains
unclear. While additional studies will be required to confirm
whether this putative mutation causes atopy in the general
population, data collected so far suggests this polymorphism is
unlikely to represent a frequent cause of atopy.
[0015] Because serum IgE levels are so closely associated with the
onset and severity of allergy and asthma as clinical disorders,
attention has focused on studies of the genetic regulation of serum
total IgE levels. While past studies have provided evidence for
Mendelian inheritance for serum total IgE levels,.sup.34-38 an
indication of the existence of one regulatory gene, others have
found evidence for polygenic inheritance of IgE, i.e., existence of
several responsible genes..sup.39
[0016] Artisans have found several genes that may be important in
the regulation of IgE and the development or progression of
bronchial inflammation associated with asthma on chromosome 5q.
They include genes encoding several interleukins, such as IL-3,
IL-4, IL-5, IL-9, IL-13, granulocyte macrophage colony stimulating
factor [GM CSF], a receptor for macrophage colony stimulating
factor [CSF-1R], fibroblast growth factor acidic [FGFA], as well as
others..sup.40 Recent evidence from family studies suggests genetic
linkage between serum IgE levels and DNA markers in the region of
these candidate genes on chromosome 5q..sup.41,42 Together, these
investigations suggest that one or more major genes in the vicinity
of the interleukin complex on chromosome 5q regulates a significant
amount of the observed biologic variability in serum IgE that is
likely to be important in the development of atopy and asthma.
[0017] Linkage [sib-pair analyses] was also used previously to
identify a genetic localization for BHR..sup.79 Because BHR was
known to be associated with a major gene for atopy, chromosomal
regions reported to be important in the regulation of serum IgE
levels were examined..sup.42 Candidate regions for atopy have been
identified by linkage analyses. These studies identified the
existence of a major gene for atopy on human chromosome
5q31-q33..sup.42
[0018] Therefore, to determine the chromosomal location of a
gene[s] providing susceptibility to BHR, which would be coinherited
with a major gene for atopy, experiments were carried out using
linkage analyses between BHR and genetic markers on chromosome
5q..sup.42,79,82 Individuals with BHR were identified by
responsiveness to histamine. Markers useful for mapping
asthma-related genes are shown in FIG. 1.
[0019] Specifically, gene candidates for asthma, bronchial
hyperresponsiveness, and atopy are shown [right] in their
approximate location relative to the markers shown. The map
includes the interleukin genes IL-4, IL-13, IL-5, and IL-3; CDC25,
cell division cycle-25; CSF2, granulocyte-macrophage colony
stimulating factor [GMCSF]; EGR1 early growth response gene-1;
CD14, cell antigen 14; ADRB2, the .beta.2-adrenergic receptor;
GRL1, lymphocyte-specific glucocorticoid receptor; PDGFR,
platelet-derived growth factor receptor. Bands 5q31-q33 extend
approximately from IL-4 to D5S410. The distances reported are
sex-averaged recombination fractions.
[0020] Affected sib-pair analyses demonstrated statistically
significant evidence for linkage between BHR and D5S436, D5S658,
and several other markers located nearby on chromosome
5q31-q33..sup.79 These data strongly supported the hypothesis that
one or more closely spaced gene[s] on chromosome 5q31-q33 determine
susceptibility to BHR, atopy, and asthma..sup.79,80,81,82
[0021] Recently linkage has also been demonstrated between the
asthma phenotype and genetic markers on chromosome 5q31-q33..sup.83
This region of the human genome was evaluated for linkage with
asthma because of the large number of genes representing reasonable
positional candidates for providing genetic susceptibility for
atopy and BHR.
[0022] Linkage was demonstrated using the methods described
above..sup.42,83 Specifically, 84 families were analyzed from the
Netherlands with both sib-pair and LODs for markers from this same
region of chromosome 5q previously shown to be linked to BHR and
atopy..sup.42,83 An algorithm was used to categorize obstructive
airways disease in the asthmatic probands and their families. This
classification scheme was based, as described previously, on the
presence or absence of BHR to histamine, respiratory symptoms,
significant smoking history [>5 pack years], atopy as defined by
skin test response, airway obstruction [FEV1% predicted <95% CI]
and reversibility to a bronchodilator [>9% predicted].
[0023] Evidence was found for linkage between asthma and markers on
chromosome 5q by affected sib pair analysis (N=10, P<0.05) and
by maximum likelihood analysis with a dominant model for the asthma
phenotype..sup.83
[0024] Asthma was linked to D5S658 with a maximal LOD of 3.64 at
.theta.=0.03, using a dominant model [class 1 affected, class 2-4
uncertain, class 5 unaffected] with a gene frequency of 0.015
[prevalence of 3%]. A maximal LOD of 2.71 at .theta.=0.0 was
observed for D5S470 which is approximately 5 cM telomeric, or away
from IL-9, relative to D5S436..sup.83
[0025] Subsequent to the original filing of this application, IL-9
or a gene nearby was suggested as likely to be important use atopy
and asthma..sup.43 The IL-9 suggestion was based on a strong
correlation in a randomly ascertained population between log serum
total IgE levels and alleles of a genetic marker in the IL-9
gene..sup.43 This type of association with one or more specific
alleles of a marker is termed "linkage disequilibrium", and
generally suggests that a nearby gene determines the biologic
variability under study..sup.44
[0026] The IL-9 gene has been mapped to the q31-q33 region of
chromosome 5..sup.40 Only a single copy of the gene is found in the
human genome..sup.45,46 Structural similarity has been observed for
the human and murine IL-9 genes..sup.45,46 Each gene consists of
five exons and four introns extending across approximately four Kb
of DNA. Expression of the gene appears to be restricted to
activated T cells..sup.45,46
[0027] The functions of IL-9 now extend well beyond those
originally recognized. While IL-9 serves as a T cell growth factor,
this cytokine is also known to mediate the growth of erythroid
progenitors, B cells, mast cells, and fetal thymocytes..sup.45,46
IL-9 acts synergistically with IL-3 in causing mast cell activation
and proliferation..sup.47 This cytokine also potentiates the IL-4
induced production of IgE, IgG, and IgM by normal human B
lymphocytes..sup.48 IL-9 also potentiates the IL-4 induced release
of IgE and IgG1 by murine B lymphocytes..sup.49 A critical role for
IL-9 in the mucosal inflammatory response to parasitic infection
has also been demonstrated..sup.50,51
[0028] In addition to IL-9, chromosome 5q bears numerous other gene
candidates including IL-3, IRF1, EGR1, ITK, GRL1, ADRB2, CSF1R,
FGFA, ITGA2, CD14, PDGFR, CDC25, CSF2, IL-4, IL-5, IL-12B, and
IL-13. These may all be important in atopic allergy and as
potential targets for therapeutic development. Moreover, the art
lacks any knowledge regarding how the sequence of IL-9 or the
function of IL-9 specifically correlates with atopic allergy,
asthma, or bronchial hyperresponsiveness. Without such knowledge,
artisans would not know how or whether to use IL-9 to either
diagnose or treat these disorders.
[0029] The art does provide that IL-9 is a novel cytokine having an
apparent molecular weight of approximately between 20 to 30 kD as
determined by sodium dodecyl sulfate polyacrylamide gel
electrophoresis under reducing conditions. It is produced as a 144
amino acid protein, that is processed to a 126 amino acid
glycoprotein. Yang et al..sup.85 disclose that the DNA sequence
encoding IL-9 comprises approximately 630 nucleotides, with
approximately 450 nucleotides in the proper reading frame for the
protein.
[0030] It is also known in the art that multiple protein isoforms
may be generated from a single genetic locus by alternative
splicing. Alternative splicing is an efficient mechanism by which
multiple protein isoforms may be generated from a single genetic
locus. Alternative splicing is used in terminally differentiated
cells to reversibly modify protein expression without changing the
genetic content of the cells. These protein isoforms are
preferentially expressed in different tissues or during different
states of cell differentiation or activation. Protein isoforms may
have different functions and Alms and White have cloned and
expressed a naturally occurring splice variant of IL-4, formed by
the omission of exon 2, thus called IL-462..sup.86 It was observed
that IL-462 inhibits T-cell proliferation induced by IL-4.
[0031] However, the art lacks any knowledge about IL-9 protein
isoforms which are formed by deletions of exons 2 and 3 or the
regulatory functions exhibited by these truncated proteins.
Specifically, their role in regulating the biological activity,
namely, the down-regulation of IL-9 expression or activity is
unclear. Moreover, the formation of such isoforms by alternative
splicing has not been previously observed or used to provide
variants of IL-9 which function as agonists or antagonists of the
native cytokine.
[0032] The art also lacks any knowledge about the role of the IL-9
receptor with asthma-related disorders. It is known that IL-9 binds
to a specific receptor expressed on the surface of target
cells..sup.46,52,53 The receptor actually consists of two protein
chains: one protein chain, known as the IL-9 receptor, binds
specifically with IL-9 and the other protein chain is the chain,
which is shared in common with the IL-2 receptor..sup.46 In
addition, the human IL-9 receptor cDNA has been
cloned..sup.46,52,53 This cDNA encodes a 522 amino acid protein
which exhibits significant homology to the murine IL-9 receptor.
The extracellular region of the receptor is highly conserved, with
67% homology existing between the murine and human proteins. The
cytoplasmic region of the receptor is less highly conserved. The
human cytoplasmic domain is much larger than the corresponding
region of the murine receptor..sup.46
[0033] The IL-9 receptor gene has also been characterized..sup.53
It is thought to exist as a single copy in the mouse genome and is
composed of nine exons and eight introns..sup.53 The human genome
contains at least four IL-9 receptor pseudogenes. The human IL-9
receptor gene has been mapped to the 320 kb subtelomeric region of
the sex chromosomes X and Y..sup.46 Nonetheless, despite these
studies, the art lacks any knowledge of a relation between the IL-9
receptor and atopic allergy, asthma, or bronchial
hyperresponsiveness.
[0034] Thus, the art lacks any knowledge of how the IL-9 gene, its
receptor, and their functions, are related to atopic allergy,
asthma, bronchial hyperresponsiveness, and related disorders.
Therefore, there is a specific need in the art for genetic
information on atopic allergy, asthma, bronchial
hyperresponsiveness, and for elucidation of the role of IL-9 in the
etiology of these disorders. There is also a need for elucidation
of the role of the IL-9 receptor and the IL-9 receptor gene in
these disorders. Furthermore, most significantly, based on this
knowledge, there is a need for the identification of agents which
are capable of regulating the interaction between IL-9 and its
receptor for treating these disorders.
SUMMARY OF THE INVENTION
[0035] Applicant has satisfied the long felt need for a treatment
for atopic allergy including asthma and related disorders by
providing information demonstrating the role of IL-9 (also known as
Asthma Associated Factor 1, or AAFI) in the pathogenesis of these
disorders which information has led to compounds that are capable
of regulating the activity of IL-9. Applicant has also demonstrated
conserved linkage and synteny homologies between mice and humans
for a gene that determines biologic variability in airway
hyperresponsiveness. These relationships specifically identify IL-9
as a gene candidate. In addition, applicant has determined that
IL-9 is critical to a number of antigen-induced responses in mice
including bronchial hyperresponsiveness, eosinophilia and elevated
cell counts in bronchial lavage, and elevated serum total IgE.
These findings typify the allergic inflammation associated with
asthma.
[0036] Furthermore, applicant has determined that a C to T nucleic
acid variation at position 3365 in exon 5 of the human IL-9 gene
produces the predicted amino acid substitution of a methionine for
a threonine at codon 117 of IL-9. When this substitution occurs in
both alleles in one individual, it is associated with less evidence
of atopic allergy including asthma, fewer abnormal skin test
responses, and a lower serum total IgE. Thus, applicant has
identified the existence of a nonasthmatic, nonatopic phenotype
characterized by methionine at codon 117 when it occurs in both
IL-9 gene products in one individual. As an additional significant
corollary, applicant has identified the existence of susceptibility
to an asthmatic, atopic phenotype characterized by a threonine at
codon 117. Thus, the invention includes purified and isolated DNA
molecules having such a sequence as well as the peptides encoded by
such DNA.
[0037] The biological activity of IL-9 results from its binding to
the IL-9 receptor and the consequent propagation of a regulatory
signal in specific cells. Therefore, IL-9 functions can be
interrupted or regulated by the interaction of IL-9 agonists or
antagonists with IL-9 or its receptor. Down regulation, i.e.
reduction of the functions controlled by IL-9, is achieved in a
number of ways. Administering agonists or antagonists that can
interrupt the binding of IL-9 to its receptor is one key mechanism
and such agonists and antagonists are within the claimed invention.
Examples include administration of polypeptide products encoded by
the DNA sequences of IL-9 or IL-9 receptor wherein the DNA
sequences contain various mutations. These mutations may be point
mutations, insertions, deletions, or spliced variants of IL-9 or
its receptor.
[0038] A further embodiment of this invention includes the
regulation of the activity of IL-9 by administering "agonists and
antagonists." The skilled artisan will readily recognize that all
molecules containing the requisite 3-dimensional structural
conformation and which contain the residues essential or critical
for receptor binding are within the scope of this invention.
Specifically, residues 43-60 and 71-90 of the mature protein appear
to be important for receptor binding. Applicant has shown that
peptides KP-16 (residues 43-60) and KP-20 (residues 71-90) act as
receptor antagonists. In addition, these residues in the native
IL-9 molecule are predicted to form anti-parallel helical
structures. The three dimensional structure of the protein suggests
that specifically serine 52 and/or glutamic acid 53 interact with
lysine 85, serine 56 interacts with lysine 82, and threonine 59
interacts with valine 78. The three dimensional coordinates of
these anti-parallel helices and the related, functional groups
represent the requisite 3-dimensional conformation critical for
receptor binding and compounds which simulate these relationships
are within the scope of this invention.
[0039] The biological activity of the IL-9 receptor (also called
Asthma Associates Factor 2, AAF2) can also be modulated by using
soluble IL-9 receptor molecules. Such a molecule prevents the
binding of IL-9 to the cell-bound receptor and acts as an
antagonist for IL-9, and is also within the scope of this
invention.
[0040] Polyclonal and monoclonal antibodies which block the binding
of IL-9 to its receptor are also within the scope of this invention
and are useful therapeutic agents in treating atopic allergy
including asthma and related disorders.
[0041] Another embodiment of this invention relates to the use of
isolated DNA sequences containing various mutations such as point
mutations, insertions, deletions, or spliced mutations of IL-9 or
the IL-9 receptor in gene therapy.
[0042] Expression of IL-9 and IL-9 receptor is also down-regulated
by administering an effective amount of synthetic antisense
oligonucleotide sequences. The oligonucleotide compounds of the
invention bind to the mRNA coding for human IL-9 and IL-9 receptor
thereby inhibiting expression of these molecules.
[0043] The structure of both IL-9 and the IL-9 receptor have been
examined and analyzed in great detail and amino acid residues of
IL-9 critical for receptor binding have been identified. Based on
structural studies and the binding characteristics of this specific
binding pair, this invention further includes small molecules
tailored such that their structural conformation provides the
residues essential for blocking the interaction of IL-9 with the
IL-9 receptor. Such blockade results in modulation of the activity
of the receptor and these molecules are, therefore, useful in
treating atopic allergies.
[0044] Another embodiment of this invention is directed to the
regulation of downstream signaling pathways necessary for IL-9
function. IL-9 induces tyrosine phosphorylation of Stat3 which
appears to be unique to the IL-9 signaling pathway.sup.58 and is
useful as a target for inhibitors. Specific and nonspecific
inhibitors of tyrosine kinase such as tyrophostins are, therefore,
useful in downstream regulation of the physiological activity of
IL-9, and are part of the invention.
[0045] In a further embodiment aminosterol compounds are also
useful in treating atopic allergies and related disorders because
they are also involved in blocking signal transduction of the IL-9
signal transduction pathway.
[0046] The products discussed above represent various effective
therapeutic agents in treating atopic allergies, asthma and other
related disorders.
[0047] This invention also includes the truncated polypeptides
encoded by the DNA molecules described above. These polypeptides
are capable of regulating the interaction of IL-9 with the IL-9
receptor.
[0048] Thus, applicant has identified the critical role of the IL-9
pathway in pathogenesis of atopic allergy, including bronchial
hyperresponsiveness, asthma, and related disorders. More
specifically, applicant has provided antagonists and methods of
identifying antagonists that are capable of regulating the
interaction between IL-9 and its receptor. Applicant also provides
methods for regulating the activity of IL-9 by: 1) administering a
compound having activity comparable to IL-9 containing methionine
at codon 117 and the ability to bind to a receptor for IL-9 in an
amount sufficient to down-regulate the activity of IL-9; and 2) by
administering truncated protein products encoded by isolated
nucleic acid sequences comprising deletions of any one or more of
exons 1, 2, 3, 4, or 5.
[0049] Having identified the critical role of the IL-9 pathway in
atopic allergy, bronchial hyperresponsiveness, and asthma,
applicant also provides a method for the diagnosis of
susceptibility to atopic allergy, asthma, and related disorders.
Lastly, applicant provides a method for assaying the functions of
IL-9 and its receptor to identify compounds or agents that may be
administered in an amount sufficient to down-regulate either the
expression or functions of IL-9 and the IL-9 receptor.
[0050] 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 DRAWINGS
[0051] FIG. 1: Map showing the relative order and distance in
centiMorgans [cM] between the polymorphic genetic markers useful
for mapping asthma-related genes.
[0052] FIG. 2: Illustration of the genetic map of human chromosome
5q31-q33 and syntenic regions in the mouse.
[0053] FIG. 3: The LOD score curve on mouse chromosome 13 for
atracurium-induced airway responsiveness in mice with increased
susceptibility to bronchoconstrictor stimuli.
[0054] FIG. 4: Alignment of amino acid sequences corresponding to
exon 5 of the human and murine IL-9 genes. The first sequence is
translated from the Thr allele of the human gene. The middle
sequence is translated from the Met allele of the human gene. The
final sequence is translated from the murine gene.
[0055] FIG. 5: Histogram of the correlation between human IL-9 gene
alleles and serum total IgE titers measured in international units.
S/S denotes Thr/Thr individuals, S/R denotes Thr/Met individuals
and R/R denotes Met/Met individuals.
[0056] FIG. 6: Illustration the simple sequence repeat polymorphism
at the IL-9 locus.
[0057] FIG. 7: Translated cDNA sequence of Thr117 version of
IL-9.
[0058] FIG. 8: Translated cDNA sequence of Met117 version of
IL-9.
[0059] FIG. 9: Map of pFlag expression construct with Thr117.
[0060] FIG. 10: Sequence of pFlag expression construct for the
Thr117 version of the cDNA from the region surrounding the site of
ligation.
[0061] FIG. 11: Map of pFlag expression construct with Met117.
[0062] FIG. 12: Sequence of pFlag expression construct for the
Met117 version of the cDNA from the region surrounding the site of
ligation.
[0063] FIG. 13: Western blot of recombinant IL-9 proteins
[0064] FIG. 14: Amino acid sequences for inhibitory peptides.
[0065] FIG. 15: Inhibition by KP-16 of IL-9 mediated MO7e
proliferation.
[0066] FIG. 16: Inhibition by KP-20 of IL-9 mediated MO7e
proliferation.
