U.S. patent application number 11/834206 was filed with the patent office on 2009-05-21 for immunization-free methods for treating antigen-stimulated inflammation in a mammalian host and shifting the host's antigen immune responsiveness to a th1 phenotype.
Invention is credited to Eyal RAZ.
Application Number | 20090131347 11/834206 |
Document ID | / |
Family ID | 25454211 |
Filed Date | 2009-05-21 |
United States Patent
Application |
20090131347 |
Kind Code |
A1 |
RAZ; Eyal |
May 21, 2009 |
IMMUNIZATION-FREE METHODS FOR TREATING ANTIGEN-STIMULATED
INFLAMMATION IN A MAMMALIAN HOST AND SHIFTING THE HOST'S ANTIGEN
IMMUNE RESPONSIVENESS TO A TH1 PHENOTYPE
Abstract
The invention relates to methods for preventing or reducing
antigen-stimulated, granulocytemediated inflammation in tissue of
an antigen-sensitized mammal host by delivering an
immunostimulatory oligonucleotide to the host. In addition, methods
for using the immunostimulatory oligonucleotides to boost a mammal
host's immune responsiveness to a sensitizing antigen (without
immunization of the host by the antigen) and shifting the host's
immune responsiveness to a Th1 phenotype to achieve various
therapeutic ends are provided. Kits for practicing the methods of
the invention are also provided.
Inventors: |
RAZ; Eyal; (Del Mar,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE, SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
25454211 |
Appl. No.: |
11/834206 |
Filed: |
August 6, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10229208 |
Aug 26, 2002 |
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11834206 |
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09235742 |
Jan 21, 1999 |
6498148 |
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10229208 |
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08927120 |
Sep 5, 1997 |
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09235742 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 21/00 20180101;
A61P 27/02 20180101; A61P 27/16 20180101; A61P 29/00 20180101; A61P
37/08 20180101; A61K 2039/543 20130101; A61K 39/35 20130101; A61K
2039/55561 20130101; A61P 11/06 20180101; A61K 2039/53 20130101;
A61P 37/02 20180101 |
Class at
Publication: |
514/44 |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61P 37/02 20060101 A61P037/02 |
Goverment Interests
STATEMENT OF GOVERNMENT RIGHTS
[0001] This invention was made with Government support under Grant
No. AI37350, awarded by the National Institutes of Health. The
Government may have certain rights in this invention.
Claims
1.-37. (canceled)
38. A method for treating antigen-stimulated inflammation in a
mammal, comprising: administering to a mammal sensitized to an
antigen an immunostimulatory polynucleotide comprising an
immunostimulatory sequence (ISS), wherein the ISS comprises the
sequence 5'-cytosine-guanine-3', wherein the ISS is from about 6 to
about 200 nucleotides in length, wherein the immunostimulatory
polynucleotide does not comprise a nucleotide sequence encoding the
antigen, and wherein the immunostimulatory polynucleotide is
administered without the antigen, including without a
polynucleotide encoding the antigen, and in an amount sufficient to
treat the antigen-stimulated inflammation.
39. The method of claim 38, wherein the ISS comprises the sequence
5'-purine-purine-cytosine-guanine-pyrimidine-pyrimidine-3'.
40. The method of claim 39, wherein the ISS comprises a nucleotide
sequence selected from AGCGTC, GACGTT, GGCGTT, AACGTC, GACGTC,
GGCGTC, AGCGCC, GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, AACGCT,
AACGTT, AGCGTT, and AACGCC.
41. The method of claim 38, wherein the mammal is a human.
42. The method of claim 38, wherein the immunostimulatory
polynucleotide is administered intramuscularly.
43. The method of claim 38, wherein the immunostimulatory
polynucleotide is administered to skin.
44. The method of claim 38, wherein the immunostimulatory
polynucleotide is administered to mucosal tissue.
45. The method of claim 44, wherein the mucosal tissue is
respiratory tissue.
46. The method of claim 45, wherein said administration is
intranasal.
47. The method of claim 38, wherein the antigen-stimulated
inflammation is an allergic condition.
48. The method of claim 38, wherein IgE production in response to
the sensitizing antigen is reduced.
49. The method of claim 38, wherein the antigen-stimulated
inflammation is Th2 associated inflammation.
50. A method for treating antigen-stimulated inflammation in a
mammal, comprising: administering to a mammal sensitized to an
antigen an immunostimulatory polynucleotide comprising an
immunostimulatory sequence (ISS), wherein the ISS comprises the
sequence
5'-purine-purine-cytosine-guanine-pyrimidine-pyrimidine-3', wherein
the immunostimulatory polynucleotide does not comprise a nucleotide
sequence encoding the antigen, and wherein the immunostimulatory
polynucleotide is administered without the antigen, including
without a polynucleotide encoding the antigen, and in an amount
sufficient to treat the antigen-stimulated inflammation.