[0067] FIG. 17: Inhibition by KP-23 of IL-9 mediated MO7e
proliferation.
[0068] FIG. 18: Inhibition by various tyrophostins of IL-9 mediated
MO7e proliferation.
[0069] FIG. 19: Inhibition by various aminosterols of IL-9 mediated
MO7e proliferation.
[0070] FIG. 20: Characterization of the role of IL-9 in the antigen
response in vivo.
[0071] FIG. 21: Histologic examination of lungs from control, ova
challenged, and anti-IL-9 pretreated animals.
[0072] FIG. 22: Inhibition of the antigen response in vivo by
blocking antibodies to the murine IL-9 receptor.
[0073] FIG. 24: Expression of human Met117 IL-9 and Thr117
IL-9.
[0074] FIG. 25: Binding of the human recombinant Met117 and Thr117
forms of IL-9 to a soluble receptor.
[0075] FIG. 26: Steady state levels of IL-9 in unstimulated and
stimulated murine splenocytes.
[0076] FIG. 27: An appendix of chemical moieties.
[0077] FIG. 28: Aminosterols isolated from the dog fish shark.
DETAILED DESCRIPTION OF THE INVENTION
[0078] Applicant has resolved the needs in the art by elucidating
an IL-9 pathway and compositions that affect that pathway that 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 (BHR), 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. BHR refers to bronchial hyperresponsiveness, a heightened
bronchoconstrictor response to a variety of stimuli.
[0079] By analyzing the DNA of families that exhibit asthma-related
disorders, applicant has identified a polymorphism in the IL-9 gene
that correlates with the biologic variability of serum total IgE as
one measurable expression of atopy. The IL-9 gene (also known as
Asthma Associated Factor 1 or AAF1) refers to the genetic locus of
interleukin-9, a cytokine exhibiting a variety of functions
involving the regulation of human myeloid and lymphoid systems. The
IL-9 gene of the present invention is found in the q31-q33 region
of human chromosome 5 and chromosome 13 in the mouse.
[0080] By polymorphism, applicant means a change in a specific DNA
sequence, termed a "locus", from the prevailing sequence. In
general, a locus is defined as polymorphic when artisans have
identified two or more alleles encompassing that locus and the
least common allele exists at a frequency of 1% or more.
[0081] The polymorphism of the present invention leads to an amino
acid substitution at residue 117 of IL-9. Specifically, instead of
the hydrophilic amino acid threonine, the IL-9 of the present
invention exhibits the hydrophobic amino acid methionine (Met
IL-9). On a genetic level, the polymorphism of the present
invention is a substitution of a thymine residue for a cytosine
residue at nucleotide position 3365 in the human IL-9 gene as it is
described by Renauld and colleagues (1990) [GenBank accession
numbers M30135 and M30136],.sup.54 or at the comparable nucleotide
position 4244 of the human IL-9 gene sequence reported by Kelleher
et al., (1991) [GenBank accession number M86593]..sup.55
[0082] Individuals with a threonine (Thr) at amino acid 117 of IL-9
in either one or both of their alleles (Thr/Thr or Thr/Met)
generally exhibit susceptibility to an asthmatic or atopic allergic
phenotype, and these genotypes are characterized by higher mean
serum total IgE levels in the populations studied. In contrast,
those individuals with a methionine (Met) at codon 117 of IL-9 in
both alleles (Met/Met) exhibit a lack of asthma, fewer abnormal
skin test responses, and a lower serum total IgE. Thus, the Met/Met
genotype of IL-9 appears to protect against asthma or atopic
allergy.
[0083] Accordingly, the invention provides a purified and isolated
DNA molecule comprising a nucleotide sequence encoding human
interleukin 9 containing methionine at position 117 (Met IL-9), or
a fragment thereof. The invention also includes degenerate
sequences of the DNA as well as sequences that are substantially
homologous. The source of the IL-9 of the invention is human.
Alternatively, the DNA or fragment thereof may be synthesized using
methods known in the art. It is also possible to produce the
compound by genetic engineering techniques, by constructing DNA by
any accepted technique, cloning the DNA in an expression vehicle
and transfecting the vehicle into a cell which will express the
compound. See, for example, the methods set forth in Sambrook et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring
Harbor Laboratory Press [1985].
[0084] Airway hyperresponsiveness is found in virtually all
asthmatics and in some strains of inbred mice (DBA/2)..sup.84
Airway hyperresponsiveness is a risk factor for the development of
asthma in humans and is used in animal models of asthma as a
physiologic measure to assess the efficacy of treatment for asthma.
These data along with human.sup.79 and murine genetic mapping
results (see Examples 1 and 2) suggest a critical role for the
murine IL-9 gene product in the airway response of the mouse. In
particular, the hyperresponsive DBA/2(D2) mice differ from the
C57BL/6(B6) hyporesponsive mice.sup.84 in their expression of
steady state levels of IL-9 (See Example 14, FIG. 26). Furthermore,
pretreatment with blocking antibodies to IL-9/IL-9 receptor can
optionally provide complete protection from antigen induced airway
hyperresponsiveness and inflammation in mice demonstrating a
critical regulatory role for IL-9 in these immune responses. Thus,
these data demonstrate that although different molecular changes
produce biologic variability in airway responsiveness in humans and
mice, these changes arise in the same gene(s) (IL-9/IL-9R) that
regulate this pathway. Taken together, these observations confirm
the critical role of IL-9 and the IL-9 receptor in airway
hyperresponsiveness, asthma, and atopic allergy. Moreover, these
data demonstrate that agents of the convention, which block the
interaction of IL-9 with its receptor, protect against an antigen
induced response such as those detailed above.
[0085] Further evidence defining the critical role of IL-9 in the
pathogenesis of atopic allergy, bronchial hyperresponsivenss,
asthma, and related disorders derives directly from the applicants
observation that IL-9 is critical to a number of antigen induced
responses in mice. When the functions of IL-9 are down regulated by
antibody pretreatment prior to aerosol challenge with antigen, the
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 total IgE. Thus, the treatment of such responses,
which are critical to the pathogenesis of atopic allergy and which
characterize the allergic inflammation associated with asthma, by
the down regulation of the functions of IL-9, are within the scope
of this invention.
[0086] Applicant also teaches the regulation of the activity of
IL-9 by administering "agonists and antagonists" to the IL-9
receptor. The skilled artisan will readily recognize that all
molecules containing the requisite 3-dimensional structural
conformation and which contain the residues essential or critical
for receptor binding are within the scope of this invention.
Applicant has shown that peptides KP-16 (IL-9 residues 43-60) and
KP-20 (IL-9 residues 71-90) (produced using standard peptide
automated synthesis techniques, for example, the Applied Biosystems
Model 431A Peptide Synthesizer) act as IL-9 antagonists.
Specifically, applicant demonstrates that residues 43-60 and 71-90
of the mature protein appear to be important for receptor binding.
In addition, these residues include most of exon 4 (amino acids
44-88) and are predicted to form anti-parallel helical structures.
The three dimensional structure of the protein suggests that
specifically serine 52 and/or glutamic acid 53 interact with lysine
85, serine 56 interacts with lysine 82, and threonine 59 interacts
with valine 78. The three dimensional coordinates of these parallel
helices and the related functional groups represent the requisite
3-dimensional conformation critical for receptor binding and
compounds that simulate these relationships are within the scope of
this invention.
[0087] The demonstration of an IL-9 sequence associated with an
asthma-like phenotype, and one associated with the lack of an
asthma-like phenotype, indicates that the lungs' inflammatory
response to antigen is dependent on IL-9, and therefore, that down
regulating the function of IL-9 should protect against the antigen
induced response. Furthermore, applicant also provides methods of
diagnosing susceptibility to atopic allergy and related disorders
and for treating these disorders based on the relationship between
IL-9 and its receptor.
[0088] A receptor is a soluble or membrane bound component that
recognizes and binds to molecules, and the IL-9 receptor (also
known as Asthma Associated Factor 2 or AAF2) of the invention is
the component that recognizes and binds to IL-9. The functions of
the IL-9 receptor consist of binding an IL-9-like molecule and
propagating its regulatory signal in specific cells..sup.57-60 An
interruption of that function will lead to a down regulation, i.e.,
reduction, of either the expression of IL-9 or of the functions
controlled by IL-9. Accordingly, by virtue of this interaction
between. IL-9 and the IL-9 receptor, certain functions of the
organism are modulated or controlled. For a general discussion of
receptors, see Goodman and Gilman's The Pharmacologic Basis of
Therapeutics (Seventh Edition, MacMillan Publishing Company, N.Y.
USA, 1985).
[0089] One diagnostic embodiment involves the recognition of
variations in the DNA sequence of IL-9. One method involves the
introduction of a nucleic acid molecule (also known as a probe)
having a sequence complementary to the IL-9 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 the Met117 IL-9 or to Thr117 IL-9, and in another embodiment
will bind to both Met117 IL-9 and Thr117 IL-9. Another method of
recognizing DNA sequence variation associated with these disorders
is direct DNA sequence analysis by multiple methods well known in
the art..sup.77 Another embodiment involves the detection of DNA
sequence variation in the IL-9 gene associated with these
disorders..sup.73-77 These include the polymerase chain reaction,
restriction fragment length polymorphism (RFLP) analysis and single
stranded conformational analysis. In a preferred embodiment,
applicant provides specifically for a method to recognize, on a
genetic level, the polymorphism in IL-9 associated with the Thr and
Met alleles using a StyI RFLP as described herein. In other
embodiments Nla, Pfim1, PflM1, and Nco1 RFLPs may be used to
distinguish these two alleles of IL-9 genes.
[0090] Another embodiment involves treatment of atopic allergy and
related disorders. In a preferred embodiment, the applicant
provides a method of administering a compound having activity
comparable to Met IL-9 and the ability to bind to an IL-9 receptor
in an amount sufficient to down regulate the activity of IL-9. A
compound having activity comparable to Met IL-9 is a compound that
functions similarly but not necessarily identically. Thus, it may
bind to the IL-9 receptor but without the same physiological
effects. Examples include amino acid sequences of IL-9 containing
various point mutations and/or deletions and sequences
substantially homologous thereto. For example, such a compound may
interrupt the binding of Thr IL-9 to the IL-9 receptor as measured
by techniques known in the art. The invention also encompasses
functionally effective fragments of the above amino acid sequences.
In one such technique, the Thr IL-9 may be considered a "ligand"
for the IL-9 receptor, and binding between the two may be assessed
by ligand-binding assays which are well known in the art as set
forth in Goodman and Gilman's The Pharmacologic Basis of
Therapeutics. (Seventh Edition, MacMillan Publishing Company, N.Y.
USA, 1985).
[0091] In another embodiment, the compound may resemble the Met
allele of IL-9 in structure. Thus, such a compound may incorporate
a methionine in codon 117 of IL-9 or may incorporate another
hydrophobic amino acid. Thus, included within the scope of this
invention are IL-9 variants comprising substitutions of Thr at
position 117 by amino acids selected from the group consisting of
alanine, valine, leucine, isoleucine, proline, phenylalanine,
tryptophan, and methionine. Alternatively, the compound of the
invention may exist as a fragment of IL-9 with a structural
composition similar to Met IL-9. In another embodiment of the
invention, the compound may retain functions comparable to Met
IL-9, but may not resemble Met IL-9 in structure. For example, the
composition of the compound may include molecules other than amino
acids. This example is merely illustrative and one of ordinary
skill in the art would readily recognize that other substitutions
and/or deletion analogs of IL-9 resulting in effective antagonists
are also within the scope of this invention. As discussed above all
molecules containing the requisite 3-dimensional structural
conformation and which contain the residues essential or critical
for receptor binding are within the scope of this invention.
[0092] Specific assays may be based on IL-9's known regulation, in
part, of the proliferation of T lymphocytes, IgE synthesis, and
release from mast cells..sup.54-60 Another assay involves the
ability of human IL-9 to specifically induce the rapid and
transient tyrosine phosphorylation of multiple proteins in MO7e
cells..sup.57 Because this response is dependent on the expression
and activation of the IL-9 receptor, it represents a simple method
or assay for the characterization of potentially valuable
compounds. The tyrosine phosphorylation of Stat3 transcriptional
factor appears to be specifically related to the actions of
IL-9,.sup.58 and this response represents a simple method or assay
for the characterization of compounds within the invention. Still
another method to characterize the function of IL-9 and IL-9-like
molecules involves the well known murine TS1 clone and the D10
clone available from ATCC used to assess human IL-9 function with a
cellular proliferation assay..sup.59 The Met IL-9 that forms a part
of the invention may be viewed as a "weak agonist" of the IL-9
receptor. Such weak agonists are another preferred embodiment of
the invention. The term agonist, according to this invention,
includes compounds that mimic at least some of the effects of
endogenous compounds by interacting or binding with a receptor.
Agonists that interact or bind to the IL-9 receptor on the surface
of certain cells initiate a series of biochemical and physiological
changes that are characteristic of this cytokine's
actions..sup.45-51,54-60 To identify other weak agonists of the
invention, one may test for binding to the IL-9 receptor or for
IL-9-like functions as described herein and in the cited
literature..sup.2,45-51,54-60
[0093] The present invention also includes antagonists of IL-9 and
its 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 a known agonist or for
down-regulation of IL-9-like functions as described herein and in
the cited literature..sup.2,45-51,54-60
[0094] The binding of either the agonist or 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
receptor.
[0095] In a further embodiment, these compounds may be analogs of
IL-9. IL-9 analogs 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..sup.62 This invention also includes spliced
variants of IL-9 and discloses isolated nucleic acid sequences of
interleukin-9, which contain deletions of one or more of its five
exons. The term "spliced variants" as used herein denotes a
purified and isolated DNA molecule encoding human IL-9 comprising
at least one exon. There is no evidence of naturally expressed
spliced mutants in the art. Thus, the present invention provides an
isolated nucleic acid containing exons 1, 4 and 5 of human IL-9.
Other variants within the scope of this invention include sequences
comprising exons 1, 2, 3, 4, and 5; exons 1, 2, 3, and 4; exons 1,
2, 4, and 5 and exons 1, 3, 4, and 5. It must be understood that
these exons may contain various point mutations.
[0096] Specific examples of antagonistic peptides derived from IL-9
include KP-16 (SEQ. ID No. 13) and KP-20 (SEQ. ID NO. 14) which are
derived from exon 4. Exon 4 encodes 44 amino acids while the
peptides mentioned above contain 16 and 20 amino acids respectively
and they do not overlap. These peptides exhibit considerable
inhibitory activity both individually and when assayed in
combination. Additionally, KP-23 (SEQ ID NO. 15) and KP-24 (SEQ ID
NO 16) are derived from exon 5 and also exhibit similar activity.
Splice variants of IL-9 can be formed by deletion of any one or
more of the IL-9 exons 1 through 5. As shown above, peptides
derived from these exons show regulatory capability and,
accordingly, are useful in treating atopic allergies, including
asthma.
[0097] It is known in the art that, in multienzyme systems, the
first or regulatory enzyme can be activated or inhibited by the end
product of the multi-enzyme system. When the concentration of the
end product increases over the steady state concentration, the end
product will act as a specific activator or inhibitor of the
regulatory enzyme in the sequence. Such feedback mechanism is also
relevant to the IL-9 system and it is observed that the various
polypeptides of this invention are capable of exerting such
activation or inhibitory control on the activity of the IL-9
receptor and possibly the expression or function of other cytokines
and their receptors that play a role in the pathogenesis of
asthma.
[0098] The invention also includes modifications of agonists or
antagonists that can be made using knowledge that is routine to
those in this art. For example, the affinity of a compound for a
receptor is generally closely related to the chemical structure of
the compound. Thus, structure-activity relationships may be used to
modify the agonists and antagonists of the invention. For example,
the techniques of crystallography/X-ray diffraction and NMR may be
used to make modifications of the invention.
[0099] For example, one can create a three dimensional structure of
human IL-9 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 mutant IL-9 molecule
with affinity for the IL-9 receptor comparable to IL-9, but with a
lower biologic activity. What is meant by lower biologic activity
is 2 to 100,000 fold less than IL-9, preferably 100 to 1,000 fold
less than IL-9.
[0100] In still another embodiment, these compounds also may be
used as dynamic probes for receptor structure and to develop
receptor antagonists using IL-9 dependent cell lines.
[0101] In addition, this invention also provides compounds that
prevent the synthesis or reduce the biologic stability of IL-9 or
the IL-9 receptor. 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.sup.89 therapeutic may be prolonged by using D-amino acids,
or shortened by altering its sequence to make it more susceptible
to enzymatic degradation.
[0102] In another embodiment, the agonists and antagonists of the
invention are antibodies to IL-9 and the IL-9 receptor. The
antibodies to IL-9 and its receptor 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, 1988). They can be used to block IL-9
from binding to the receptor. In one embodiment the antibodies
interact with IL-9. In another embodiment the antibodies interact
with the IL-9 receptor. The IL-9 used to elicit these antibodies
can be any of the IL-9 variants discussed above.
[0103] Antibodies are also produced from peptide sequences of IL-9
or the IL-9 receptor using standard techniques in the art (see
Protocols in Immunology, Chapt. 9, Wiley). The peptide sequences
from the murine IL-9 receptor that can be used to produce blocking
antisera have been identified as: GGQKAGAFTC (residues 1-10) (SEQ
ID NO:19); LSNSIYRIDCHWSAPELGQESR (residues 11-32) (SEQ ID NO:20);
and CESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue
added to the N-terminus for coupling the peptide to the carrier
protein) (SEQ ID NO:21). In addition, an epitope that binds to a
blocking antibody directed to the human IL-9 receptor has been
identified as residues 8-14 of the mature human IL-9 receptor.
(TCLTNNI) (SEQ ID NO:22) and two epitopes that bind to blocking
antibodies directed to human IL-9 have also been identified as
residues 50-67 (CFSERLSQMTNTTMQTRY) (SEQ ID NO:23) and residues
99-116 (TAGNALTFLKSLLEIFQK) (SEQ ID NO:16) The human epitopes as
well as the human peptides that correspond to the peptides that
produce blocking antibodies in the murine sequences are most likely
to be useful for the production of therapeutic antibodies.
[0104] In still another embodiment, the compounds of the invention
may be coupled to chemical moieties, including proteins that alter
the functions or regulation of the IL-9 pathway for therapeutic
benefit in atopic allergy and asthma..sup.61 These proteins may
include in combination other cytokines and growth factors
including.sup.67 L-4, IL-5, IL-3, IL-2, IL-13, and IL-10 that may
offer additional therapeutic benefit in atopic allergy and asthma.
In addition, the IL-9 of the invention may also be conjugated
through phosphorylation and conjugated to biotinylate, thioate,
acetylate, iodinate, and any of the crosslinking reagents shown in
FIG. 27 (Pierce).
[0105] In a further embodiment, the invention includes the down
regulation of IL-9 expression or function by administering soluble
IL-9 receptor molecules that bind IL-9. Renauld et al..sup.59 have
shown the existence of a soluble form of the IL-9 receptor. This
molecule can be used to prevent the binding of IL-9 to cell bound
receptor and act as an antagonist of IL-9. Soluble receptors have
been used to bind cytokines or other ligands to regulate their
function..sup.87 A soluble receptor is a form of a membrane bound
receptor that occurs in solution, or outside of the membrane.