51. The method of claim 50, wherein the mammal is a human.
52. The method of claim 50, wherein the immunostimulatory
polynucleotide is administered intramuscularly.
53. The method of claim 50, wherein the immunostimulatory
polynucleotide is administered to skin.
54. The method of claim 50, wherein the immunostimulatory
polynucleotide is administered to mucosal tissue.
55. The method of claim 54, wherein the mucosal tissue is
respiratory tissue.
56. The method of claim 55, wherein said administration is
intranasal.
57. The method of claim 50, wherein the antigen-stimulated
inflammation is an allergic condition.
58. The method of claim 50, wherein IgE production in response to
the sensitizing antigen is reduced.
59. The method of claim 50, wherein the antigen-stimulated
inflammation is Th2 associated inflammation.
60. The method of claim 50, wherein the ISS is from about 6 to
about 200 nucleotides in length.
61. A method of shifting an immune response to an antigen away from
a Th2 phenotype and toward a Th1 phenotype in a mammal, the method
comprising administering to a mammal sensitized to an antigen an
immunostimulatory polynucleotide comprising an immunostimulatory
sequence (ISS), wherein the ISS comprises the sequence
5'-cytosine-guanine-3', wherein the ISS is from about 6 to about
200 nucleotides in length, wherein the immunostimulatory
polynucleotide does not comprise a nucleotide sequence encoding the
antigen, and wherein the immunostimulatory polynucleotide is
administered without the antigen, including without a
polynucleotide encoding the antigen, and in an amount sufficient to
shift the immune response toward a Th1 phenotype.
Description
FIELD OF THE INVENTION
[0002] The invention relates to methods and oligonucleotide
compositions for use in reducing, or suppressing
granulocyte-mediated inflammation in a host tissue and in
modulating the host's immune responsiveness to an antigen.
HISTORY OF THE RELATED ART
[0003] In vertebrates, endothelial cell adhesion by granulocytes
(eosinophils, basophils, neutrophils and mast cells) is followed by
the release of inflammatory mediators, such as leukotrienes, major
basic protein and histamine. In susceptible individuals, the
resulting inflammation can damage affected host tissues.
[0004] The most common pathologic inflammatory condition is asthma,
which is characterized by marked eosinophil infiltration into
respiratory airways, followed by inflammation-induced tissue
damage. Other pathologic inflammatory conditions associated with
granulocyte infiltration into affected tissues include nasal
polyposis, allergic rhinitis, allergic conjunctivitis, atopic
dermatitis, eosinophilic fasciitis, idiopathic hypereosinophilic
syndrome and cutaneous basophil hypersensitivity, as well as
inflammation and fibrosis resulting from increased production of
granulocyte-stimulatory cytokines, such as interleukin (IL)-5 and
certain interferons (INF).
[0005] Routine treatment of such conditions is typically directed
toward inhibiting the activity of inflammatory mediators released
after granulocyte adhesion to endothelia (e.g., by delivering a
corticoid composition to the affected tissues). Where the identity
of an inflammation inducing antigen is known, some immune
protection against further antigen challenge can be provided
through immunization. However, although effective in stimulating
production of neutralizing antibodies, canonical immunization does
not effectively stimulate longer term cellular immunity. Moreover,
antigen immunization stimulates host production of IL-4 and IL-5.
IL-5 encourages granulocyte adhesion to endothelia while IL-4
induces immunoglobulin switching to the IgE isotype at the risk of
anaphylaxis.
SUMMARY OF THE INVENTION
[0006] The invention provides means to rapidly suppress
antigen-stimulated inflammation in a mammalian host by suppressing
granulocyte infiltration into a host tissue. The invention also
provides immunization-free means to provide protection to an
antigen-sensitized mammalian host against subsequent antigen
challenge without risk of anaphylaxis. These aims are achieved by
the invention through delivery of an immunostimulatory
oligonucleotide (ISS-ODN) to the host without codelivery of an
immunizing antigen.
[0007] Surprisingly, ISS-ODN have anti-inflammatory properties in
addition to their immunostimulatory properties. ISS-ODN are
therefore useful in the treatment and prevention of inflammation
associated with antigen-stimulated granulocyte infiltration of
tissue, such as occurs in the respiratory passages of asthmatics
during an asthma attack. Advantageously, delivery of ISS-ODN
according to the invention suppresses antigen-stimulated
granulocyte infiltration into host tissue even before the ISS-ODN
affect the host's immune response to the antigen. Thus, the
invention provides an antigen-independent method to reduce
antigen-stimulated inflammation by suppressing cellular adhesion,
thereby avoiding the release of inflammatory mediators which would
be stimulated through granulocyte-binding of endothelial cells.
[0008] An example of a therapeutic application for the invention is
in the control of asthma, whereby the ISS-ODN are delivered into
pulmonary tissue intranasally or by systemic routes. In asthmatics,
eosinophil infiltration of lung tissue occurs mainly during the
late phase of an allergic response to a respiratory allergen.
Canonical immunotherapy can modulate the host immune response to
the allergen and eventually stem the tide of eosinophils into the
host airways. However, practice of the invention suppresses
eosinophil infiltration of host airways well before the host immune
system responds to the respiratory allergen, thereby providing a
form of protection against the airway narrowing and respiratory
tissue damage which characterize an acute asthma attack.
[0009] In another aspect, the invention provides means to shift a
present host cellular immune response to an antigen away from a Th2
phenotype and into a Th1 phenotype. To this end. ISS-ODN are
delivered by any route through which antigen-sensitized host
tissues will be contacted with the ISS-ODN. ISS-ODN administered in
this fashion boost both humoral (antibody and cellular (Th1 type)
immune responses of the host. Unlike canonical immunotherapy,
immunity is stimulated by this method of the invention even when no
additional antigen is introduced into the host. Thus, use of the
method to boost the immune responsiveness of a host to subsequent
challenge by a sensitizing antigen without immunization avoids the
risk of immunization-induced anaphylaxis, suppresses IgE production
in response to the antigen challenge and eliminates the need to
identify the sensitizing antigen for use in immunization. An
especially advantageous use for this aspect of the invention is
treatment of localized allergic responses in target tissues where
the allergens enter the body, such as the skin and mucosa.
[0010] Suppression of the Th2 phenotype according to the invention
is also a useful adjunct to canonical immunotherapy to reduce
antigen-stimulated IL-4 and IL-5 production. Thus, the invention
encompasses delivery of ISS-ODN to a host to suppress the Th2
phenotype associated with conventional antigen immunization (e.g.,
for vaccination or allergy immunotherapy).
[0011] The shift to a Th1 phenotype achieved according to the
invention is accompanied by increased secretion of IFN .alpha.,
.beta., and .gamma., as well as IL-12 and IL-18. Each of these
cytokines enhance the host's immune defenses against intracellular
pathogens, such as viruses. Thus, the invention encompasses
delivery of ISS-ODN to a host to combat pathogenic infection.
[0012] Angiogenesis is also enhanced in the Th1 phenotype
(ostensibly through stimulation by IL-12). Thus, the invention
encompasses delivery of ISS-ODN to a host to stimulate therapeutic
angiogenesis to treat conditions in which localized blood flow
plays a significant etiological role, such as in diabetic
retinopathy.
[0013] Pharmaceutically acceptable compositions of ISS-ODN are
provided for use in practicing the methods of the invention. The
ISS-ODN of the invention include DNA or RNA oligonucleotides which
are enriched with CpG dinucleotides, including those which are
comprised of the primary structure
5'-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3'.
[0014] Where appropriate to the contemplated course of therapy, the
ISS-ODN may be administered with other anti-inflammatory or
immunotherapeutic agents. Thus, a particularly useful composition
for use in practicing the method of the invention is one in which
an anti-inflammatory agent (e.g., a glucocorticoid) or
immunotherapeutic agent (e.g., an antigen, cytokine or adjuvant) is
mixed with, or conjugated to, an ISS-ODN.
[0015] The ISS-ODN can also be provided in the form of a kit
comprising ISS-ODN and any additional medicaments, as well as a
device for delivery of the ISS-ODN to a host tissue and reagents
for determining the biological effect of the ISS-ODN on the treated
host.
BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 is a chart which summarizes aspects of the mammalian
immune system.
[0017] FIG. 2 is a graph of data which confirm a shift from a Th2
to a Th1 phenotype (as measured by IgG2A production) in mice
treated with an ISS-ODN 3 days before antigen challenge.
[0018] FIG. 3a and 3b are graphs of data which confirm the
induction of a Th2 phenotype (as measured by IgG1 production) in
mice treated with a mutant, inactive ISS-ODN 3 days before antigen
challenge.
[0019] FIG. 4 is a graph of data which confirm Th1-associated
suppression of antigen-specific IgE IF in antigen-sensitized,
ISS-ODN (pCMV-LacZ, a plasmid containing two copies of the DY1018
ISS-ODN) treated mice as compared to antigen-sensitized (control)
mice.
[0020] FIG. 5 is a graph of data which confirm suppression of IL-4
secretion by ISS-ODN as compared to a control.
[0021] FIG. 6 is a graph of data which confirm suppression of IL-5
secretion by ISS-ODN as compared to a control.