Soluble receptors may occur because the segment of the molecule
which commonly associates with the membrane is absent. This segment
is commonly referred to in the art as the transmembrane domain of
the gene, or membrane binding segment of the protein. Thus, in one
embodiment of the invention, a soluble receptor may represent a
fragment or an analog of a membrane bound receptor. In another
embodiment of the invention, the structure of the segment that
associates with the membrane may be modified (e.g. DNA sequence
polymorphism or mutation in the gene) so the receptor is not
inserted into the membrane, or the receptor is inserted, but is not
retained within the membrane. Thus, a soluble receptor, in contrast
to the corresponding membrane bound form, differs in one or more
segments of the gene or receptor protein that are important to its
association with the membrane..sup.52,53
[0106] These compounds may be known forms of a soluble IL-9
receptor that act to bind IL-9. Alternatively, these compounds may
resemble known forms of the IL-9 receptor, but may exist as
fragments. In another embodiment of the invention, the compound may
retain functions comparable to soluble IL-9 receptor, but may not
resemble soluble IL-9 receptor in composition. For example, the
composition of the compound may include molecules other than amino
acids. Thus, these compounds will bind IL-9 and prevent IL-9 from
acting at its cell surface receptor.
[0107] A further embodiment of the invention relates to antisense
or gene therapy. It is now known in the art that altered DNA
molecules can be tailored to provide a specific selected effect,
when provided as antisense or gene therapy. The native DNA segment
coding for IL-9 receptor, has, as do all other mammalian DNA
strands, two strands; a sense strand and an antisense strand held
together by hydrogen bonding. 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 the receptor. 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 strands 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.
[0108] The sequence may be complementary to any portion of the
sequence of the mRNA, i.e., 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.
[0109] In the practice of the invention, expression of the IL-9
receptor is down-regulated by administering an effective amount of
synthetic antisense oligonucleotide sequences described above. The
oligonucleotide compounds of the invention bind to the mRNA coding
for human IL-9 or IL-9 receptors thereby inhibiting expression
(translation) of these proteins. See Gruss et al., "Interleukin 9
is expressed by primary and cultural Hodgkin and Reed-Sternberg
cells." Cancer Res. 52:1026-31 (Feb. 15, 1992)
[0110] The isolated DNA sequences containing various mutations such
as point mutations, insertions, deletions, or spliced mutations of
IL-9 are useful in gene therapy as well.
[0111] In addition to the direct regulation of the IL-9 receptor,
this invention also encompasses methods of downstream regulation
which involve inhibition of signal transduction. In particular, a
further embodiment of this invention is drawn to inhibition of
tyrosine phosphorylation. 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. IL-9 induces
tyrosine phosphorylation of multiple proteins; it is known in the
art that in addition to the activation of JAK1 and JAK3 tyrosine
kinases, IL-9 also induces tyrosine phosphorylation of
Stat3..sup.58 Phoshorylation of Stat3 is unique to the IL-9 signal
transduction pathway and hence is a perfect target for
inhibitors..sup.58 This invention includes within its scope
tyrphostins which are specific inhibitors of protein tyrosine
kinases. Thus, tyrphostins (obtained for example from Calbiochem)
and other similar inhibitors of these kinases are useful in the
modulation of signal transduction and are useful in the treatment
of atopic allergies and asthma.
[0112] In still another aspect of the invention, it was
surprisingly, found that aminosterol compounds are also useful in
the inhibition of signal transduction due to IL-9 stimulation.
Aminosterol compounds which are useful in this invention are
described in U.S. patent application Ser. No. 08/290,826 and its
related applications Ser. Nos. 08/416,883 and 08/478,763 as well as
in Ser. No. 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.
[0113] In addition, the invention 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 injectionable solutions. Suitable
pharmaceutical carriers are described in Martin, E. W., Remington's
Pharmaceutical Sciences, specifically incorporated herein by
reference.
[0114] 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.
[0115] 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 as are well described in the art of pharmaceutical
formulations as exemplified, for example, by Remington's
Pharmaceutical Science, Edition 17, Mack Publishing Company,
Easton, Pa. 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 a administered systemically, it may be confected as a
powder, pill, tablets 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 intermuscular injection. In a preferred embodiment, the
compounds of this invention 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.
[0116] An effective amount is that amount which will down regulate
either the expression of IL-9 or the functions controlled by IL-9.
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 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.
[0117] Applicant also provides for a method to screen for the
compounds that down regulate the expression of IL-9 or the
functions controlled by IL-9. One may determine whether the
functions expressed by IL-9 are down-regulated using techniques
standard in the art..sup.57-60 In a specific embodiment, applicant
provides for a method of identifying compounds with functions
comparable to Met IL-9. Thus, in one embodiment, serum total IgE
may be measured using techniques well known in the art.sup.42 to
assess the efficacy of a compound in down regulating the functions
of IL-9 in vivo. In another embodiment, bronchial
hyperresponsiveness, bronchoalveolar lavage, and eosinophilia may
be measured using techniques well known in the art.sup.42 to assess
the efficacy of a compound in down regulating the functions of IL-9
in vivo. In yet another embodiment, the functions of IL-9 may be
assessed in vitro. As is known to those in the art, human IL-9
specifically induces the rapid and transient tyrosine
phosphorylation of multiple proteins in MO7e cells. The tyrosine
phosphorylation of Stat3 transcriptional factor appears to be
specifically related to the actions of IL-9. Another method to
characterize the function of IL-9 and IL-9-like molecules that
depends on the "stable expression" of the IL-9 receptor uses the
well known murine TS1 clones to assess human IL-9 function with a
cellular proliferation assay..sup.59
[0118] The invention also includes a simple screening assay for
saturable and specific ligand binding based on cell lines that
express the IL-9 receptor..sup.46,59 The IL-9 receptor is expressed
in on a wide variety of cell types, including K562, C8166-45,B
cells, T cells, mast cells, neutrophils, megakaryocytes (UT-7
cells),.sup.53 the human megakaryoblastic leukemia cell lines
MO7e.sup.57, TF1,.sup.59 macrophages, fetal thymocytes, the human
kidney cell line 293,.sup.53 and murine embryonic hippocampal
progenitor cell lines..sup.46,52,53 In another embodiment, soluble
IL-9 receptor may be used to evaluate ligand binding and potential
receptor antagonists.
[0119] 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. See, for example, Sambrook et
al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. Cold Spring
Harbor Laboratory Press [1985], or Ausubel et al., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc.
[1994].
[0120] Nonetheless, we offer the following basic background
information. The body's genetic material, or DNA, is arranged on 46
chromosomes, which each comprises two arms joined by a centromere.
Each chromosome is divided into segments designated p or q. The
symbol p is used to identify the short arm of a chromosome, as
measured from the centromere to the nearest telomere. The long arm
of a chromosome is designated by the symbol q. Location on a
chromosome is provided by the chromosome's number (i.e., chromosome
5) as well as the coordinates of the p or q region (i.e., q31-q33).
In addition, the body bears the sex chromosomes, X and Y. During
meiosis, the X and Y chromosomes exchange DNA sequence information
in areas known as the pseudoautosomal regions.
[0121] DNA, deoxyribonucleic acid, consists of two complementary
strands of nucleotides, which include the four different base
compounds, adenine [A], thymine [T], cytosine [C], and guanine [G].
A of one strand bonds with T of the other strand while C of one
strand bonds to G of the other to form complementary "base pairs,"
each pair having one base in each strand.
[0122] A sequential grouping of three nucleotides [a "codon"] codes
for one amino acid. Thus, for example, the three nucleotides CAG
codes for the amino acid Glutamine. The 20 naturally occurring
amino acids, and their one letter codes, are as follows:
TABLE-US-00001 Alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic Acid Asp D Asparagine or Asx B Aspartic acid Cysteine Cys
C Glutamine Gln Q Glutamine Acid Glu E Glutamine or Glx Z Glutamic
acid Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L
Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine
Val V
[0123] Amino acids comprise proteins. Amino acids may be
hydrophilic, i.e., displaying an affinity for water, or
hydrophobic, i.e., having an aversion to water. Thus, the amino
acids designated as G, A, V, L, I, P, F, Y, W, C and M are
hydrophobic and the amino acids designated as S, Q, K, R, H, D, E,
N and T are hydrophilic. In general, the hydrophilic or hydrophobic
nature of amino acids affects the folding of a peptide chain, and
consequently the three dimensional structure of a protein.
[0124] DNA is related to protein as follows:
##STR00001##
Genomic DNA comprises all the DNA sequences found in an organism's
cell. It is "transcribed" into messenger RNA ["mRNA"].
Complementary DNA ["cDNA"] is a complementary copy of mRNA made by
reverse transcription of mRNA. Unlike genomic DNA, both mRNA and
cDNA contain only the protein-encoding or polypeptide-encoding
regions of the DNA, the so-called "exons." Genomic DNA may also
include "introns," which do not encode proteins.
[0125] In fact, eukaryotic genes are discontinuous with proteins
encoded by them, consisting of exons interrupted by introns. After
transcription into RNA, the introns are removed by splicing to
generate the mature messenger RNA (mRNA). The splice points between
exons are typically determined by consensus sequences that act as
signals for the splicing process. Splicing consists of a deletion
of the intron from the primary RNA transcript and a joining or
fusion of the ends of the remaining RNA on either side of the
excised intron. Presence or absence of introns, the composition of
introns, and number of introns per gene, may vary among strains of
the same species, and among species having the same basic
functional gene. Although in most cases, introns are assumed to be
nonessential and benign, their categorization is not absolute. For
example, an intron of one gene can represent an exon of another. In
some cases, alternate or different patterns of splicing can
generate different proteins from the same single stretch of DNA. In
fact, structural features of introns and the underlying splicing
mechanisms form the basis for classification of different kinds of
introns.
[0126] As to the exons, these can correspond to discrete domains or
motifs, as for example, functional domains, folding regions, or
structural elements of a protein; or to short polypeptide
sequences, such as reverse turns, loops, glycosylation signals and
other signal sequences, or unstructured polypeptide linker regions.
The exon modules of the present combinatorial method can comprise
nucleic acid sequences corresponding to naturally occurring exon
sequences or naturally occurring exon sequences which have been
mutated (e.g. point mutations, truncations, fusions).
[0127] Returning now to the manipulation of DNA, DNA can be cut,
spliced, and otherwise manipulated using "restriction enzymes" that
cut DNA at certain known sites and DNA ligases that join DNA. Such
techniques are well known to those of ordinary skill in the art, as
set forth in texts such as Sambrook, et al., MOLECULAR CLONING: A
LABORATORY MANUAL, 2d ed. Cold Spring Harbor Laboratory Press
[1985] or Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, Inc. [1994].
[0128] DNA of a specific size and sequence can then be inserted
into a "replicon," which is any genetic element, such as a plasmid,
cosmid, or virus, that is capable of replication under its own
control. A "recombinant vector" or "expression vector" is a
replicon into which a DNA segment is inserted so as to allow for
expression of the DNA, i.e., production of the protein encoded by
the DNA. Expression vectors may be constructed in the laboratory,
obtained from other laboratories, or purchased from commercial
sources.
[0129] The recombinant vector [known by various terms in the art]
may be introduced into a host by a process generically known as
"transformation." Transformation means the transfer of an exogenous
DNA segment by any of a number of methods, including infection,
direct uptake, transduction, F-mating, microinjection, or
electroporation into a host cell.
[0130] Unicellular host cells, known variously as recombinant host
cells, cells, and cell culture, include bacteria, yeast, insect
cells, plant cells, mammalian cells and human cells. In
particularly preferred embodiments, the host cells include E. coli,
Pseudonas, Bacillis, Streptomyces, Yeast, CHO, R1-1, B-W, LH,
COS-J, COS-7, BSC1, BSC40, BMT10, and S69 cells. Yeast cells
especially include Saccharomyces, Pichia, Candida, Hansenula, and
Torulopis.
[0131] As those skilled in the art recognize, the expression of the
DNA segment by the host cell requires the appropriate regulatory
sequences or elements. The regulatory sequences vary according to
the host cell employed, but include, for example, in prokaryotes, a
promoter, ribosomal binding site, and/or a transcription
termination site. In eukaryotes, such regulatory sequences include
a promoter and/or a transcription termination site. As those in the
art well recognized, expression of the polypeptide may be enhanced,
i.e., increased over the standard levels, by careful selection and
placement of these regulatory sequences.
[0132] In other embodiments, promoters that may be used include the
human cytomegalovierus (CMV) promoter, tetracycline inducible
promoter, simian virus (SV40) promoter, moloney murine leukemia
long terminal repeat (LTR) promoter, glucocorticoid inducible
murine mammary tumor virus (MMTV) promoter, Herpes thymidine kinase
promoter, murine and human .beta.-actin promoters, HTLV1 and HIV
IL-9 5' flanking region, human and mouse IL-9 receptor 5' flanking
region, bacterial tac promoter and drosophila heat shock scaffold
attachment region (SAR) enhancer elements.
[0133] The DNA may be expressed as a polypeptide of any length such
as peptides, oligopeptides, and proteins. Polypeptides also include
translational modifications such as glycosylations, acetylations,
phosphorylations, and the like.
[0134] Another molecular biologic technique of interest to the
present invention is "linkage analysis." Linkage analysis is an
analytic method used to identify the chromosome or chromosomal
region that correlates with a trait or disorder..sup.44 Chromosomes
are the basic units of inheritance on which genes are organized. In
addition to genes, artisans have identified "DNA markers" on
chromosomes. DNA markers are known sequences of DNA whose identity
and sequence can be readily determined. Linkage analysis
methodology has been applied to the mapping of disease genes, for
example, genes relating to susceptibility to asthma, to specific
chromosomes..sup.42,44
[0135] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed. It is intended that the
specifications and examples be considered exemplary only with a
true scope of the invention being indicated by the claims.
Methods
[0136] In conducting the experiments described in the Examples
below, applicant used the following methods:
[0137] Patient Populations
[0138] Asthma families were recruited from two
sources..sup.27,42,79-83,88 In each case patients were genotyped
with respect to the polymorphism at position 3365 of the human IL-9
gene [GenBank accession number M30136].
[0139] A third population of 74 individuals was ascertained
randomly with respect to asthma and atopy from the East Coast of
the United States. The frequency of the Met substitution at codon
117 was used as an unbiased estimate of the prevalence of this
variant in the general population.
[0140] A fourth population of 49 individuals was ascertained
randomly with respect to asthma and atopy from the Philadelphia,
Pa. area. Total serum IgE were assayed by enzyme-linked
immunosorbent test [ELISA, Genzyme, Cambridge, Mass.]. DNA was
extracted from the WBC in peripheral blood from each individual.
Analyses of genetic markers (genotyping) and candidate genes were
performed on the genomic DNA extracted. Once again, the frequency
of the Met substitution at codon 117 was used as an unbiased
estimate of the prevalence of this variant in the general
population.
[0141] Oligonucleotide Primers.
[0142] All primers were designed using OLIGO 4.0. Characterization
of the IL-9 gene was carried out using primers surrounding each of
the 5 exons of the reported sequence. The primer sequences
surrounding each exon were: exon 1 [upper] [5' GCT CCA GTC CGC TGT
CAA 3'] and [lower] [5' CTC CCC CTG CAG CCT ACC 3'][product size
150 bp]; exon 2 [upper] [5' CGG GGC TGA CTA AAG GTT CT 3'] and
[lower] [5' GTT CTT AAA GAG CAT TCA CT 3'][product size 99 bp];
exon 3 [upper] [5' ATT TTC ACA TCT GGA ATC TTC ACT 3'] and [lower]
[5' AAT CCA AGG TCA ACA TTA TG 3'] [product size 113 bp]; exon 4
[upper] [5' TTT CTT TGA ATA AAT CCT TAC 3'] and [lower] [5' GAA ATC
ACC AAC AGG AAC ATA 3'] [product size 206 bp]; and exon 5 [upper]
[5' ATC AAC TTT CAT CCC CAC AGT 3'] and [lower] [5' GGA TAA ATA ATA
TTT CAT CTT CAT 3']. Each exon was examined first by a single
strand conformational polymorphism assay [SSCP]..sup.72,77 The
primers for exon 5 produced a 160 bp product after polymerase chain
reaction [PCR] amplification which was also examined by direct
solid phase sequence analysis..sup.72,77 The upper primer was
synthesized with a 5' biotin label and, following amplification,
the PCR product was captured by a streptavidin-linked paramagnetic
bead [Dynal] and characterized by Sanger sequencing as described
elsewhere..sup.77 Sequence polymorphisms were distinguished from
artifact by repeated analyses.
[0143] SSCP Analysis.
[0144] SSCP, a method for detection of polymorphisms on the basis
of changes in migration of single-stranded DNA exposed to an
electric field,.sup.72 was carried out as set forth in Schwengel et
al., (1994) at room temperature with and without 10% glycerol using
6% polyacrylamide gel electrophoresis at a cross-linking monomer
concentration of 2.67%..sup.77 Four .mu.l of PCR product was mixed
with 5 .mu.l 2.times. stop buffer [95% formamide, 20 mM EDTA, 0.05%
BPB, 0.05% xylene cyanol], and 1 .mu.l 0.5% SDS and 50 .mu.M EDTA,
denatured at 85-90.degree. C. for 8 minutes, and then immediately
placed on ice. Electrophoresis was carried out at 12 watts for
approximately 24 hours for glycerol containing gels and 12 hours
for non-glycerol gels. The gels were then dried and exposed to
Kodak XAR.RTM. film.
[0145] DNA Sequencing.
[0146] Direct DNA sequencing of the PCR products was accomplished
using solid phase techniques after verifying the presence of the
correct size PCR product on a 1% agarose gel stained with ethidium
bromide as set forth in Schwengel et al., 1994..sup.77 Twenty .mu.l
of PCR product was incubated with 40 .mu.l of Dynabeads.RTM. m-280
[Dynal] for 15 minutes. The beads were washed and diluted as
suggested by the manufacturer. Each sample was subsequently washed
with B&W buffer containing 10 mM tris-HCl pH 7.5, 1 mM EDTA, 2
M NaCl, denatured with 0.1 N NaOH, and then washed with 0.1 N NaOH,
B&W buffer, and 10 mM Tris-HCl pH 8 and 1 mM EDTA [TE]. The
pellet of beads was resuspended with 10 .mu.l of H2O.
[0147] Sanger sequencing reactions were carried out using Sequenase
[United States Biochemical Co.]. .sup.35S-dATP or .sup.33P-dATP was
incorporated into the sequencing reactions, and the products were
electrophoresed through either 5% or 6% polyacrylamide gels
containing 7 M urea. Gels were dried without fixing and exposed to
X-ray film. Alleles were determined by comparing the genotypes of
parents and offspring. Infrequent artifacts were easily
distinguished from true sequence polymorphisms by repetition.
[0148] DNA was available and extracted from peripheral leukocytes.