[0022] FIG. 7 is a graph of data which confirm suppression of IL-10
secretion by ISS-ODN as compared to a control.
[0023] FIG. 8 is a graph of data which confirm stimulation of
INF-.gamma. secretion by ISS-ODN as compared to a control.
[0024] FIG. 9 is a graph of data demonstrating an ISS-ODN mediated
shift to a Th1 phenotype (as indicated by IFN.gamma. levels) in
animals treated with ISS-ODN before antigen challenge (asterisked
bars) or after antigen challenge.
[0025] FIG. 10) is a graph of data demonstrating an ISS-ODN
mediated boost in immune responsiveness (as indicated by increases
in CD4+ lymphocyte proliferation) in animals treated with ISS-ODN
before antigen challenge (asterisked bars) or after antigen
challenge.
DETAILED DESCRIPTION OF THE INVENTION
A. Anti-Inflammatory and Immunotherapeutic Methods of the
Invention
[0026] 1. Therapeutic Effects of the Methods of the Invention
[0027] The main therapeutic goals which may be achieved through
practice of the methods of the invention are treatment of
inflammation and boosting of host immune responsiveness with a Th1
phenotype against a sensitizing antigen. Both goals are achieved by
delivering ISS-ODN to an antigen-sensitized host; i.e., a mammal
whose immune system has been primed to respond to challenge by a
sensitizing antigen. For purposes of this disclosure, "sensitizing
antigen" refers to an exogenous, immunogenic protein, peptide,
glycoprotein, lipid or polysaccharide. For reference, a chart
summarizing aspects of mammal antigen immunity is appended as FIG.
1.
[0028] The anti-inflammatory method of the invention is useful in
suppressing the onset of, and in reducing, acute
granulocyte-mediated inflammation in an antigen-sensitized host.
Specifically, treatment of an antigen-sensitized (primed) host
before subsequent antigen challenge suppresses antigen-stimulated
infiltration of host tissue by granulocytes (especially,
eosinophils and basophils). Similarly, treatment of an
antigen-sensitized host on or after antigen challenge reduces
antigen-stimulated infiltration of host tissue by granulocytes.
Advantageously, the anti-inflammatory impact of ISS-ODN delivered
according to the invention is rapid, taking effect even before the
ISS-ODN would be expected to impact the host's immune
responsiveness to the sensitizing antigen. The invention therefore
provides the host with fairly immediate protection against tissue
damage from granulocyte-mediated inflammation.
[0029] For example, as shown by the data in Example II,
antigen-sensitized animal models of allergic asthma treated with
ISS-ODN without concurrent antigen challenge experienced as much as
a 90% reduction of eosinophil infiltration into respiratory tissue
as compared to control animals and animals treated only with an
inactive ISS-ODN mutant. Significantly, reduction of eosinophil
infiltration in previously challenged mice, or suppression of
eosinophil infiltration in primed, unchallenged mice, was obtained
within as little as 24 hours of delivery of the ISS-ODIN. The
effect of the ISS-ODN on eosinophil infiltration is therefore
independent of the later-developing host immune response to the
sensitizing antigen. Being antigen independent, the ISS-ODN can be
utilized as inflammation suppressors before antigen challenge or
during a period when the risk of antigen challenge is present
(e.g., during an allergy season). Importantly, as shown in Examples
IV and VI, ISS-ODN can be used according to the invention to
prevent inflammation or an immune response on subsequent antigen
challenge in an antigen-primed host as well as to reduce
inflammation or other antigen-stimulated immune responses after
antigen challenge.
[0030] Although the invention is not limited to any mechanism of
action, it is probable that the anti-inflammatory activity of
ISS-ODN is at least in part a consequence of IL-5 suppression.
However, suppression of granulocyte accumulation in host tissue is
achieved more rapidly (within 24 hours) than immune activation of
cytokine-secreting lymphocytes would be expected to occur. It is
therefore also possible that ISS-ODN administered according to the
invention physically interfere with granulocyte adhesion to
endothelial, perhaps by blocking VCAM-1 endothelial receptors,
their eosinophilic ligand (VLA-4) or by lysing granulocytes.
Whatever the mechanism, ISS-ODN suppression of granulocyte
accumulation according to the invention appears to be independent
of ISS-ODN stimulation of the host immune system.
[0031] The immunotherapeutic method of the invention produces a
vaccination-like immune response to challenge by a sensitizing
antigen without concurrent exposure of the host to the antigen.
Immune stimulation achieved through practice of the invention is
comparable to the immune stimulation which occurs on vaccination of
a host with a sensitizing antigen. Thus, the methods of the
invention provides means to immunize a host against a sensitizing
antigen without deliberate antigen challenge.
[0032] Advantageously, the immune response stimulated according to
the invention differs from an immunization response in that the
latter develops in a Th2 phenotype while the former develops in a
Th1 phenotype. In this regard, it is helpful to recall that CD4+
lymphocytes generally fall into one of two distinct subsets; i.e.,
the Th1 and Th2 cells. Th1 cells principally secrete IL-2,
IFN.gamma. and TNF.beta. (the latter two of which mediate
macrophage activation and delayed type hypersensitivity) while Th2
cells principally secrete IL-4 (which stimulates production of IgE
antibodies), IL-5 (which stimulates granulocyte infiltration of
tissue), IL-6 and IL-10. These CD4' subsets expert a negative
influence on one another; i.e., secretion of Th1 lymphokines
inhibits secretion of Th2 lymphokines and vice versa.
[0033] Factors believed to favor Th1 activation resemble those
induced by viral infection and include intracellular pathogens,
exposure to IFN-.beta., IFN-.alpha., IFN.gamma., IL-12 and IL-18
and exposure to low doses of antigen. Th1 type immune responses
also predominate in autoimmune disease. Factors believed to favor
Th2 activation include exposure to IL-4 and IL-10, APC activity on
the part of .beta.-lymphocytes and high doses of antigen. Active
Th1 (IFN.gamma.) cells enhance cellular immunity and are therefore
of particular value in responding to intracellular infections,
while active Th2 cells enhance antibody production and are
therefore of value in responding to extracellular infections (at
the risk of anaphylactic events associated with IL-4 stimulated
induction of IgE antibody production). Thus, the ability to shift
host immune responses from the Th1 to the Th2 repertoire and vice
versa has substantial clinical significance for controlling host
immunity against antigen challenge (e.g., in infectious and
allergic conditions).
[0034] To that end, the methods of the invention nut the host
immune response to a sensitizing antigen toward a Th1 phenotype
(Example IV). Consequently, antigen-stimulated/Th2 associated IL-4,
IL-5 and IL-10 secretion (Example VI), IL->stimulated
granulocyte infiltration of antigen-sensitized tissue (Examples II
and III) and IL-4 stimulated production of IgE (Example V) are
suppressed, thereby reducing the host's risk of prolonged allergic
inflammation and minimizing the risk of antigen-induced
anaphylaxis. Although the invention is not limited to any
particular mechanism of action, it is conceivable that ISS-ODN
facilitate uptake of exogenous antigen by antigen presenting cells
for presentation through host MHC Class I processing pathways.
Whatever the mechanism of action, use of ISS-ODIN to boost the
host's immune responsiveness to a sensitizing antigen and shift the
immune response toward a Th1 phenotype avoids the risk of
immunization-induced anaphylaxis, suppresses IgE production in
response to a sensitizing antigen and eliminates the need to
identify the sensitizing antigen for use in immunization.
[0035] With reference to the invention, ISS-ODN mediated "reduction
of inflammation" (in a primed, antigen-challenged host),
"prevention of inflammation" (in a primed host before antigen
challenge) and "boosting of immune responsiveness in a Th1
phenotype" in an ISS-ODN treated host are evidenced by any of the
following events: [0036] (1) a reduction in levels of IL-4 measured
before and after antigen-challenge; or detection of lower (or even
absent) levels of IL-4 in a treated host as compared to an
antigen-primed, or primed and challenged, control; [0037] (2) an
increase in levels of IL-12, IL-18 and/or IFN (.alpha., .beta. or
.gamma.) before and after antigen challenge; or detection of higher
levels of IL-12, IL-18 and/or IFN (.alpha., .beta. or .gamma.) in
an ISS-ODN treated host as compared to an antigen-primed or, primed
and challenged, control; [0038] (3) IgG2a antibody production in a
treated host; or [0039] (4) a reduction in levels of
antigen-specific IgE as measured before and after antigen
challenge; or detection of lower (or even absent) levels of
antigen-specific IgE in an ISS-ODN treated host as compared to an
antigen-primed, or primed and challenged, control.
[0040] Also, with respect to reduction and prevention of
inflammation in particular, an especially meaningful indicia of the
efficacy of the inventive method in a treated host is: [0041] (5) a
reduction in granulocyte counts (e.g., of eosinophils or basophils,
depending on which cell type is most involved in the condition
affecting the host) in inflammatory infiltrate of an affected host
tissue as measured in an antigen-challenged host before and after
ISSODN administration, or detection of lower (or even absent)
levels of eosinophil or basophil counts in a treated host as
compared to an antigen-primed, or primed and challenged,
control.
[0042] Exemplary methods for determining such values are described
further in the Examples.