Genomic DNA was diluted to a concentration of 200 .mu.g/ml for
amplification..sup.27,42 Simple sequence repeats [SSR] including
DXYS154 were selected from the Genome Data Base [GDB; Welch
library, Johns Hopkins University, Baltimore, Md.]. Genotyping of
the sKK-1 marker was carried out using the following primers sKK-1U
[5' CAA ATC TGA AGA GCA AAC TAT 3'] and sKK-1L [5' TTA AAA AAT TCA
TTT CAG TAT TCT 3'] which produce a 90 bp product. Each SSR product
was amplified by PCR.sup.72 and sized according to methods
previously described..sup.27,42 Sample handling was carried out as
described by Weber et al. with minor modifications..sup.71,27,42
Genotypes were determined from two independent readings of each
autoradiograph. Individuals genotyping the families were blinded to
the clinical data.
[0149] RFLP Analysis.
[0150] As a result of the C to T polymorphism at position 3365, a
StyI restriction fragment length polymorphism [RFLP] was produced
at position 52 of the IL-9 exon 5 PCR product. To test for the
presence of this DNA sequence variant the lower primer from exon 5
was end-labeled prior to PCR amplification. The PCR product was
then digested with StyI producing two fragments 108 bp [labeled]
and 52 bp [unlabeled] in length. This RFLP was used along with SSCP
to confirm the presence of this polymorphism in families and
individuals.
[0151] Linkage Analyses and Data Management.
[0152] Linkage analyses were performed using affected sib-pair
methods [SIBPAL, S.A.G.E.].sup.78 an established approach for the
investigation of the genetic basis of complex traits, such as BHR,
atopy, and asthma. Affected sib-pairs are usually tested first,
since a proportion of unaffected sib-pairs may still be gene
carriers but do not express the trait. In contrast to LOD score
methods where the model of inheritance [dominant, recessive, etc.]
must be specified exactly, analysis by sib-pair methods makes no
explicit assumptions in this regard. Thus, in sib-pair analyses the
parents' clinical information is not used in testing for linkage.
The pertinent observation in these methods is how often two
affected offspring share copies of the same parental marker
allele..sup.44 If the same copy of a parental marker allele is
observed in different offspring, they are said to be inherited
"identical by descent." Linkage is suggested when affected
sib-pairs are identical by descent for a marker allele
significantly more often than expected by chance [50%]. When the
same marker allele is transmitted with the disease gene in
different offspring, this implies that the marker locus is linked,
or must be located close enough on the same chromosome, to the
disease gene so they cosegregate during meiosis. The trait is then
mapped by knowing the chromosomal localization of the marker.
[0153] Linkage in humans may also be established by the method of
likelihood ratios. This method involves comparison of the
probability that observed family data would arise under one
hypothesis, for instance, linkage between two DNA markers, to the
probability that it would arise under an alternative hypothesis,
typically, nonlinkage. The ratio of these probabilities is called
the odds ratio for one hypothesis relative to the other. By
convention, mammalian geneticists prefer the log of the odds
ration, or the LOD score. Generally, linkage is considered proved
when the odds in favor of linkage versus nonlinkage become
overwhelming, or reach 1,000:1 [LOD=3]. Linkage is rejected when
the odds drop to 100:1 against this hypothesis [LOD=-2]. The
maximum likelihood estimate is the recombination fraction where the
likelihood ratio is largest. LODs from multiple pedigrees are thus
added until the score grows to 3 [signifying 1,000:1 odds] or falls
to -2 [indicating 100:1 odds].
[0154] All clinical and genotype data is managed using EXCELL.RTM.
on a Macintosh.RTM. or Sun Microsystems.RTM. computer. Statistical
analyses were preformed using JMP [SAS Institute, Inc. Cary, N.C.].
The Wilcoxon/Kruskal-Wallis Tests [rank sums] was used to test
whether individuals who were homozygous [Met/Met], heterozygous
[Met/Thr], or homozygous [Thr/Thr] at codon 117 differ in their
serum total IgE. All P-values are two-tailed except affected
sib-pair analyses, where a one-tailed test was used because only an
increased sharing of alleles was expected.
[0155] Having provided this background information, applicant now
describes preferred aspects of the invention.
EXAMPLE 1
Linkage Analysis Between BHR and Murine Chromosome 13
[0156] As an aid in dissecting the complex genetic determinants of
BHR, applicant has developed murine models that differ in their
genetic susceptibility to various bronchoconstrictor stimuli.
Inbred animal models using recombinant inbred strains [BXD] can
facilitate ongoing studies in humans to determine the number of
genes regulating susceptibility to BHR, the magnitude of their
affect, and their precise chromosomal location. In particular,
localizing in an animal model a gene determining susceptibility to
a critical risk factor for asthma may aid in the positional cloning
of this gene in humans.
[0157] Although the gene[s] predisposing to BHR and atopy had not
yet been identified prior to this invention, chromosome 5q31-q33
was known to be syntenic with portions of mouse chromosomes 11, 13,
and 18. FIG. 2 illustrates the syntenic regions containing numerous
positional candidates that may potentially play a role in airway
inflammation associated with BHR, atopy, and asthma. Specifically,
the region of human chromosome 5q31-q33 demonstrating significant
evidence for linkage with BHR is homologous to portions of mouse
chromosomes 11, 13, and 18 which contain numerous candidate
genes..sup.84
[0158] In particular, IL-9 or a nearby gene have recently been
suggested as likely candidates on the basis of linkage
disequilibrium between log serum total IgE levels and a marker in
this gene using a randomly ascertained population..sup.43
[0159] Despite comparisons with four candidate intervals, evidence
for linkage was found for only one region, designated Aib 1
[atracurium induced bronchoconstriction 1]. FIG. 3 provides the
results. Specifically, FIG. 3 sets forth the LOD score curve on
mouse chromosome 13 for atracurium-induced airway responsiveness in
24 BXD RI strains which are derived from the hyporesponsive
C57BL/6J and the hyperresponsive DBA/2J progenitor strains [solid
line]. The LOD score curve resulting from the selective genotyping
of 20 BXD strains is also shown [dashed line]. BXD strains -2, -6,
-18, and -32 were not used in the second analysis since they were
intermediate in phenotype displaying a mean response greater than 1
standard deviation below the DBA/2 and above the C57BL/6 mean
responses. The bronchoconstrictor response to atracurium, 20 mg/kg
given intravenously, was assessed by the change in peak inspiratory
pressure integrated over time [4 min],termed the airway pressure
time index [APTI]. Atracurium-induced APTI was measured in 2-8
animals per RI strain. Marker data were obtained from the RWE data
base in the Map Manager data analysis program. The genetic distance
[cM] between markers is indicated on the abscissa. LOD scores were
calculated by the MAPMAKER/QTL linkage program. A QTL was detected
in this region and termed atracurium-induced bronchoconstriction-1
[Aib1].
[0160] FIG. 3 indicates that this quantitative trait locus [QTL] is
located on the midportion of murine chromosome 13 and attained this
interval a maximum likelihood log of the odds [LOD] of 2.42.
Forty-four percent of the total variance in atracurium-induced
bronchoconstriction was explained at Aib1 when all of the markers
in the BXD map were analyzed. The LOD for chromosome 13 increased
to 2.85 when QTL analyses were run after excluding the four strains
[BXD-2, -6, -18, and -32] that were intermediate responders to
atracurium. The known positional candidates in the linked region of
chromosome 13 include: D1 dopamine receptor [Drd1], fibroblast
growth factor receptor 4 [Fgfr4], lymphocyte antigen-28 [Ly28],
thiopurine methyltransferase [Tpmt], and IL-9.
[0161] Because the applicant was specifically testing for linkage
to four candidate regions in the mouse, based on previous mapping
data in the human, the data presented here are highly significant.
As stated in the classic paper by Lander and Botstein,.sup.67 a
false positive rate for linkage will result if the LOD threshold
(T) is chosen so that T=1/2(log 10 e)(Z .alpha./n)2 (where n is
equal to the number of intervals tested). Typically a minimum LOD
of 3.3 is required as evidence of linkage..sup.67 However, this
threshold is based on the assumption that one is searching the
entire genome. These same authors point out that a LOD of 0.83 is
sufficient when only one region is examined. In this case, with
four candidate regions, a P value (.alpha.) of 0.0125 for each
region is required to obtain a true P.ltoreq.0.05, when one
corrects for multiple independent comparisons. Adopting a 5% error
rate that even a single false positive finding will occur, as
suggested by Soller and Brody,.sup.68 and solving the equation
1/2(log 10 e)(Z .alpha./n).sup.2, yields a LOD threshold of at
least 1.36. A maximal LOD of 1.48 was obtained for the Il-9 gene
candidate. Restricting the acceptable false positive error rate to
.ltoreq.0.1%, increases the LOD threshold to 2.36. Thus, the
maximal LOD generated of 2.42 for the candidate interval on
chromosome 13 (Aib1) is highly significant.
[0162] These LOD threshold data provide evidence of a conserved
linkage for BHR in humans and mice. BHR in humans links to the
region on chromosome 5q containing a number of growth factors and
cytokines including the IL-9 gene and the Aib1 locus maps to the
IL-9 region of murine chromosome 13.
EXAMPLE 2
Identification of an IL-9 Gene Polymorphism
[0163] Applicant demonstrated conserved linkage between the mouse
and humans for BHR. These data suggest that variation in the
functions of this gene or DNA sequence may be important in
regulating bronchial responsiveness in the mouse. Using the methods
described above, a unique product of the correct size was
identified by gel electrophoresis for each of the exons of human
IL-9 after PCR. A single polymorphism was identified by SSCP in
exon 5 of the human IL-9 gene. Direct DNA sequence analysis
demonstrated a C to T nucleotide substitution at position 3365
[GenBank accession number M30136] of the human IL-9 gene as the
cause of the novel SSCP conformer. This DNA sequence change
predicts a nonconservative substitution of a methionine
[hydrophobic] for a threonine [hydrophilic] at amino acid 117 of
the IL-9 protein.
[0164] Exon 5 codes for this segment of the protein which is within
the most highly conserved interval of human IL-9 as compared to the
mouse IL-9 sequence (see FIG. 4).
[0165] Individuals were genotyped from various populations to
examine the frequency of these alleles by direct analyses of the
nucleotide substitution in the coding sequence of human IL-9. Two
of 394 individuals from a group of asthmatic families were
homozygous [Met/Met] at codon 117 [0.5%]. There were 91 [23.1%]
heterozygous, and 301 [76.4%] homozygous [Thr/Thr] individuals. The
true prevalence for this IL-9 variant is likely to be significantly
higher because the Italian population of families was ascertained
through symptomatic patients with asthma. From a separate
ethnically diverse population ascertained randomly with respect to
atopy and asthma, there were 1 of 49 individuals homozygous for
[Met/Met] at codon 117 [2.0%]. There were 11 [22.4%] heterozygous,
and 37 [75.5%] homozygous [Thr/Thr] individuals. The prevalence of
the Met/Thr heterozygotes was 18.9% in a fourth population
ascertained randomly with respect to atopy and asthma. Thus,
approximately 20% of the population are likely to represent
carriers of the T allele at position 3,365 as compared to the
reported sequence [GenBank accession number M30136]. Because it is
well known in the art that the frequency of any allele in the
population is p2+2pq+q2, then, approximately 4% of the population
is expected to be Met/Met homozygous at codon 117 of IL-9.
[0166] Overall, serum total IgE averaged 44.5 I.U. for homozygous
individuals [Met/Met], which was significantly different from those
who were homozygous wild type [Thr/Thr] [351.7 I.U.], or
heterozygous [Met/Thr] [320.9 I.U.]. See FIG. 5. The homozygous
protected individuals [Met/Met] failed to demonstrate evidence of
atopic allergy except for a single positive skin test in one
individual. These data indicate that this novel DNA polymorphism,
when inherited in the homozygous state, is associated with
protection from atopic allergy, including lower serum total
IgE.
[0167] FIG. 6 illustrates the PCR amplification of the IL-9 simple
sequence repeat polymorphism. This marker is compared with genotype
for these individuals for the restriction fragment length
polymorphism produced by the nucleotide polymorphism at position
3,365 as compared to the reported sequence [GenBank accession
number M30136]. Two families are shown. The individuals in lanes 1
and 2 are the parents (Thr/Met) of individuals in lanes 3 (Met/Thr)
and 4 (Met/Met); lanes 5 (Thr/Thr) and 6 (Met/Met) are parents for
offspring in lanes 7 (Met/Thr) and 8 (Met/Thr). The smallest allele
for the IL-9 simple sequence repeat polymorphism (the lowest band
in each figure is 248 nucleotides in length) is in complete linkage
disequilibrium with the Met117 allele (nucleotide substitution at
position 3,365 as compared to the reported sequence [GenBank
accession number M30136]) in these individuals and in all
individuals from populations tested world wide. This was true in
both the Italian and all random ascertained ethnically diverse
individuals studied, and therefore, this marker may be used
diagnostically to detect the presence of the Met117 allele. These
data are most consistent with the hypothesis that this variant is
widely distributed in populations worldwide and arose before many
of these populations separated.
EXAMPLE 3
IL-9 Receptor Expression and Ligand Binding Assay
[0168] Purified recombinant Thr IL-9, Met IL-9, and compounds
potentially resembling Met IL-9 in structure or function are
radiolabelled using the Bolton and Hunter reagent as described in
Bolton A E, and Hunter W M, Biochem J. 133:529-539(1973). This
material is labeled to high specific activity of 2,300 cpm/fmol or
greater. Human K562 and MO7e cells are grown and resuspended at
30.degree. C. in 0.8 ml of Dulbecco's modified Eagle's medium
supplemented with 10% (vol/vol) fetal bovine serum, 50 mM
2-mercaptoethanol, 0.55 mM L-arginine, 0.24 mM L-asparagine, and
1.25 mM L-glutamine. K562 or MO7e cells are used as is or after
transfection with the IL-9 receptor gene as described below.
Plasmid DNA containing the full length IL-9 receptor is cloned into
pRC/RSV plasmid (In Vitrogen, San Diego) and purified by
centrifugation through CsCl2. Plasmid DNA (50 micrograms) is added
to the cells in 0.4 cm cuvettes just before electroporation. After
a double electric pulse (750V/74 ohms/40 microFaradays and 100 V/74
ohms/2100 microFaradays) the cells are immediately diluted in fresh
medium supplemented with IL-9. After 24 h the cells are washed and
incubated in G418 (2.5 mg/ml, GIBCO) with either no ligand, or
various concentrations of 125I-labeled ligand at 20.degree. C. for
3 h. An excess of unlabeled ligand is used in parallel experiments
to estimate nonspecific binding. The cells are then washed,
filtered, and collected for counting. Specific incorporation is
calculated by Scatchard analysis. Similar competitive assays are
run using 125I-labeled Thr117 IL-9 and various amounts of putative
cold ligands to assess specific binding.
[0169] Soluble IL-9 receptor including amino acids 44 to 270
(R&D Systems) was incubated with different forms of human
recombinant IL-9. Varying amounts of FlagMet117 and FlagThr117
(described in Example 7) were incubated in PBS at room temperature
for 30 minutes with 0.5 .mu.gs of soluble receptor. EBC buffer (50
mM Tris pH 7.5; 0.1 M NaCl; 0.5% NP40) was added (300 .mu.l) was
added along with 1 .mu.g of anti-FLAG monoclonal antibody (IBI) and
incubated for 1 hour on ice. Forty microlitres of protein A
sepharose solution was added to each sample and mixed for 1 hour at
4.degree. C. Samples were centrifuged for 1 minute 11,000.times.G
and pellets were washed 4 times with 500 .mu.l of EBC. Pellets were
dissolved in 26 .mu.l of 2.times. SDS buffer, boiled for 4 minutes,
and electrophoresed through an 18% SDS polyacrylamide gel. Western
blots were performed as described in Example 15 except the blots
were probed with an anti-IL-9 receptor antibody (R&D
Systems).
[0170] FIG. 25 demonstrates the binding of the IL-9 recombinant
proteins soluble IL-9 receptor. Lane 1 is molecular weight markers,
lane 2 is the IL-9 FlagMet117 incubated with the receptor, lane 3
is the IL-9 FlagThr117 incubated with the receptor. These data
demonstrate that both forms of the recombinant IL-9 protein are
bound to the soluble receptor. Moreover, these data along with
those of Example 2 (where heterozygotes do not differ in serum Ig-E
from homozygous Thr117 individuals) are consistent with the IL-9
Met117 form representing a weak agonist.
EXAMPLE 4
IL-9 Receptor Expression and Ligand Functional Assay in K562,
C8166-45, and MO7e Cells
[0171] Recombinant Thr117 IL-9, Met117 IL-9, and compounds
potentially resembling Met IL-9 in structure or function were
purified and prepared for use in Dulbecco's modified Eagle's
medium. K562, C8166-45 or MO7e cells are used as is or after
transfection with the IL-9 receptor gene as described in Example 3.
After 24 h of deprivation from growth factors the cells are
incubated without (control) or with variable amounts of purified
Thr117 IL-9, Met117 IL-9, and compounds potentially resembling
Met117 IL-9 in structure or function. Cellular proliferation is
assessed by measuring acid phosphotase activity. Briefly,
quadruplicate samples of MO7e cells are cultured in flat-bottom
microtiter plates (150 or 200 microliter wells) with or without
ligand for 72 to 96 hours at 37 degrees C. Acid phosphatase is
measured as suggested by the manufacturer (Clontech, Palo Alto,
Calif.). All experiments are repeated at least twice.
EXAMPLE 5
Cell Isolation and Culture
[0172] Human peripheral blood mononuclear cells {PBMC} were
isolated from healthy donors by density gradient centrifugation
using endotoxin tested Ficoll-Paque PLUS according to the
manufacturer (Pharmacia Biotech, AB Uppsala Sweden). PBMC
(5.times.10.sup.6), mouse spleen cells (5.times.10.sup.6), or
5.times.10.sup.6 MO7e cells were cultured in 7 ml of RPMI-1640
(Bethesda Research Labs (BRL), Bethesda, Md.) supplemented to a
final concentration of 10% with either isogenic human serum or
heat-inactivated FBS. Cells were cultured for 24 hrs at 37.degree.
C. either unstimulated, or stimulated with either PMA 5
.mu.g/ml/PHA 5 .mu.g/ml, or PHA 5 .mu.g/ml/rhIL2 50U (R&D
Systems, Minneapolis, Minn.).
EXAMPLE 6
RNA Isolations, RT-PCR, Cloning and Sequencing of RT-PCR
Products
[0173] Total cellular RNA was extracted after 24 hrs from cultured
PBMC, mouse spleen cells, and MO7e cells using RNA PCR corekit
(Perkin-Elmer Corp, Foster City, Calif.) according to the supplier.
One pg of RNA from each source was denatured for 5 minutes at
65.degree. C. and then reverse transcribed into cDNA using a 20 41
reaction mixture (RNA PCR corekit, Perkin-elmer Corp, Foster City,
Calif.) containing 50U of MuMLV Reverse Transcriptase, 1U/.mu.l
RNAse Inhibitor, 2.5 mM oligo d(T)16 primer, 1 mM each of dATP,
dCTP, dGTP, dTTP, 50 mM KCl, 10 mM Tris-HCL, pH 7.0, 25 mM MgCl2.
The reaction mixture was pipetted into thermocycler tubes, placed
in a PCR thermal cycler and subjected to 1 cycle (15 minutes at
42.degree. C., 5 minutes at 99.degree. C., and 5 minutes at
4.degree. C.). A mock reverse transcription reaction was used as a
negative control.