[0043] 2. Methods and Routes for Administration of ISS-ODN to a
Host
[0044] The ISS-ODN of the invention are administered to a host
using any available method and route suitable for drug delivery,
including ex vivo methods (e.g., delivery of cells incubated or
transfected with an ISS-ODN) as well as systemic or localized
routes. However, those of ordinary skill in the art will appreciate
that methods and localized routes which direct the ISS-ODN into
antigen-sensitized tissue will be preferred in most circumstances
to systemic routes of administration, both for immediacy of
therapeutic effect and avoidance of oligonucleotide degradation in
vivo.
[0045] The entrance point for many exogenous antigens into a host
is through the skin or mucosa. Thus, delivery methods and routes
which target the skin (e.g., for cutaneous and subcutaneous
conditions) or mucosa (e.g., for respiratory, ocular, lingual or
genital conditions) will be especially useful. Those of ordinary
skill in the clinical arts will be familiar with, or can readily
ascertain, means for drug delivery into skin and mucosa. For
review, however, exemplary methods and routes of drug delivery
useful in the invention are briefly discussed below.
[0046] Intranasal administration means are particularly useful in
addressing respiratory inflammation, particularly inflammation
mediated by antigens transmitted from the nasal passages into the
trachea or bronchioli. Such means include inhalation of aerosol
suspensions or insufflation of the polynucleotide compositions of
the invention. Nebulizer devices suitable for delivery of
polynucleotide compositions to the nasal mucosa, trachea and
bronchioli are well-known in the art and will therefore not be
described in detail here. For general review in regard to
intranasal drug delivery, those of ordinary skill in the art may
wish to consult Chien, Novel Drug Delivery Systems, Ch. 5 (Marcel
Dekker, 1992).
[0047] Dermal routes of administration, as well as subcutaneous
injections, are useful in addressing allergic reaction and
inflammation in the skin. Examples of means for delivering drugs to
the skin are topical application of a suitable pharmaceutical
preparation, transdermal transmission, injection and epidermal
administration.
[0048] For transdermal transmission, absorption promoters or
iontophoresis are suitable methods. For review regarding such
methods, those of ordinary skill in the art may wish to consult
Chien, supra at Ch. 7. Iontophoretic transmission may be
accomplished using commercially available "patches" which deliver
their product continuously via electric pulses through unbroken
skin for periods of several days or more. Use of this method allows
for controlled transmission of pharmaceutical compositions in
relatively great concentrations, permits infusion of combination
drugs and allows for contemporaneous use of an absorption
promoter.
[0049] An exemplary patch product for use in this method is the
LECTRO PATCH trademarked product of General Medical Company of Los
Angeles, Calif. This product electronically maintains reservoir
electrodes at neutral pH and can be adapted to provide dosages of
differing concentrations, to dose continuously and/or to dose
periodically. Preparation and use of the patch should be performed
according to the manufacturer's printed instructions which
accompany the LECTRO PATCH product; those instructions are
incorporated herein by this reference.
[0050] Epidermal administration essentially involves mechanically
or chemically irritating the outermost layer of the epidermis
sufficiently to provoke an immune response to the irritant. An
exemplary device for use in epidermal administration employs a
multiplicity of very narrow diameter, short tynes which can be used
to scratch ISS-ODN coated onto the tynes into the skin. The device
included in the MONO-VACC old tuberculin test manufactured by
Pasteur Merieux of Lyon, France is suitable for use in epidermal
administration of ISS-ODN. Use of the device is according to the
manufacturer's written instructions included with the device
product; these instructions regarding use and administration are
incorporated herein by this reference to illustrate conventional
use of the device. Similar devices which may also be used in this
embodiment are those which are currently used to perform allergy
tests.
[0051] Ophthalmic administration (e.g., for treatment of allergic
conjunctivitis) involves invasive or topical application of a
pharmaceutical preparation to the eye. Eye drops, topical creams
and injectable liquids are all examples of suitable milieus for
delivering drugs to the eye.
[0052] Systemic administration involves invasive or systemically
absorbed topical administration of pharmaceutical preparations.
Topical applications as well as intravenous and intramuscular
injections are examples of common means for systemic administration
of drugs.
[0053] 3. Dosing Parameters for ISS-ODN
[0054] A particular advantage of ISS-ODN of the invention is their
capacity to exert anti-inflammatory and immunotherapeutic activity
even at relatively minute dosages. Although the dosage used will
vary depending on the clinical goals to be achieved, a suitable
dosage range is one which provides up to about 1-1000 .mu.g of
ISS-ODN/ml of carrier in a single dosage. In view of the teaching
provided by this disclosure, those of ordinary skill in the
clinical arts will be familiar with, or can readily ascertain,
suitable parameters for administration of ISS-ODN according to the
invention.