[0174] Then this mixture was added to a second tube containing 2 mM
MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 7.0, 65.5 .mu.l of DI water,
2.5U Amplitaq DNA polymerase, and 1 .mu.l (20 .mu.M) each of
oligonucleotides representing human cDNA IL-9 exon 1 (forward) and
exon 5 (reverse), for a final volume of 100 .mu.l. The reaction
mixture was subjected to the following PCR conditions: 120 seconds
at 98.degree. C., then 30 cycles at: 30 seconds at 94.degree. C.;
40 seconds at 55.degree. C.; 40 seconds at 72.degree. C. Finally,
the reaction mixture was cycled one time for 15 minutes at
72.degree. C. for extension;
[0175] PCR products representing hIL-9 cDNA were subjected to gel
electrophoresis through 1.5% agarose gels and visualized using
ethidium bromide staining. Products of a mock reverse transcriptase
reaction, in which H.sub.2O was substituted for RNA, and used as
negative control amplification in all experiments.
[0176] The PCR oligonucleotide primer pairs used in these
experiments to amplify cDNA include: human interleukin 9 (hIL-9)
exon 1 forward 5'-TCT CGA GCA GGG GTG TCC AAC CTT GGC G-3' (SEQ ID
NO: 1) and exon 5 reverse 5'GCA GCT GGG ATA AAT AAT ATT TCA TCT TCA
T-3' (SEQ ID NO: 2); mouse interleukin 9 (mIL-9) exon 1 forward
5'-TCT CGA GCA GAG ATG CAG CAC CAC ATG GGG C-3' (SEQ ID NO: 3) and
mouse exon 5 reverse 5'-GCA GCT GGT AAC AGT TAT GGA GGG GAG GTT
T-3' (SEQ ID NO: 4); XhoI and PvuII restriction enzyme recognition
sequences are underlined in the human and mouse IL-9 primers. PCR
products were subcloned into the TA Cloning vector (Invitrogen, San
Diego, Calif.). Amplification of the mouse cDNA gave a 438 bp
product and amplification of the human cDNA gave a 410 bp
product.
[0177] Complementary DNAs for human IL-9 and murine mIL-9 were
generated and amplified by RT-PCR using IL-9 exon 1 and 5 specific
primers containing digestion sites for XhoI and PvuII restriction
endonucleases. Amplification products for hIL-9 and mIL-9 were
isolated from 2.5% agarose gels using silica (Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor
Laboratory Press, New York) (incorporated herein by reference in
its entirety). After recovery, the cDNA products were ligated into
the TA Cloning vector (Invitrogen Corp., San Diego, Calif.) and
then used to transform INV.alpha.F' competent cells, according to
the manufacturer's instructions. Plasmids containing hIL-9 and
mIL-9 cDNA inserts were isolated by conventional techniques
(Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual
Cold Spring Harbor Laboratory Press, New York). After amplification
the DNA sequence including and surrounding each insert was analyzed
for PCR-induced or cloning-induced errors.
[0178] hIL-9 and mIL-9 cDNA inserts were sequenced by the
dideoxy-mediated chain termination method (Sanger et al. (1977)
Proc. Natl. Acad. Sci. USA 74:5463), using the M13 (-20) forward
primer (5'-GTA AAA CGA CGG CCA GT-3') (SEQ ID NO: 17) and
Sequenase.TM. (USB), and analyzed by gel electrophoresis (Sambrook,
J. et al. (1989) Molecular cloning: a laboratory manual Cold Spring
Harbor Laboratory Press, New York). hIL-9 and mIL-9 cDNA inserts
without cloning and/or Taq polymerase-induced sequence errors (see
translated cDNA sequences FIGS. 7 and 8) were subcloned into
expression vectors (see FIGS. 9-12) or used to create missense
mutations and deletion mutants.
EXAMPLE 7
Cloning and Expression of IL-9 Constructs in Vitro
General Cloning Methods for Constructs
[0179] hIL-9 was subcloned into procaryotic expression vectors. The
TA2AAF1 met and thr vectors were digested by EcoRI and the 0.420 kB
fragment (containing an XhoI site at the 5' end of the hIL9 cDNA)
was cloned into the EcoRI site contained with the polylinker of
pBluescript (PBS) (Stratagene). Clones in the sense orientation to
the T3 promoter were then digested with XhoI (the fragment
contained a 5' XhoI site from the IL-9 cDNA insert from TA vectors
and a 3' XhoI site from the PBS polylinker) and inserts were
subcloned into the XhoI sites of the procaryotic expression vectors
pGEX and pFLAG.
Cloning and Expression of IL-9 Constructs in the pGEX-4T-1
Glutathione s-Transferase Gene Fusion Vector
[0180] For the expression, purification, and detection of IL-9
protein, IL-9 cDNA inserts were subcloned into the XhoI site within
the multiple cloning cassette of the 4.9 Kb pGEX-4T-1 glutathione
s-transferase gene fusion vector (Pharmacia Biotech, Piscataway,
N.J.) by standard techniques. Briefly, TA clones containing intact
IL-9 cDNA sequences, and the pGEX-4T-1 vector were digested for one
hour at 37.degree. C. using XhoI and PvuII restriction endonuclease
in the presence of 1.times. React 2 buffer (New England Biolabs,
Beverly, Mass.) (total volume 50 .mu.l). Products were
electrophoresed in a 1.5% preparative agarose gel with 10 .mu.g/ml
ethidium bromide. The appropriate sized DNA band was excised, the
agarose was melted at 45.degree. C. for 10 minutes in 3 volumes of
NaI stock solution. A silica matrix solution in DI H.sub.2O
(Geneclean II, LaJolla, Calif.) was added to the solution at 5
.mu.l per 5 .mu.g of DNA and 1 .mu.l per 0.5 .mu.g of DNA above 5
.mu.g. The slurry was incubated at 4.degree. C. and occasionally
shaken during 30 minutes. The slurry was then pelleted via
microcentrifugation, washed 3 times in low-salt buffer and
resuspended in 10 .mu.l of DI H.sub.2O to elute the DNA from the
silica. A final microcentrifugation provided the 10 .mu.l solution
containing purified DNA.
[0181] Products were resuspended in 50 .mu.l of DI H.sub.2O and
precipitated by the addition of 2 volumes of ethanol and 1/10
volume 3M sodium acetate. Samples were centrifuged at RT at 14,000
rpm for 10 minutes, air dried under negative pressure and
resuspended in an appropriate volume of DI H.sub.2O. Ligations and
transformations of DH5a bacteria (GIBCO/BRL, Gaithersburg, Md.)
with mIL-9 and hIL-9 cDNA inserts in the pGEX-4T-1 vector were
performed using standard techniques.
[0182] To confirm that the hIL-9 cDNA inserts contained in the
pGEX-4T-1 vector were of the correct nucleotide sequence, plasmids
containing candidate IL-9 cDNA were sequenced via the
dideoxy-mediated chain termination method using the aforementioned
mIL-9 and hIL-9 cDNA-specific oligonucleotides (exon 1 forward,
exon 5 reverse primers).
[0183] Recombinant fusion proteins were obtained from large scale
cultures. The overnight culture of transformed E. coli (50 ml) was
inoculated into fresh LB/amp broth. The culture was incubated for 4
hr at 37.degree. C. with vigorous shaking, isopropyl
.beta.-D-thiogalactopyranoside was then added to a final
concentration of 1 mM, and the culture was incubated for an
additional 1.5 h. The cells were harvested by centrifugation at
500.times.g at 4.degree. C. and recombinant variants were purified
by making use of affinity chromatography on glutathione-sepharose
4B column (Pharmacia) for GST-fusion proteins. Some variants were
expressed as inclusion bodies and were purified from insoluble
inclusion bodies by the procedure described by Marston (1987 The
purification of eukaryotic polypeptides expressed in E. coli in
Clover D. M. ed. DNA cloning: A practical approach, IRL Press,
Oxford, 59-88). Briefly, the cells were lysed with lysozyme
followed by treatment with deoxycholic acid. Contaminating nucleic
acids were removed by treatment with DNase I. The insoluble
material was washed once with 2 M urea and finally solubilized in
lysis buffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 100 mM NaCl)
containing 8 M urea. The solubilized components from the inclusion
bodies were dialyzed stepwise against decreasing concentrations of
urea (starting with 8, 6, 4 and 2 M of urea) in lysis buffer to
allow for refolding of the denatured protein. Finally, the sample
was dialyzed against 2 M urea and 2.5% .beta.-mercaptoethanol
(.beta.-ME) and centrifuged at 10,000 g for 15 min. The fusion
protein was finally dialyzed against 0.01 M Tris-Cl, pH 8.0. Fusion
proteins expressed in pGEX-4T vector were cleaved with 100 U of
Thrombin for 6 hr at 37.degree. C. and recovered in flow through
fractions after chromatography on glutathione-Sepharose 4B column.
Final purification was achieved by chromatography on Sephadex G-100
column (100.times.1.5 cm), packed and equiliberated with 0.05 M
ammonium bicarbonate buffer.
[0184] Cloning and Expression of IL-9 Constructs in the pFLAG-1.TM.
Expression Vector
[0185] For the expression, purification and detection of human IL-9
protein, IL-9 cDNA inserts were subcloned into the Xho2 site of the
multiple cloning site (XhoI) of the 5.37 Kb Flag vector. FLAG
technology is centered on the fusion of a low molecular weight (1
kD), hydrophilic, FLAG marker peptide to the N-Terminus of a
recombinant protein expressed by the pFLAG-1.TM. Expression
Vector(1) (obtained from IBI Kodak). Each bacterial colony was
grown in LB broth containing 50 .mu.g ampicillin per ml until the
optical density at 590 nm reached O.6. IPTG was then added to a
final concentration of 1 mM, and the cultures incubated for an
additional 1 hr to induce protein synthesis. The cells were
harvested by centrifugation, and the cell pellet was boiled in 50
.mu.l of Laemmli buffer (Laemmli, 1970) for 10 min and
electrophoresed on 10% polyacrylamide gels. The Anti-FLAG.TM. M1
monoclonal antibody was used for specific and efficient detection
of the FLAG fusion protein on western (see FIG. 13) slot or dot
blots throughout its expression, affinity purification, and FLAG
marker removal. The FLAG fusion protein was rapidly purified under
mild, non-denaturing conditions in a single step by affinity
chromatography with the murine Anti-FLAG.TM. IgG M1 monoclonal
antibody covalently attached to agarose. Following affinity
purification the fusion protein may be used after removal from the
affinity column or the authentic protein may be recovered in
biologically active form by specific and efficient proteolytic
removal of the FLAG peptide with enterokinase. Final purification
was achieved by chromatography on Sephadex G-100 column
(100.times.1.5 cm), packed and equiliberated with 0.05 M ammonium
bicarbonate buffer. The promoters described above in this example
may also be used with FLAG technology.
[0186] SDS-PAGE and Immunoblot Analysis
[0187] SDS-PAGE was performed by the method of Laemmli (Laemmli
U.K. (1970) Nature 227, 680-685) (incorporated herein by reference
in its entirety) by using a 12.5% polyacrylamide gel in a mini-gel
system (SE 280 vertical gel unit, Hoefer). For immunoblot analysis,
the proteins separated by SDS-PAGE were transferred to
nitrocellulose membranes by using the TE 22 Mighty small transfer
unit (Hoefer) in 25 mM Tris-glycine buffer, pH 8.3, containing 15%
methanol (Towbin H., et al., (1979) Proc. Natl. Acad. Sci. U.S.A.
76, 4350-4354). The unoccupied binding sites on the membrane were
blocked by incubating for 1 h with 20 mM Tris-HCl buffer, pH 8.0,
containing 2% bovine serum albumin. The membranes were then
incubated with 1:200 dilution of antibodies overnight at 4.degree.
C. The membranes were washed and treated with 1:2000 diluted goat
anti-rabbit IgG conjugated with either peroxidase or alkaline
phosphotase for 1 h. After washing, the bound antibodies were
visualized by addition of the super-substrate chemiluminescent
reagent (Pierce) or the 4-chloro-1-naphthol color developing
reagent. The reaction was stopped by immersing the membranes in
distilled water. FIG. 13 demonstrates that the purified recombinant
human FLAG IL-9 fusion proteins (Met117 and Thr117) are the correct
size and in the correct reading frame because they are recognized
by the Anti-FLAG.TM. M1 monoclonal antibody.
[0188] Analytical Methods
[0189] The molecular mass of the purified proteins was confirmed by
matrix assisted laser desorption mass spectrometry using Perceptive
Biosystems Voyager Biospectrometry workstation. Amino acid analyses
were performed after hydrolysis of the sample in 6N HCl at 110-C
for 24 h in evacuated sealed glass bulbs.
[0190] Automated Edman Degradation
[0191] The partial amino acid sequence of the purified proteins is
determined by automated step-wise sequencing on an Applied
Biosystems model 477A gas-phase sequencer with an on-line model 20A
PTH analyzer.
EXAMPLE 8
Deletion of Exon 2 and/or Exon 3: Mutagenesis and Sequencing of
Constructs
[0192] Human exon 2 and exon 3 deletions are created using ExSite
PCR-based site-directed mutagenesis kit as suggested by the
manufacturer (Stratagene, La Jolla, Calif.). The PCR primers are as
follows: h9CD1U forward 5'-GTG ACC AGT TGT CTC TGT TTG-3' (SEQ ID
NO: 5); h9CD1L reverse 5'-CTG CAT CTT GTT GAT GAG GAA-3' (SEQ ID
NO: 6); h9CD2U forward 5'-GAC AAC TGC ACC AGA CCA TGC-3' (SEQ ID
NO: 7); h9CD2L reverse 5'-ATT AGC ACT GCA GTG GCA CTT-3' (SEQ ID
NO: 8). Exon 2 deletions are created by using the primer pair
h9CD1L forward and h9CD1L reverse. Exon 3 deletions are created by
using h9CD2U forward and h9CD2L reverse. Deletions that included
exon 2 and exon 3 use the primer pair h9CD2U forward h9CD1L
reverse.
[0193] Mouse exon 2 and exon 3 deletions are created using ExSite
PCR-based site-directed mutagenesis kit as suggested by the
manufacturer (Stratagene, La Jolla, Calif.). The PCR primers are as
follows: m9CD1U forward 5'-GTG ACC AGC TGC TTG TGT CTC-3' (SEQ ID
NO: 9); m9CD1L reverse 5'-CTT CAG ATT TTC AAT AAG GTA-3' (SEQ ID
NO: 10); m9CD2U forward 5'-GAT GAT TGT ACC ACA CCG TGC-3' (SEQ ID
NO: 11); m9CD2L reverse 5'-GTT GCC GCT GCA GCT ACA TTT-3' (SEQ ID
NO: 12). Exon 2 deletions are created by using the primer pair
m9CD1U forward and m9CD1L reverse. Exon 3 deletions are created by
using m9CD2U forward and m9CD2L reverse. Deletions that included
exon 2 and exon 3 use the primer pair m9CD2U forward m9CD1L
reverse.
[0194] Mutagenized constructs of the hIL-9 and mIL-9 cDNA inserts
are sequenced by the dideoxy-mediated chain termination method
(Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463)
(incorporated herein by reference in its entirety), using the M13
(-20) forward primer (5'-GTAAAACGACGGCCAGT-3') (SEQ ID NO: 18) and
Sequenase.TM. (USB), with analysis by gel electrophoresis
(Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual
Cold Spring Harbor Laboratory Press, New York). Mutants that lack
exon 2, exon 3, or both exon 2 and exon 3, and are without Taq
polymerase-induced sequence errors can be used to create expression
vectors.
EXAMPLE 9
Cell Lines, Cellular Proliferation Assays and Inhibition of IL-9
Activity
[0195] Cell lines were used to assess the function of peptides,
aminosterols, tyrophostins, rhIL-9, rmIL-9, and recombinant mutant
forms of these proteins as well as all other compounds that block
IL-9 function. A proliferative response was measured and compared
to each of the other cytokines, variant or mutant forms of 11-9, or
IL-9 antagonists. In addition, compounds were tested for their
ability to antagonize the baseline proliferative response. Once a
baseline proliferative response was established for a cytokine a
statistically significant loss of response in assays repeated three
times in triplicate was considered evidence for antagonism. A true
antagonistic response was differentiated from cellular toxicity by
direct observation, trypan blue staining (a technique well known to
one of normal skill in the art), and loss of acid phosphatase
activity. Specificity was assessed for the antagonist by evaluating
whether the activity was substantially expressed against other
proliferative agents such as steel factor, interleukin 3, or
interleukin 4.
[0196] The MO7e line is a human megakaryoblastic cell line,
cultured in RPMI 1640 (GIBCO/BRL, Gaithersburg, Md.), 20% Fetal
Bovine Serum (Hyclone) and 10 ng/ml IL-3 (R&D Systems,
Minneapolis, Minn.). The MJ line is a cytokine independent human
lymphoblastoid cell line grown in RPMI 1640 (GIBCO/BRL) K562 is a
human erthroleukemia cell line, cultured in RPMI 1640 (GIBCO/BRL)
and 10% fetal bovine serum (Hyclone). C8166-45 is a IL-9 receptor
bearing line, cultured in RPMI 1640 GIBCO/BRL) and 10% Fetal bovine
serum (Hyclone). All the cell lines respond to cytokines including
IL-9. The cell lines are fed and reseeded at 2.times.10.sup.5
cells/ml every 72 hours.
[0197] The cells were centrifuged for 10 minutes at 2000 rpm and
resuspended in RPMI 1640 with 0.5% Bovine Serum Albumin (GIBCO/BRL,
Gaithersburg, Md.) and insulin-transferrin -selenium (ITS)
cofactors (GIBCO/BRL, Gaithersburg, Md.). Cells were counted using
a hemocytometer and diluted to a concentration of 1.times.10.sup.5
cells/ml and plated in a 96-well microtiter plate. Each well
contained 0.15 or 0.2 ml giving a final concentration of
2.times.10.sup.4 cells per well.
[0198] MO7e cells were stimulated with 50 ng/ml Stem Cell Factor
(SCF) (R&D Systems, Minneapolis, Minn.) alone, 50 ng/ml SCF
plus 50 ng/ml IL-3 (R&D Systems, Minneapolis, Minn.), or 50
ng/ml SCF plus 50 ng/ml IL-9. A control was included which contains
cells and basal media only. Serial dilutions of test compounds
(i.e, recombinant IL-9 proteins, peptides, small molecules) were
added to each test condition in triplicate. The MJ cell line was
used as an independent control for nonspecific cytotoxicity.
Cultures were incubated for 72-96 hours at 37.degree. C. in 5%
CO.sub.2.
[0199] Cell proliferation was assayed using the Abacus Cell
Proliferation Kit (Clontech, Palo Alto, Calif.) which determines
the amount of intracellular acid phosphatase present as an
indication of cell number. The substrate p-nitrophenyl phosphate
(pNPP) was converted by acid phosphatase to p-nitrophenol which was
measured as an indicator of enzyme concentration. pNPP was added to
each well and incubated at 37.degree. C. for one hour. 1N sodium
hydroxide was then added to stop the enzymatic reaction, and the
amount of p-nitrophenol was quantified using a Dynatech 2000 plate
reader (Dynatech Laboratories, Chantilly, Va.) at 410 nm
wavelength. Standard curves that compare cell number with optical
absorbance were used to determine the linear range of the assay.
Assay results were only used when absorbance measurements are
within the linear range of the assay.
[0200] FIG. 14 illustrates the amino acid sequence of three peptide
antagonists of IL-9 function. Each peptide was incubated with MO7e
cells and inhibition of cellular growth induced by IL-9 was
determined by comparison with control conditions (no peptide) (see
FIGS. 15-17). There was no evidence for cytotoxicity with any of
the peptides. Peptides KP-16 and KP-20 are predicted to lie within
two anti-parallel alpha-helicies and define a critical IL-9
receptor binding domain for the IL-9 ligand. The protein
polymorphism at codon 117 lies within KP-23 and KP-24 which also
exhibited antagonistic properties, further demonstrating the
importance of this region surrounding the site of genetic
variation.
[0201] FIG. 18 illustrates the effect of tyrophostins (obtained
from Calbiochem) on the IL-9 dependent growth of MO7e cell in
vitro. Each tyrophostin was incubated with MO7e cells and
inhibition of cellular growth induced by IL-9 was determined by
comparison with control conditions (no treatment). There was no
evidence for cytotoxicity with any of the treatments. Tyrophostins
B46 and B56 provided the greatest inhibition suggesting a common
structure activity relationship.
[0202] FIG. 19 illustrates the effect of aminosterols isolated from
the shark liver as set forth in U.S. Ser. Nos. 08/290,826,
08/416,883, 08/478,763, and/or 08/483,059, incorporated herein by
reference on the IL-9 dependent growth of MO7e cell in vitro. Each
aminosterol was incubated with MO7e cells at 20 .mu.g/ml of the
culture media and inhibition of cellular growth induced by IL-9 was
determined by comparison with control conditions (no treatment).
There was no evidence for cytotoxicity with any of the treatments.
Aminosterols 3 and 6 consistently provided the greatest inhibition
of growth.
EXAMPLE 10
Assay for Proliferation of IgE Secreting Cells
[0203] B cell lines can be used to assess the function of rIL-9 and
recombinant mutant forms of these proteins as well as other IL-9
antagonists. The proliferation of IgE secreting cells is measured
for rIL-9 and compared to other cytokines or variant forms of
rIL-9. In addition, compounds are tested for their ability to
antagonize the baseline proliferative response of IgE secreting
cells to rIL-9. Once a baseline IgE response is established for a
cytokine, a statistically significant (P<0.05) loss of response
in assays repeated three times in triplicate is considered evidence
for antagonism. A true antagonistic response is differentiated from
cellular toxicity by trypan blue staining (a technique well known
to one of normal skill in the art).
[0204] Cell Preparation and Cultures
[0205] Peripheral blood lymphocytes (PBL) are isolated from
heparinized blood of healthy donors or by mincing the spleens of
mice. Mononuclear cells are separated by centrifugation on
Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) gradients.
Semi-purified human B lymphocytes are obtained by rosetting with
neuraminidase (Behring, Marburg, FRG)--treated sheep red blood
cells and plastic adherence for 1 hour at 37.degree. C. B cells are
also purified using paramagnetic separation with anti-CD20 coated
magnetic beads (DYNAL) according to the manufacturer's
recommendations.
[0206] The relative proportion of B cells, T cells and monocytes is
determined by flow cytometry using monoclonal antibodies specific
for CD23, CD3 and CD14, respectively (Becton Dickinson, Mountain
View, Calif.). Briefly, 10.sup.6 cells/ml are incubated with a
1:1000 dilution of phycoerythrin conjugated anti-CD23 and
fluorescein-conjugated anti-CD3 and anti-CD14 antibodies for 30
minutes at 4.degree. C. After 3 spin washes with sterile PBS and 1%
bovine serum albumin (Sigma) fluorescence is measured with a
cytofluorograph (FACSTAR Plus, Becton Dickinson, Grenoble, France).
Typically, there are 45% CD20+, 35% CD3+ and 10% CD14+ cells in a
count of 5000 cells per sample.
[0207] Cells are cultured at a density of 2.times.10.sup.6 cells/ml
RPM1 1640 supplemented with 10% heat-inactivated fetal calf serum
(FCS), 2 mM glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin and 20 mM HEPES (RPM1-FCS) at 37.degree. C. under a 5%
CO2/95% air humidified atmosphere. Cultures are incubated with
increasing concentrations of IL-4, rhIL-9, rmIL-9, or recombinant
mutant forms of these proteins, alone, or in combination.
Competition experiments are run with mixtures of one or more of
these recombinant molecules or other IL-9 antagonists. The cultures
are maintained for 9-13 days.
[0208] Frequency of IgE-Secreting B Cells
[0209] The frequency of IgE-secreting human B cells in response to
human or murine IL-9 is determined using an ELISA-spot assay (Dugas
et al., 1993, Eur J Immunol 23:1687-1692; Renz, H. et al., 1990. J.
Immunology. 145:3641. Nitrocellulose flat-bottomed 96-well plates
are coated overnight at 4.degree. C. with purified goat antihuman
IgE mAb diluted in 0.1 M NaHCO3 buffer (2.5 .mu.g/m1). After one
PBS-Tween 20 wash, plates are incubated for 1 hour with RPMI-FCS to
saturate nonspecific binding sites. B cells obtained after 9-13
days of culture are collected, washed thrice and resuspended at
10.sup.5 cells/ml RPMI-FCS, then transferred onto the
anti-IgE-coated plates followed by an 18 hour incubation at
37.degree. C. A peroxidase-conjugated mouse antihuman IgE mab at
various dilutions is added for 2 hours at 37.degree. C. after
washing. Spots are visualized after addition of diamino-benzidine
diluted in 0.1 M Tris-HCl containing 0.03% H.sub.2O.sub.2. After 24
hours spots are counted with an inverted microscope at 25.times.
magnification. Data are expressed as the number of IgE-secreting
cells per 10.sup.6 cells.
EXAMPLE 11
ELISA for IgE Secreted by Cells Co-Stimulated with IL-9
[0210] Cells are isolated, prepared and stimulated as described
above in Example 10. Flat bottom microtiter plates (Nunc) are
coated with rabbit anti-human IgE (1:2000, final dilution; Serotec,
Oxford, GB), in 200 .mu.l of 10 mM bicarbonate buffer (pH 9.6).
After overnight incubation at 4.degree. C., the plates are washed
four times with phosphate-buffered saline (PBS) containing 0.05%
Tween (PBS-Tween; Merck, Hohenbrunn, FRG) and are incubated for 1 h
at room temperature with RPMI-FCS to saturate nonspecific
protein-binding sites. After washing, 200 .mu.l serial dilutions of
human IgE (Eurobio, Les Ulis, France), standards in PBS-Tween are
added to the respective plates to establish calibration curves.
Dilutions of culture supernatants to be tested are then added and,
after 2 h at room temperature, the plates are washed and 200 .mu.l
of diluted specific alkaline phosphatase-conjugated anti-IgE
(1:250; Serotec), anti-IgG or anti-IgM (Behring) is added in the
appropriate plates. After 2 h at room temperature, the plates are
washed and 200 .mu.l (0.5 mg/ml) p-nitrophenylphosphate (Sigma) in
citrate buffer is added. Plates are incubated at 37.degree. C., and
absorbance (A) is measured at 405 nm using an autoreader (Dynatech
Laboratories Inc, Chantilly, Va.). The threshold sensitivities of
the assays are 100 pg/ml for IgE, 1 ng/ml for IgG, and 2 ng/ml for
IgM and the variation between duplicate determinations of samples
typically does not exceed 10%.
EXAMPLE 12
The Role of IL-9 in Murine Models of Asthma: The Airway Response of
Unsensitized Animals
[0211] Animals
[0212] Certified virus-free male mice ranging in age from 5 to 6
weeks were obtained from the Jackson Laboratory (Bar Harbor, Me.).
Animals were housed in high-efficiency particulate filtered air
(HEPA) laminar flow hoods in a virus and antigen free facility and
allowed free access to pelleted rodent chow and water for 3 to 7
days prior to experimental manipulation. The animal facilities were
maintained at 22.degree. C. and the light:dark cycle was
automatically controlled (10:14 h light:dark). Male and female
DBA/2 (D2), C57BL/6 (B6), and (B6D2)F1 (F1) mice 5 to 6 weeks of
age were purchased from the Jackson Laboratory, Bar Harbor, Me., or
the National Cancer Institute, Frederick, Md. BXD mice were
purchased from the Jackson Laboratory, Bar Harbor, Me. Food and
water were present ad libitum.
[0213] Phenotyping and Efficacy of Pretreatment
[0214] To determine the bronchoconstrictor response, respiratory
system pressure was measured at the trachea and recorded before and
during exposure to the drug. Mice were anesthetized and
instrumented as previously described. (Levitt R C, and Mitzner W,
FASEB J 2:2605-2608 (1988); Levitt R C, and Mitzner W, J Appl
Physiol 67(3): 1125-1132 (1989); Kleeberger S, Bassett D, Jakab G
J, and Levitt R C, Am J Physiol 258(2) L313-320 (1990); Levitt R C;
Pharmacogenetics 1:94-97 (1991); Levitt R C, and Ewart S L; Am J of
Respir Crit Care tiled 151:1537-1542(1995); Ewart S, Levitt R C,
and Mitzner W, In press, J Appl Phys (1995). Airway responsiveness
was measured to one or more of the following: 5-hydroxytryptamine
(5HT) (sigma). An additional branch construction that can be used
is acetylcholine (sigma), atracurium (Glaxo welcome). A simple and
repeatable measure of the change in P.sub.pi following
bronchoconstrictor challenge was used and which has been termed the
Airway Pressure Time Index (APTI) (Levitt R C, and Mitzner W, FASEB
J 2:2605-2608 (1988); Levitt R C, and Mitzner W, J Appl Physiol
67(3): 1125-1132 (1989). The APTI was assessed by the change in
peak inspiratory pressure (P.sub.pi) integrated from the time of
injection till the peak pressure returned to baseline or plateaued.
The APTI was comparable to airway resistance (R.sub.rs), however,
the APTI includes an additional component related to the recovery
from bronchoconstriction.
[0215] The strain distribution of bronchial responsiveness was
identified in multiple inbred mouse strains in previous studies
(Levitt R C, and Mitzner W, FASEB J 2:2605-2608 (1988); Levitt R C,
and Mitzner W, J Appl Physiol 67(3): 1125-1132 (1989). The R.sub.rs
and/or APTI was determined in A/J, C3H/HeJ, DBA/2J, C57BL/6J
mice.
[0216] Prior to sacrifice whole blood was collected for serum IgE
measurements by needle puncture of the inferior vena cava in
completely anesthetized animals. The samples were spun to separate
cells and serum was collected and used to measure total IgE levels.
Samples not measured immediately were frozen at -20.degree. C.
[0217] Bronchoalveolar lavage and cellular analyses was preformed
as described elsewhere (Kleeberger et al., 1990).
[0218] All IgE serum samples were measured using an ELISA
antibody-sandwich assay. Microtiter plates (Corning #2585096,
Corning, N.Y.) were coated, 50 .mu.l per well, with rat anti-mouse
IgE antibody (Southern Biotechnology #1130-01, Birmingham, Ala.) at
a concentration of 2.5 .mu.g/ml in coating buffer of sodium
carbonate-sodium bicarbonate with sodium azide (Sigma #S-7795,
#S-6014 and #S-8032, St Louis, Mo.). Plates were covered with
plastic wrap and incubated at 4.degree. C. for 16 hours. The plates
were washed three times with a wash buffer of 0.05% Tween-20 (Sigma
#P-7949) in phosphate-buffered saline (BioFluids #313, Rockville,
Md.), incubating for five minutes for each wash. Blocking of
nonspecific binding sites was accomplished by adding 200 .mu.l per
well 5% bovine serum albumin (Sigma #A-7888) in PBS, 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 were added to the wells. Test samples were assayed after
being diluted 1:10, 1:50, and 1:100 with 5% BSA in wash buffer. In
addition to the test samples a set of IgE standards (PharMingen
#03121D, San Diego, Calif.) at concentrations from 0.8 ng/ml to 200
ng/ml in 5% BSA in wash buffer were assayed to generate a standard
curve. A blank of no sample or standard was used to zero the plate
reader (background). After adding samples and standards, the plate
was covered with plastic wrap and incubated for 2 hours at room
temperature. After washing three times with wash buffer, 50 ul of
second antibody rat anti-mouse IgE-horseradish peroxidase conjugate
(PharMingen #02137E) was added at a concentration of 250 ng/ml in
5% BSA in wash buffer. The plate was covered with plastic wrap and
incubated 2 hours at room temperature. After washing three times
with wash buffer, 100 ul of the substrate 0.5 mg/ml
O-phenylaminediamine (Sigma #P-1526) in 0.1 M citrate buffer (Sigma
#C-8532) was added to every well. After 5-10 minutes the reaction
was stopped with 50 .mu.l of 12.5% H.sub.2SO.sub.4 (VWR #3370-4,
Bridgeport, N.J.) and absorbance was measured at 490 nm on a
Dynatech MR-5000 plate reader (Chantilly, Va.). A standard curve
was 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 was
interpolated from the standard curve.
EXAMPLE 13
The Role of IL-9 in Murine Models of Asthma: The Airway Response of
Sensitized Animals
[0219] Animals, Phenotyping, and Optimization of Antigen
Sensitization
[0220] Animals and handling were essentially as described in
Example 12. Sensitization with turkey egg albumin (OVA) and aerosol
challenge was carried out to assess the effect on BHR, BAL, and
serum IgE. OVA was injected I.P. (25 .mu.g) day 0 prior to OVA or
saline aerosolization. Mice were challenged with OVA or saline
aerosolization which was given once daily for 5 to 7 days starting
on either day 13 or 14. Phenotypic measurements of serum IgE, BAL,
and BHR was carried out on day 21. The effect of a 7 day OVA
aerosol exposure on bronchoconstrictor challenge with 5-HT and
acetylcholine were evaluated along with serum total IgE, BAL total
cell counts and differential cell counts, and bronchial
responsiveness. The effect of antibody (Ab) or saline pretreatment
on saline aerosol or OVA aerosol induced lung inflammation was
examined by measuring BHR, BAL, and serum IgE. Ab were administered
I.P. 2-3 days prior to aerosolization of saline or OVA.
[0221] Lung histology was carried out after the lungs are removed
during deep anesthesia. Since prior instrumentation may introduce
artifact, separate animals were used for these studies. Thus, a
small group of animals was treated in parallel exactly the same as
the cohort undergoing various pretreatments except these animals
were not used for other tests aside from bronchial responsiveness
testing. After bronchial responsiveness testing, the lungs were
removed and submersed in liquid nitrogen. Cryosectioning and
histologic examination were carried out in a routine fashion.
[0222] Polyclonal neutralizing antibodies for murine IL-9 were
purchased from R & D systems, Minneapolis, Minn. and blocking
antibodies for murine IL-9 receptor were produced for Magainin
Pharmaceuticals Inc. by Lampine Biological Labatories, Ottsville,
Pa. using peptide conjugates produced at Magainin. The polyclonal
antisera were prepared in rabbits against peptide sequences from
the murine IL-9 receptor. The peptides used to produce the antisera
were: GGQKAGAFTC (residues 1-10) (SEQ ID NO:19);
LSNSIYRIDCHWSAPELGQESR (residues 11-32) (SEQ ID NO:20); and
CESYEDKTEGEYYKSHWSEWS (residues 184-203 with a Cys residue added to
the N-terminus for coupling the peptide to the carrier protein)
(SEQ ID NO:21). The antisera were generated using techniques
described in Protocols in Immunology, Chapter 9, Wiley. Briefly,
the peptides were coupled to the carrier protein, Keyhole Limpet,
hemocyanin, (Sigma) through the side chain of the Cys residue using
the bifunctional cross-linking agent MBS (Pierce). Peptide
conjugates were used to immunize rabbits with appropriate adjuvants
and useful antisera was obtained after several booster injections
of the peptide conjugate. The antibodies were used therapeutically
to down regulate the functions of IL-9 and assess the importance of
this pathway to baseline lung responsiveness, serum IgE, and BAL in
the unsensitized mouse. After Ab pretreatment on baseline BHR, BAL,
and serum IgE levels relative to controls was determined. In
additional experiments, recombinant human and murine IL-9 were
administered I.P. 1 day before and daily during antigen
sensitization (days 13-18). The animals were then phenotyped as
described.
[0223] The phenotypic response of a representative animal treated
with saline I.P. on day zero and challenged on days 14-20 with
saline (as described in Example 12) is shown in FIG. 20 panel 1
(top). Baseline (control) serum total IgE was 9.2 ng/ml.
Bronchoalveolar lavage (BAL) total cell counts showed 182,500 cells
per milliliter of BAL. These animals did not demonstrate bronchial
hyperresponsiveness when compared to historical controls (Levitt R
C, and Mitzner W, J Appl Physiol 67(3): 1125-1132; 1989).
[0224] FIG. 20 panel 2 (top middle) shows a representative animal
from a group presensitized with OVA I.P on day zero and challenged
with saline on days 14-20. These animals did not differ in their
response to bronchoconstrictor, serum IgE, or BAL cell counts from
the unsensitized mice (FIG. 7 top panel).
[0225] FIG. 20 panel 3 (bottom middle) shows a representative
animal from those presensitized with OVA I.P on day zero and
challenged with antigen (OVA) on days 14-20. These animals
developed bronchial hyperresponsiveness (approximately two to
three-fold over controls), elevated serum IgE (nearly one
thousand-fold over controls), and increased numbers of inflammatory
cells in the airway as demonstrated by elevated BAL cell counts
(approximately thirty-fold) as compared to controls (FIG. 20 top 2
panels). Most of the cells recruited to the airway as a result of
this antigen challenge were eosinophils.
[0226] FIG. 20 panel 4 (bottom) shows a representative animal from
those presensitized with OVA I.P on day zero, pretreated with
polyclonal neutralizing antibodies for murine IL-9 (approximately
200 .mu.g/mouse I.P. in 0.5 ml of PBS), and challenged with antigen
(OVA) on days 14-20. These animals were protected from the response
to antigen. They did not differ significantly in their bronchial
responsiveness, serum IgE, or BAL cell counts from controls (FIG.
20 top 2 panels).
[0227] FIG. 21 illustrates the effect of antigen challenge to OVA
(as described above) with and without pretreatment with polyclonal
neutralizing antibodies to murine IL-9 I.P. three days prior in
representative animals. The left figure (A1-2-1B) is a histologic
section from the lungs of control animals (sensitized to OVA but
exposed only to a saline aerosol challenge). The middle figure
(A1-3-5) is a histologic section from the lungs of animals
sensitized to OVA and exposed to an OVA aerosol challenge. The
right figure (A1-4-5) is a histologic section from the lungs of
animals sensitized to OVA and exposed to an OVA aerosol challenge
who were pretreated three days prior with polyclonal neutralizing
antibodies to murine IL-9. Pretreatment with neutralizing antibody
produced histological confirmation of complete protection from
antigen challenge.