[0055] In this respect, it should be noted that the
anti-inflammatory and immunotherapeutic activity of ISS-ODN in the
invention is essentially dose-dependent. Therefore, to increase
ISS-ODN potency by a magnitude of two, each single dose is doubled
in concentration. Clinically, it may be advisable to administer the
ISS-ODN in a low dosage (e.g., about 1 .mu.g/ml to about 50
.mu.g/ml), then increase the dosage as needed to achieve the
desired therapeutic goal. Alternatively, a target dosage of ISS-ODN
can be considered to be about 1-10 .mu.M in a sample of host blood
drawn within the first 24-48 hours after administration of ISS-ODN.
Based on current studies, ISS-ODN are believed to have little or no
toxicity at these dosage levels.
B. ISS-ODN Anti-Inflammatory Compositions
[0056] 1. ISS-ODN Structure
[0057] Functionally, ISS-ODN enhance the cellular and humoral
immune responses in a host, particularly lymphocyte proliferation
and the release of cytokines (including IFN) by host monocytes and
natural killer (NK) cells. Immunostimulation by synthetic ISS-ODN
in vivo occurs by contacting host lymphocytes with, for example,
ISS-ODN oligonucleotides, ISS-ODN oligonucleotide-conjugates and
ISS-containing recombinant expression vectors (data regarding the
activity of ISS-ODN conjugates and ISS-ODN vectors are set forth in
co-pending, commonly assigned U.S. patent application Ser. Nos.
60/028,118 and 08/593,554; data from which is incorporated herein
by reference to demonstrate ISS-ODN immunostimulatory activity in
vivo). Thus, while native microbial ISS-ODN stimulate the host
immune system to respond to infection, synthetic analogs of these
ISS-ODN are useful therapeutically to modulate the host immune
response not only to microbial antigens, but also to tumor
antigens, allergens and other substances.
[0058] Structurally, ISS-ODN are non-coding oligonucleotides 6 mer
or greater in length which may include at least one unmethylated
CpG motif. The relative position of each CpG sequence in ISS-ODN
with immunostimulatory activity in certain mammalian species (e.g.
rodents) in 5'-CG3' (i.e., the C is in the 5' position with respect
to the G in the 3 position). Many known ISS-ODN flank the CpG motif
with at least two purine nucleotides (e.g. GA or AA) and at least
two pyrimidine nucleotides
(5'-Purine-Purine[C]-[G]-Pyrimidine-Pyrimidine-3'). CpG
motif-containing ISS-ODN are believed to stimulate B lymphocyte
proliferation (see, e.g., Krieg, et al., Nature, 374:546-549,
1995).
[0059] The core hexamer structure of the foregoing ISS-ODN Slav be
flanked upstream and/or downstream by any number or composition of
nucleotides or nucleosides. However, ISS-ODN are at least 6 mer in
length, and preferably are between 6 and 200 mer in length, to
enhance uptake of the ISS-ODN into target tissues. Those of
ordinary skill in the art will be familiar with, or can readily
identify, reported nucleotide sequences of known ISS-ODN. For ease
of reference in this regard, the following sources are especially
helpful: [0060] Yamamoto, et al., Microbiol. Immunol., 36:983
(1992) [0061] Ballas, et al. J. Immunol., 157:1840 (1996) [0062]
Klinman, et al., J. Immunol., 158:3635 (1997) [0063] Sato, et al.,
Science, 273:352 (1996)
[0064] Each of these articles are incorporated herein by reference
for the purpose of illustrating the level of knowledge in the art
concerning the nucleotide composition of ISS-ODN.
[0065] In particular, ISS-ODN useful in the invention include those
which have the following hexameric nucleotide sequences: [0066] 1.
ISS-ODN having "CpG" dinucleotides; and, [0067] 2. Inosine and/or
uracil substitutions for nucleotides in the foregoing hexamer
sequences for use as RNA ISS-ODN.
[0068] For example, DINA based ISS-ODN useful in the invention
include those which have the following hexameric nucleotide
sequences:
TABLE-US-00001 (respectively, SEQ ID.Nos. 1-18) AACGTT, AGCGTC,
GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, GGCGTC, AACGCC, AGCGCC,
GACGCC, GGCGCC, AGCGCT, GACGCT, GGCGCT, TTCGAA, GGCGTT and
AACGCC.
[0069] ISS-ODN may be single-stranded or double-stranded DNA,
single or double-stranded RNA and/or oligonucleosides. The ISS-ODN
may or may not include palindromic regions. If present, a
palindrome may extend only to a CpG motif, if present, in the core
hexamer sequence, or may encompass more of the hexamer sequence as
well as flanking nucleotide sequences.