[0228] FIG. 22 panel 1 (top) shows a representative animal from
mice presensitized with OVA I.P on day zero and challenged with
antigen (OVA) on days 13-18. These animals developed bronchial
hyperresponsiveness (approximately two to three-fold over
controls), and increased numbers of inflammatory cells including
eosinphils in the airways as demonstrated by elevated BAL cell
counts as compared to controls (FIG. 20 top 2 panels). Many of the
cells recruited to the airway as a result of this antigen challenge
were eosinophils.
[0229] FIG. 22 panel 2 (bottom) shows a representative animal from
those presensitized with OVA I.P on day zero, pretreated with
polyclonal neutralizing antibodies to the murine IL-9 receptor
(approximately 1 mg/mouse I.P. in 0.5 ml of PBS), and challenged
with antigen (OVA) on days 13-18. This representative animal was
protected from the response to antigen. This response did not
differ significantly bronchial responsiveness, BAL cell counts from
controls (FIG. 20 top 2 panels). These data demonstrate the
potential effectiveness of treating atopic allergy with antibodies
to the IL-9 receptor.
EXAMPLE 14
Murine Spleen Isolation and Culture
[0230] Mice were anesthetized and spleens were removed aseptically.
Spleens were minced with scissors and gently passed through a wire
mesh (autoclaved) [#60 sieve]. Cells were resuspended in 40 mls of
RPMI-1640 [GIBCO, BRL, Rockville, Md.], and spun for 5 min. at
250.times.G twice. The pellet was resuspended in 10 mls of lysing
butter to remove RBCs [4.15 gm NH4Cl, 0.5 gm KHCO3; 019 g EDTA to
500 mls with ddH20]. Cells were incubated for about 5 minutes at
37.degree. C. and 40 mls of RPMI-FCS [RPMI-1640, 10% AFBS, 50 .mu.M
BME 2 mM glutamine, containing penicillin and streptomycin]. These
cells were spun again for 5 minutes at 250.times.G and resuspended
in 20 mls RPMI-FCS with or without 5 .mu.g/ml of concanavalin A
[Sigma #C5275]. IL-9 was assessed at 48 hours in untreated
splenocytes and after concanavalin A stimulation from DBA/2J (D2)
and C57BL/6J (B6) mice. IL-9 was amplified by RT-PCR (as set forth
in Example 6), and probed with an IL-9 specific murine probe after
Southern transfer. Southern blots were performed by "standard"
techniques. Briefly, RT-PCR products were electrophoresed in 2%
agarose gels. Gels were stained with ethidium bromide and
photographed with a ruler to determine molecular weight of DNA in
southern blot. Gels were then soaked in 0.5N NaOH for 30 minutes
and neutralized in 0.5M Tris, pH 7.0 for 30 minutes. DNA was
transferred to zetaprobe (BioRAD) nylon membrane by capillary
transfer in 20.times.SSC overnight. The next day, the membrane was
air dried, baked at 80.degree. C. for 15 minutes and prehybridized
in 6.times.SSC and 0.1% SDS for 1 hour at 42.degree. C. A kinase
end-labelled p32 oligonucleotide probe
(5'-AATTACCTTATTGAAAATCTGAAG-3') was added to the hybridization
solution plus 0.1 mg/ml sheared salmon sperm DNA and incubated
overnight at 42.degree. C. The next day, the filter was washed in
3.times.SSC and 0.1% SDS at 37.degree. C. for 30 minutes, and the
filter was exposed to film for 1 hour. FIG. 26 illustrates steady
state levels of IL-9 after 48 hours from each strain of mice. IL-9
was observed in unstimulated D2 (D2-) splenocytes, whereas no IL-9
was detectable in B6 (B6-) mice. While there was a significant
increase of IL-9 after concanavalin A stimulation in D2 (D2+)
splenocytes, there was no detectable It-9 in B6 (B6+) mice despite
concanavalin A treatment.
EXAMPLE 15
Expression of Human Met117 IL-9 and Thr117 IL-9 in PBMCs
SDS-PAGE and Immunoblot Analysis
[0231] After obtaining proteins isolated from human PBMC of healthy
donors inhibiting either the wild type (Thr117) or Met117-IL-9
genotypes as set forth in Example 13, and SDS-PAGE was performed by
the method of Laemmli (Laemmli U.K. (1970) Nature 227, 680-685) by
using a 18% polyacrylamide gel in a mini-gel system (Xcell II
vertical gel unit, Novex). For immunoblot analysis, the proteins
separated by SDS-PAGE were transferred to nitrocellulose membranes
by using the SD transblot transfer unit (Biorad) in 25 mM
Tris-glycine buffer, pH 8.3, containing 15% methanol (Towbin H., et
al., (1979) (Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354). The
unoccupied binding cites on the membrane were blocked by incubating
for 1 hour to overnight with 20 mM Tris-HCl buffer, pH 8.0, plus
0.05% tween 20 (TBST) containing 5% dry milk. The membranes were
then incubated with 1:1000 dilution of goat anti-human IL-9
polyclonal antibody (R&D Systems) for 1 hour at room
temperature. The membranes were washed with TBST and treated with
1:10,000 dilution of mouse anti-goat TgG conjugated with
horseradish peroxidase for 1 hour. After washing with TBST, the
bound antibodies were visualized by addition of the super signal
substrate chemiluminescence system (Pierce).
[0232] FIG. 24 demonstrates the expression of human IL-9 proteins
from cultured PBMCs 48 hours after mitogen stimulation in
individuals whose genotypes have been determined by genomic
analysis of the IL-9 gene. Lane 1 is molecular weight markers, lane
2 is a Met117 homozygote, lane 3 is a heterozygote Met117/Thr117,
lane four is a Thr117 homozygote. A single product of the
approximate expected size (14 kD) was seen in each individual PBMCs
after mitogen stimulation. These data demonstrate that both forms
of the IL-9 protein are expressed and stable at steady state.
[0233] 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 material 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.
REFERENCES
[0234] 1. Gergen P J, and Weiss K B: The increasing problem of
asthma in the United States. Am Rev Respir Dis 146:823-824, 1992.
[0235] 2. Goodman and Gilman's The Pharmacologic Basis of
Therapeutics, Seventh Edition, MacMillan Publishing Company, N.Y.
USA, 1985. [0236] 3. Burrows B, Martinez F D, Halonen M, Barbee R
A, and Cline M G: Association of asthma with serum IgE levels and
skin-test reactivity to allergens. New Eng J Med 320:271-277, 1989.
[0237] 4. Clifford R D, Pugsley A, Radford M, and Holgate S T:
Symptoms, atopy, and bronchial response to methacholine in parents
with asthma and their children. Arch Dis in Childhood 62:66-73,
1987. [0238] 5. Gergen P J: The association of allergen skin test
reactivity and respiratory disease among whites in the U.S.
population. Arch Intern Med 151:487-492, 1991. [0239] 6. Burrows B,
Sears M R, Flannery E M, Herbison G P, and Holdaway M D:
Relationship of bronchial responsiveness assessed by methacholine
to serum IgE, lung function, symptoms, and diagnoses in 11-year-old
New Zealand children. J Allergy Clin Immunol 90:376-385, 1992.
[0240] 7. Johannson S G O, Bennich H H, and Berg T: The clinical
significance of IgE. Prog Clin Immunol 1:1-25, 1972. [0241] 8.
Sears M R, Burrows B, Flannery E M, Herbison G P, Hewitt C J, and
Holdaway M D: Relation between airway responsiveness and serum IgE
in children with asthma and in apparently normal children New Engl
J Med 325(15):1067-1071, 1991. [0242] 9. Halonen M, Stern D,
Taussig L M, Wright A, Ray C G, and Martinez F D: The predictive
relationship between serum IgE levels at birth and subsequent
incidences of lower respiratory illnesses and eczema in infants. Am
Rev Respir Dis 146:666-670, 1992. [0243] 10. Marsh D G, Meyers D A,
and Bias W B: The epidemiology and genetics of atopic allergy. New
Eng J Med 305:1551-1559, 1982. [0244] 11. Hopp R J, Bewtra A K,
Biven R, Nair N M, Townley R G. Bronchial reactivity pattern in
nonasthmatic parents of asthmatics. Ann Allergy 1988; 61:184-186.
[0245] 12. Hopp R J, Townley R G, Biven R E, Bewtra A K, Nair N M.
The presence of airway reactivity before the development of asthma.
Am Rev Respir Dis 1990; 141:2-8. [0246] 13. Ackerman V, Marini M,
Vittori E, et al. Detection of cytokines and their cell sources in
bronchial biopsy specimens from asthmatic patients: relationship to
atopic status, symptoms, and level of airway hyperresponsiveness.
Chest 1994; 105:687-696. [0247] 14. Hamid G, Azzawi M, Ying S, et
al. Expression of mRNA for interleukin-5 in mucosal bronchial
biopsies from asthma. J Clin Invest 1991; 87:1541-1546. [0248] 15.
Djukanovic R, Roche W R, Wilson J W, et al. Mucosal inflammation in
asthma. Am Rev Respir Dis 1990; 142:434-57. [0249] 16. Robinson D
S, Hamid Q, Ying S, et al. Predominant TH2-like bronchoalveolar T
lymphocyte population in atopic asthma. N Engl J Med 1992;
326:298-304. [0250] 17. Robinson D S, Hamid Q, Ying S, et al.
Prednisolone treatment in asthma is associated with modulation of
bronchoalveolar lavage cell interleukin-4, interleukin-5, and
interferon-_cytokine gene expression. Am Rev Respir Dis 1993;
148:401-406. [0251] 18. Robinson D S, Ying S, Bentley A, et al.
Relationship among numbers of bronchoalveolar lavage cells
expressing messenger ribonucleic acid for cytokines, asthma
symptoms, and airway methacholine responsiveness in atopic asthma.
J Allergy Clin Immunol 1993; 92:397-403. [0252] 19. Sears M,
Burrows B, Flannery E M, Herbison G P, Hewitt C J, Holdaway M D.
Relation between airway responsiveness and serum IgE in children
with asthma and in apparently normal children. N Engl J Med 1991;
325:1067-1071. [0253] 20. Burrows B, Sears M R, Flannery E M,
Herbison G P, Holdaway M D. Relationship of bronchial
responsiveness assessed by methacholine to serum IgE, lung
function, symptoms, and diagnoses in 11-year-old New Zealand
children. J Allergy Clin Immunol 1992; 90:376-385. [0254] 21.
Clifford R D, Pugsley A, Radford M, Holgate S T. Symptoms, atopy,
and bronchial response to methacholine in parents with asthma and
their children. Arch Dis Childhood 1987; 62:66-73. [0255] 22.
O'Connor G T, Sparrow D, and Weiss S T: The role of allergy and
nonspecific BHR in the pathogenesis of COPD. Am Rev Respir Dis
140:225-252, 1989. [0256] 23. Cogswell J J, Halliday D F, and
Alexander J R: Respiratory infections in the first year of life in
children at the risk of developing atopy. Brit Med J 284:1011-1013,
1982. [0257] 24. Boushey H A, Holtzman M J, Sheller J R, and Nadel
J A: BHR. Am Rev Respir Dis 121:389-413, 1980. [0258] 25. Cookson W
O C M, and Hopkin J M: Linkage between immunoglobin E responses
underlying asthma and rhinitis and chromosome 11q: Lancet
1292-1295, 1989. [0259] 26. Moffatt M F, Sharp P A, Faux J A, Young
R P, Cookson W O C M, and Hopkins J M; Factors confounding genetic
linkage between atopy and chromosome 11q. Clin Exp Allergy
22:1046-1051, 1992. [0260] 27. Amelung P, Panhuysen C, Postma D S,
Levitt R C, Koeter G H, Francomano C, Bleeker E R, and Meyers D A:
Atopy, asthma and bronchial hyperresponsiveness: Exclusion of
linkage to markers on chromosome 11q and 6p. Clin Exper Allergy
22:1077-1084, 1992. [0261] 28. Rich S S, Roitman-Johnson B,
Greenberg B, Roberts S, and Blumenthal M N: Genetic evidence of
atopy in three large kindreds: no evidence of linkage to D11S97.
Clin Exp Allergy 22:1070-1076, 1992. [0262] 29. Lympany P, Welsh K,
MacCochrane G, Kemeny D M, and Lee T H: Genetic analysis using DNA
polymorphism of the linkage between chromosome 11q13 and atopy and
BHR to methacholine. J Allergy Clin Immunol 89:619-628, 1992a.
[0263] 30. Lympany P, Welsh K I, Cochrane G M, Kemeny D M, and Lee
T H: Genetic analysis of the linkage between chromosome 11q and
atopy. Clin Exp Allergy 22:1085-1092, 1992b. [0264] 31. Hizawa N,
Yamagushi E, Ohe M, Itoh A, Furuya K, Ohnuma N, and Kawakami Y:
Lack of linkage between atopy and locus 11q13. Clinical and
Experimental Allergy 22:1065-1069, 1992. [0265] 32. Sanford A J,
Shirakawa T, Moffatt M F, Daniels S E, Ra C, Faux J A, Young R P,
Nakamura Y, Lathrop G M, Cookson W O C M, and Hopkin J M:
Localization of atopy and b subunit of high-affinity IgE receptor
(FceR1) on chromosome 11q. Lancet 341:332-334, 1993. [0266] 33.
Shirakawa T, Airong L, Dubowitz M, Dekker J W, Shaw A E, Faux J A,
Ra C, Cookson W O C M, and Hopkin J M: Association between atopy
and variants of the .beta. subunit of the high-affinity
immunoglobulin E receptor. Nature Genetics 7:125-130, 1994. [0267]
34. Marsh D G, Bias W B, and Ishizaka K: Genetic control of basal
serum immunoglobulin E level and its effect on specific reaginic
sensitivity. Proc Natl Acad Sci USA 71:3588-3592, 1974. [0268] 35.
Gerrard J W, Rao D C, and Morton N E: A genetic study of IgE. Am J
Hum Genet 30:46-58, 1978. [0269] 36. Meyers D A, Beaty T H,
Freidhoff L R, and Marsh D G: Inheritance of serum IgE (basal
levels) in man. Am J Hum Genet 41:51-62, 1987. [0270] 37. Meyers D
A, Bias W B, and Marsh D G: A genetic study of total IgE levels in
the Amish. Hum Hered 32:15-23, 1982. [0271] 38. Martinez F D,
Holberg C J, Halonen M, Morgan W J, Wright A L, and Taussig L M:
Evidence for mendelian inheritance of serum IgE levels in Hispanic
and Non-hispanic white families. Am J Hum Genet 55:555-565, 1994.
[0272] 39. Blumenthal M N, Namboordiri K K, Mendell N, Gleich G,
Elston R C, and Yunis E: Genetic transmission of serum IgE levels.
Am J Med Genet 10:219-228, 1981. [0273] 40. The Genome Data Base.
The Welch Library, The Johns Hopkins Medical Institutions,
Baltimore, Md., USA. [0274] 41. Marsh D G, Neely J D, Breazeale D
R, et al. Linkage analysis of IL4 and other chromosome 5q31.1
markers and total serum immunoglobulin E concentrations. Science
1994; 264:1152-1156. [0275] 42. Meyers D A, Postma D S, Panhuysen C
I M, et al. Evidence for a locus regulating total serum IgE levels
mapping to chromosome 5. Genomics 1994; 23:464 470.41. [0276] 43.
Doull, I., Lawrence, S., Watson, M., Begishvili, T., Beasley, R.,
Lampe, F., Holgate, S. T., Morton, N. E. Allelic association of
makers on chromosome 5q and 11q with atopy and bronchial
hyperresponsiveness. Am J Respir Crit Care Med, 1996;
153:1280-1284. [0277] 44. Ott J. Analysis of human genetic linkage.
Baltimore, Md.: The Johns Hopkins University Press, 1991. [0278]
45. Renauld, J-C, Houssiau, F, Druez, C. Interleukin-9. Int Rev Exp
Pathology 1993; 34A: 99-109. [0279] 46. Renauld, J-C, Kermouni, A,
Vink, A, Louahed, J, Van Snick, J. Interleukin-9 and its receptor:
involvement in mast cell differentiation and T cell oncogenesis. J
Leukoc Biol 1995; 57:353-360. [0280] 47. Hultner, L, Moeller, J,
Schmitt, E, Jager, G, Reisbach, G, Ring, J. Dormer, P.
Thiol-sensitive mast cell lines derived from mouse bone marrow
respond to a mast cell growth-enhancing activity different from
both IL-3 and IL-4. J Immunol 1989; 142:3440-3446. [0281] 48.
Dugas, B, Renauld, J-C, Pene, J, Bonnefoy, J, Peti-Frere, C,
Braquet, P, Bousquet, J, Van Snick, J, Mencia-Huerta, J M.
Interleukin-9 potentiates the interleukin-4-induced immunoglobulin
[IgG, IgM and IgE] production by normal human B lymphocytes. Eur J
Immunol 1993; 23:1687-1692. [0282] 49. Petit-Frere, C, Dugas, B,
Braquet, P, Mencia-Huerta, J M. Interleukin-9 potentiates the
interleukin-4-induced IgE and IgG1 release from murine B
lymphocytes. Immunology 1993; 79:146-151. [0283] 50. Behnke, J M,
Wahid, F N, Grencis, R K, Else, K J, Ben-Smith, A W, Goyal, P K.
Immunological relationships during primary infection with
Heligmosomoides polygyrus [Nematospiroides dubius]: downregulation
of specific cytokine secretion [IL-9 and IL-10) correlates with
poor mastocytosis and chronic survival of adult worms. Parasite
Immunol 1993; 15:415-421. [0284] 51. Gessner, A, Blum, H,
Rollinghoff, M. Differential regulation of IL-9 expression after
infection with Leischmania major in susceptible and resistant mice.
Immunobiology 1993; 189:419-435. [0285] 52. Renauld J-C, Druez C,
Kermouni A, et al. Expression cloning of the murine and human
interleukin 9 receptor cDNAs. Proc Natl Acad Sci
89:5690-5694(1992). [0286] 53. Chang M-S, Engel G, Benedict C et
al. Isolation and characterization of the Human interleukin-9
receptor gene. Blood 83:3199-3205(1994). [0287] 54. Renauld J-C,
Goethals A, Houssiau F, et al. Human P40/IL-9. Expression in
activated CD4+ T cells, Genomic Organization, and Comparison with
the Mouse Gene. J Immunol 144:4235-4241(1990). [0288] 55. Kelleher
K, Bean K, Clark S C, et al. Human interleukin-9: genomic sequence,
chromosomal location, and sequences essential for its expression in
human T-cell leukemia virus (HTLV-I-transformed human T cells.
Blood 77:1436-1441(1991). [0289] 56. Houssiau F A, Schandene L,
Stevens M, et al. A cascade of cytokines is responsible for IL-9
expression in human T cells. Involvement of IL-2, IL-4, and IL-10.
J of Immunol. 154:2624-2630(1995). [0290] 57. Miyazawa K, Hendrie P
C, Kim Y-J, et al. Recombinant human interleukin-9 induces protein
tyrosine phosphorylation and synergizes with steel factor to
stimulate proliferation of the human factor-dependent cell line,
M07e. Blood 80:1685-1692(1992). [0291] 58. Yin T, Tsang M L-S, Yang
Y-C. JAK1 kinase forms complexes with interleukin-4 receptor and
4PS/insulin receptor substrate-1-like protein and is activated by
interleukin-4 and interleukin-9 in T lymphocytes. J Biol Chem
269:26614-26617(1994). [0292] 59. Renauld J-C, Druez C, Kermouni A,
et al. Expression cloning of the murine and human interleukin 9
receptor cDNAs. Proc Natl Acad Sci 89:5690-5694(1992). [0293] 60.
Chang M-S, Engel G, Benedict C et al. Isolation and
characterization of the Human interleukin-9 receptor gene. Blood
83:3199-3205(1994). [0294] 61. Kreitman R J, Puri R K, Leland P, et
al. Site-specific conjugation to interleukin4 containing mutated
cysteine residues produces interleukin 4-toxin conjugates with
improved binding and activity. Biochemistry 33:11637-11644(1994)
[0295] 62. Simoncsits A, Bristulf J, Tjornhammar M L, et al.
Deletion mutants of human interleukin 1 beta significantly reduced
agonist properties: search for the agonist/antagonist switch in
ligands to the interleukin 1 receptors. Cytokine 6:206-214(1994).
[0296] 63. Zav'yalov V P, Navolotskaya E V, Isaev I S, et al.
Nonapeptide corresponding to the sequence 27-35 of the mature human
IL-2 efficiently competes with rIL-2 for binding to thymocyte
receptors. Immunol Lett 31:285-288(1992). [0297] 64. Chu J W, and
Sharom F J. Glycophorin A interacts with interleukin-2 and inhibits
interleukin-2-dependent T-lymphocyte proliferation. Cell Immunol
145:223-239(1992). [0298] 65. Alexander A G, Barnes N C, Kay A B.
Trial of cyclosporin in corticosteroid-dependent chronic severe
asthma. Lancet 339:324-328(1992). [0299] 66. Morely J. Cyclosporin
A in asthma therapy: a pharmacological rationale. J Autoimmun 5
Suppl A:265-269(1992). [0300] 67. Lander, E. S., Botstein, D.
Mapping Mendelian factors underlying quantitative traits using RFLP
linkage maps. Genetics 121, 185-199(1989). [0301] 68. Soller, M.,
Brody, T. On the power of experimental designs for the detection of
linkage between maker loci and quantitative loci in crosses between
inbred lines. Theor Appl Genet 47:35-39(1976). [0302] 69. Kvaloy K,
Galvagni F, and Brown W R A. The sequence organization of the long
arm pseudoautosomal region of the human sex chromosomes. Hum Mol
Genet 3:771-778(1994). [0303] 70. Freije D, Helms C, Watson M S, et
al. Science 258:1784-1787(1992). [0304] 71. Weber J L, May P E.
Abundant class of human DNA polymorphisms which can be typed using
the polymerase chain reaction. Am J Human Genet 1989; 44:388-396.
[0305] 72. Saiki K K, Gelfand D H, Stoffel S, et al.
Primer-directed enzymatic amplification of DNA with a thermostable
DNA polymerase. Science 1988; 239:487-491. [0306] 73. Sheffield V
C, Beck J S, Kwitek A E, Sandstrom D W, and Stone E M: The
sensitivity of single-strand conformation polymorphism analysis for
the detection of single base substitutions. Genomics 16:325-332,
1993. [0307] 74. Orita M, Suzuki Y, Sekiya T, and Hayashi K: Rapid
and sensitive detection of point mutations and DNA polymorphisms
using the polymerase chain reaction. Genomics 5:874-9, 1989. [0308]
75. Sarkar G, Yoon H-S, and Sommer S S: Dideoxy fingeringprint
(ddF): A rapid and efficient screen for the presence of mutations.
Genomics 13:441-443, 1992. [0309] 76. Cotton R G: Detection of
single base changes in nucleic acids. Biochemical Journal
263(1):1-10, 1989. [0310] 77. Schwengel D, Nouri N, Meyers D, and
Levitt R C: Linkage mapping of the human thromboxane A2 receptor
(TBXA2R) to chromosome 19p13.3 using transcribed 3' untranslated
DNA sequence polymorphisms. Genomics 18:212-215, 1993. [0311] 78.
SAGE, Statistical Analysis for Genetic Epidemiology (1992). Release
2.1. Computer program package available from the Department of
Biometry and Genetics, LSU Medical Center, New Orleans, La. [0312]
79. Postma D S, Bleecker E R, Holroyd K J, Amelung P J, Panhuysen C
I M, Xu J, Meyers D A, Levitt R C: Genetic Susceptibility to
Asthma: Bronchial Hyperresponsiveness Coinherited with a Major Gene
for Atopy. N Engl J Med 333:894-900 (1995).
[0313] 80. Xu J, Levitt R C, Panhuysen C I M, Postma D S, Taylor E
W, Amelung P J, Holroyd K J, Bleecker E R, Meyers D A: Evidence for
two-unlinked loci regulating serum total IgE levels. Am J Hum Genet
57:425-430 (1995). [0314] 81. Meyers D A, Xu J, Holroyd K J,
Panhuysen C I M, Amelung P J, Postma D S, Levitt R C, Bleecker E R.
Two locus segregation and linkage analysis for total serum IgE
levels. Clin Exp Allergy 25:113-115 (1995). [0315] 82. Bleecker E
R, Amelung P J, Levitt R C, Postma D S, Meyers D A. Evidence for
linkage of total serum IgE and bronchial hyperresponsiveness to
chromosome 5q: a major regulatory locus important in asthma. Clin
Exp Allergy 25:84-88 (1995). [0316] 83. Panhuysen C I M, Levitt R
C, Postma D S, et al., Evidence for a susceptibility locus for
asthma mapping to chromosome 5q. Journal of Investigative Medicine
43:281A (1995). [0317] 84. Levitt R C, Eleff S M, Zhang L-Y,
Kleeberger S R, and Ewart S L. Linkage homology for bronchial
hyperresponsiveness between DNA markers on human chromosome
5q31-q33 and mouse chromosome 13. Clin Exp Allergy 25:61-63 (1995).
[0318] 85. Yang et al., U.S. Pat. No. 5,414,071 Human cytokine IL-9
(May 9, 1995). [0319] 86. Alms W and White B. Human interleukin
variants generated by alternative splicing PCT/US95/04094 (WO
95/27052). [0320] 87. Cytokine handbook, Angus Thomson (1994).
[0321] 88. Martinati L C, Trabetti E, Casartelli A, Boner A L,
Pignatti P F. Affected sib-pair and mutation analyses of the high
affinity IgE receptor beta chain locus in Italian families with
atopic asthmatic children. Am J Respir Crit Care Med, 1996;
153:1682-1685. [0322] 89. Kauvar, M. Lawrence, Peptide mimetic
drugs: A comment on progress and prospects, Nature Biotechnology
Volume 14, June 1996.
[0323] Other embodiments of the invention described above and will
be apparent to those skilled in the art from consideration of the
specification and practice of the invention disclosed within. It is
intended that the specification and examples considered as
exemplary only, with true scope and spirit of the invention being
indicated by the following claims:
Sequence CWU 1
1
45128DNAArtificial sequencePCR oligonucleotide primer 1tctcgagcag
gggtgtccaa ccttggcg 28231DNAArtificial sequencePCR oligonucleotide
primer 2gcagctggga taaataatat ttcatcttca t 31331DNAArtificial
sequencePCR oligonucleotide primer 3tctcgagcag agatgcagca
ccacatgggg c 31431DNAArtificial sequencePCR oligonucleotide primer
4gcagctggta acagttatgg aggggaggtt t 31521DNAArtificial sequencePCR
oligonucleotide primer 5gtgaccagtt gtctctgttt g 21621DNAArtificial
sequencePCR oligonucleotide primer 6ctgcatcttg ttgatgagga a
21721DNAArtificial sequencePCR oligonucleotide primer 7gacaactgca
ccagaccatg c 21821DNAArtificial sequencePCR oligonucleotide primer
8attagcactg cagtggcact t 21921DNAArtificial sequencePCR
oligonucleotide primer 9gtgaccagct gcttgtgtct c 211021DNAArtificial
sequencePCR oligonucleotide primer 10cttcagattt tcaataaggt a
211121DNAArtificial sequencePCR oligonucleotide primer 11gatgattgta
ccacaccgtg c 211221DNAArtificial sequencePCR oligonucleotide primer
12gttgccgctg cagctacatt t 211318PRTArtificial sequencePeptide
sequence 13Ser Asp Asn Ala Thr Arg Pro Ala Phe Ser Glu Arg Leu Ser
Gln Met1 5 10 15Thr Asn1418PRTArtificial sequencePeptide sequence
14Phe Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Ala1
5 10 15Pro Tyr1518PRTArtificial sequencePeptide sequence 15Glu Gln
Pro Ala Asn Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu1 5 10 15Lys
Ser1618PRTArtificial sequenceResidues 99-116 of Mature hIL-9
Receptor 16Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu
Ile Phe1 5 10 15Gln Lys1717DNAArtificial sequenceOligonucleotide
primer 17gtaaaacgac ggccagt 171817DNAArtificial
sequenceOligonucleotide primer 18gtaaaacgac ggccagt 171910PRTMus
musculusmisc_featureMurine IL-9 Receptor Peptide Sequence 19Gly Gly
Gln Lys Ala Gly Ala Phe Thr Cys1 5 102022PRTMus
musculusmisc_featureMurine IL-9 Receptor Peptide Sequence 20Leu Ser
Asn Ser Ile Tyr Arg Ile Asp Cys His Trp Ser Ala Pro Glu1 5 10 15Leu
Gly Gln Glu Ser Arg 202121PRTMus musculusmisc_featureMurine IL-9
Receptor Peptide Sequence 21Cys Glu Ser Tyr Glu Asp Lys Thr Glu Gly
Glu Tyr Tyr Lys Ser His1 5 10 15Trp Ser Glu Trp Ser
20227PRTArtificial sequenceResidues 8-14 of Mature hIL-9 Receptor
22Thr Cys Leu Thr Asn Asn Ile1 52318PRTArtificial sequenceResidues
50-67 of Mature hIL-9 Receptor 23Cys Phe Ser Glu Arg Leu Ser Gln
Met Thr Asn Thr Thr Met Gln Thr1 5 10 15Arg Tyr24415DNAHomo sapiens
24tctcgagcag gggtgtccaa ccttggcggg gatcctggac atcaacttcc tcatcaacaa
60gatgcaggaa gatccagctt ccaagtgcca ctgcagtgct aatgtgacca gttgtctctg
120tttgggcatt ccctctgaca actgcaccag accatgcttc agtgagagac
tgtctcagat 180gaccaatacc accatgcaaa caagataccc actgattttc
agtcgggtga aaaaatcagt 240tgaagtacta aagaacaaca agtgtccata
tttttcctgt gaacagccat gcaaccaaac 300cacggcaggc aacgcgctga
catttctgaa gagtcttctg gaaattttcc agaaagaaaa 360gatgagaggg
atgagaggca agatatgaag atgaaatatt atttatccca gctgc 41525127PRTHomo
sapiens 25Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe
Leu Ile1 5 10 15Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys
Ser Ala Asn 20 25 30Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp
Asn Cys Thr Arg 35 40 45Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr
Asn Thr Thr Met Gln 50 55 60Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val
Lys Lys Ser Val Glu Val65 70 75 80Leu Lys Asn Asn Lys Cys Pro Tyr
Phe Phe Ser Cys Glu Gln Pro Cys 85 90 95Asn Gln Thr Thr Ala Gly Asn
Ala Leu Thr Phe Leu Lys Ser Leu Leu 100 105 110Glu Ile Phe Gln Lys
Glu Lys Met Arg Gly Met Arg Gly Lys Ile 115 120 12526415DNAHomo
sapiens 26tctcgagcag gggtgtccaa ccttggcggg gatcctggac atcaacttcc
tcatcaacaa 60gatgcaggaa gatccagctt ccaagtgcca ctgcagtgct aatgtgacca
gttgtctctg 120tttgggcatt ccctctgaca actgcaccag accatgcttc
agtgagagac tgtctcagat 180gaccaatacc accatgcaaa caagataccc
actgattttc agtcgggtga aaaaatcagt 240tgaagtacta aagaacaaca
agtgtccata tttttcctgt gaacagccat gcaaccaaac 300catggcaggc
aacgcgctga catttctgaa gagtcttctg gaaattttcc agaaagaaaa
360gatgagaggg atgagaggca agatatgaag atgaaatatt atttatccca gctgc
41527126PRTHomo sapiens 27Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu
Asp Ile Asn Phe Leu Ile1 5 10 15Asn Lys Met Gln Glu Asp Pro Ala Ser
Lys Cys His Cys Ser Ala Asn 20 25 30Val Thr Ser Cys Leu Cys Leu Gly
Ile Pro Ser Asp Asn Cys Thr Arg 35 40 45Pro Cys Phe Ser Glu Arg Leu
Ser Gln Met Thr Asn Thr Thr Met Gln 50 55 60Thr Arg Tyr Pro Leu Ile
Phe Ser Arg Val Lys Lys Ser Val Glu Val65 70 75 80Leu Lys Asn Asn
Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn 85 90 95Gln Thr Met
Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu 100 105 110Ile
Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile 115 120
12528585DNAHomo sapiens 28atgaaaaaga cagctatcgc gattgcagtg
gcactggctg gtttcgctac cgttgcgcaa 60gctgactaca aggacgacga tgacaagctt
gaattctcta gagatatcgt cgacagatct 120ctcgagcagg ggtgtccaac
cttggcgggg atcctggaca tcaacttcct catcaacaag 180atgcaggaag
atccagcttc caagtgccac tgcagtgcta atgtgaccag ttgtctctgt
240ttgggcattc cctctgacaa ctgcaccaga ccatgcttca gtgagagact
gtctcagatg 300accaatacca ccatgcaaac aagataccca ctgattttca
gtcgggtgaa aaaatcagtt 360gaagtactaa agaacaacaa gtgtccatat
ttttcctgtg aacagccatg caaccaaacc 420acggcaggca acgcgctgac
atttctgaag agtcttctgg aaattttcca gaaagaaaag 480atgagaggga
tgagaggcaa gatatgaaga tgaaatatta tttatcccag ctgccaacgg
540tagcgaaacc agccagtgcc actgcaatcg cgatagctgt ctttt
58529168PRTHomo sapiens 29Met Lys Lys Thr Ala Ile Ala Ile Ala Val
Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Tyr Lys Asp
Asp Asp Asp Lys Leu Glu Phe 20 25 30Ser Arg Asp Ile Val Asp Arg Ser
Leu Glu Gln Gly Cys Pro Thr Leu 35 40 45Ala Gly Ile Leu Asp Ile Asn
Phe Leu Ile Asn Lys Met Gln Glu Asp 50 55 60Pro Ala Ser Lys Cys His
Cys Ser Ala Asn Val Thr Ser Cys Leu Cys65 70 75 80Leu Gly Ile Pro
Ser Asp Asn Cys Thr Arg Pro Cys Phe Ser Glu Arg 85 90 95Leu Ser Gln
Met Thr Asn Thr Thr Met Gln Thr Arg Tyr Pro Leu Ile 100 105 110Phe
Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Cys 115 120
125Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn Gln Thr Thr Ala Gly Asn
130 135 140Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe Gln Lys
Glu Lys145 150 155 160Met Arg Gly Met Arg Gly Lys Ile
16530585DNAHomo sapiens 30atgaaaaaga cagctatcgc gattgcagtg
gcactggctg gtttcgctac cgttgcgcaa 60gctgactaca aggacgacga tgacaagctt
gaattctcta gagatatcgt cgacagatct 120ctcgagcagg ggtgtccaac
cttggcgggg atcctggaca tcaacttcct catcaacaag 180atgcaggaag
atccagcttc caagtgccac tgcagtgcta atgtgaccag ttgtctctgt
240ttgggcattc cctctgacaa ctgcaccaga ccatgcttca gtgagagact
gtctcagatg 300accaatacca ccatgcaaac aagataccca ctgattttca
gtcgggtgaa aaaatcagtt 360gaagtactaa agaacaacaa gtgtccatat
ttttcctgtg aacagccatg caaccaaacc 420atggcaggca acgcgctgac
atttctgaag agtcttctgg aaattttcca gaaagaaaag 480atgagaggga
tgagaggcaa gatatgaaga tgaaatatta tttatcccag ctgccaacgg
540tagcgaaacc agccagtgcc actgcaatcg cgatagctgt ctttt
58531168PRTHomo sapiens 31Met Lys Lys Thr Ala Ile Ala Ile Ala Val
Ala Leu Ala Gly Phe Ala1 5 10 15Thr Val Ala Gln Ala Asp Tyr Lys Asp
Asp Asp Asp Lys Leu Glu Phe 20 25 30Ser Arg Asp Ile Val Asp Arg Ser
Leu Glu Gln Gly Cys Pro Thr Leu 35 40 45Ala Gly Ile Leu Asp Ile Asn
Phe Leu Ile Asn Lys Met Gln Glu Asp 50 55 60Pro Ala Ser Lys Cys His
Cys Ser Ala Asn Val Thr Ser Cys Leu Cys65 70 75 80Leu Gly Ile Pro
Ser Asp Asn Cys Thr Arg Pro Cys Phe Ser Glu Arg 85 90 95Leu Ser Gln
Met Thr Asn Thr Thr Met Gln Thr Arg Tyr Pro Leu Ile 100 105 110Phe
Ser Arg Val Lys Lys Ser Val Glu Val Leu Lys Asn Asn Lys Cys 115 120
125Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn Gln Thr Met Ala Gly Asn
130 135 140Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu Ile Phe Gln Lys
Glu Lys145 150 155 160Met Arg Gly Met Arg Gly Lys Ile
1653218DNAArtificial sequencePCR oligonucleotide primer
32gctccagtcc gctgtcaa 183318DNAArtificial sequencePCR
oligonucleotide primer 33ctccccctgc agcctacc 183420DNAArtificial
sequencePCR oligonucleotide primer 34cggggctgac taaaggttct
203520DNAArtificial sequencePCR oligonucleotide primer 35gttcttaaag
agcattcact 203624DNAArtificial sequencePCR oligonucleotide primer
36attttcacat ctggaatctt cact 243720DNAArtificial sequencePCR
oligonucleotide primer 37aatccaaggt caacattatg 203821DNAArtificial
sequencePCR oligonucleotide primer 38tttctttgaa taaatcctta c
213921DNAArtificial sequencePCR oligonucleotide primer 39gaaatcacca
acaggaacat a 214021DNAArtificial sequencePCR oligonucleotide primer
40atcaactttc atccccacag t 214124DNAArtificial sequencePCR
oligonucleotide primer 41ggataaataa tatttcatct tcat
244221DNAArtificial sequenceOligonucleotide primer 42caaatctgaa
gagcaaacta t 214324DNAArtificial sequenceOligonucleotide primer
43ttaaaaaatt catttcagta ttct 244424DNAArtificial sequenceKinase
end-labeled p32 oligonucleotide probe 44aattacctta ttgaaaatct gaag
24458PRTArtificial sequencePeptide sequence 45Asp Tyr Lys Asp Asp
Asp Asp Lys1 5
* * * * *