[0070] The nucleotide bases of the ISS-ODN which flank the CpG
motif of the core hexamer and/or make up the flanking nucleotide
sequences may be any known naturally occurring bases or synthetic
nonnatural bases (e.g., TCAG or, in RNA, UACGI). Oligonucleosides
may be incorporated into the internal region and/or termini of the
ISS-ODN using conventional techniques for use as attachment points
for other compounds (e.g., peptides). The base(s), sugar moiety,
phosphate groups and termini of the ISS-ODN may also be modified in
any manner known to those of ordinary skill in the art to construct
an ISS-ODN having properties desired in addition to the described
activity of the ISS-ODN. For example, sugar moieties may be
attached to nucleotide bases of ISS-ODN in any steric
configuration.
[0071] In addition, backbone phosphate group modifications (e.g.,
methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages) can confer
anti-microbial activity on the ISS-ODN and enhance their stability
in vivo, making them particularly useful in therapeutic
applications. A particularly useful phosphate group modification is
the conversion to the phosphorothioate or phosphorodithioate forms
of the ISS-ODN oligonucleotides. In addition to their potentially
anti-microbial properties, phosphorothioates and
phosphorodithioates are more resistant to degradation in vivo than
their unmodified oligonucleotide counterparts, making the ISS-ODN
of the invention more available to the host.
[0072] 2. Synthesis of, and Screening for, ISS-ODN
[0073] ISS-ODN can be synthesized using techniques and nucleic acid
synthesis equipment which are well-known in the art. For reference
in this regard, see e.g., Ausubel, et al., Current Protocols in
Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989);
Maniatis, et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Lab., New York, 1982); U.S. Pat. No. 4,458,066 and
U.S. Pat. No. 4,650,675. These references are incorporated herein
by reference for the sole purpose of demonstrating knowledge in the
art concerning production of synthetic oligonucleotides. Because
the ISS-ODN is non-coding, there is no concern about maintaining an
open reading frame during synthesis.
[0074] ISS-ODN may be incorporated into a delivery vector, such as
a plasmid, cosmid, virus or retrovirus, which may in turn code for
therapeutically, beneficial polypeptides, such as cytokines,
hormones and antigens. Incorporation of ISS-ODN into such a vector
does not adversely affect their activity.
[0075] Alternatively, ISS-ODN may be isolated from microbial
species (especially mycobacteria) using techniques well-known in
the art, such as nucleic acid hybridization. Preferably, such
isolated ISS-ODN will be purified to a substantially pure state;
i.e., to be free of endogenous contaminants, such as
lipopolysaccharides. ISS-ODN isolated as part of a larger
polynucleotide can be reduced to the desired length by techniques
well known in the art, such as by endonuclease digestion. Those of
ordinary skill in the art will be familiar with, or can readily
ascertain, techniques suitable for isolation, purification and
digestion of polynucleotides to obtain ISS-ODN of potential use in
the invention.
[0076] Confirmation that a particular oligonucleotide has the
properties of an ISS-ODIN useful in the invention can be obtained
by evaluating whether the ISS-ODN affects cytokine secretion and
IgG; antibody isotype production as described in Section A.I,
above. Details of in vitro techniques useful in making such an
evaluation are given in the Examples; those of ordinary skill in
the art will also known of, or can readily ascertain, other methods
for measuring cytokine secretion and antibody production along the
parameters taught herein.
[0077] The techniques for making phosphate group modifications to
oligonucleotides are known in the art and do not require detailed
explanation. For review of one such useful technique, the an
intermediate phosphate triester for the target oligonucleotide
product is prepared and oxidized to the naturally occurring
phosphate triester with aqueous iodine or with other agents, such
as anhydrous amines. The resulting oligonucleotide phosphoramidates
can be treated with sulfur to yield phophorothioates. The same
general technique (excepting the sulfur treatment step) can be
applied to yield methylphosphoamidites from methylphosphonates. For
more details concerning phosphate group modification techniques,
those of ordinary skill in the art may wish to consult U.S. Pat.
Nos. 4,425,732; 4,458,066; 5,218,103 and 5,453,496, as well as
Tetrahedron Lett. at 21:4149 (1995), 7:5575 (1986), 25:1437 (1984)
and Journal Am. ChemSoc., 93:6657 (1987), the disclosures of which
are incorporated herein for the sole purpose of illustrating the
standard level of knowledge in the art concerning preparation of
these compounds.
[0078] A colloidal dispersion system may be used for targeted
delivery of the ISS-ODN to an inflamed tissue. Colloidal dispersion
systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. The preferred
colloidal system of this invention is a liposome.
[0079] Liposomes are artificial membrane vesicles which are useful
as delivery vehicles in vitro and in vivo. It has been shown that
large unilamellar vesicles (LUV), which range in size from 0.2-4.0
.mu.m can encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. RNA, DNA and intact virions can be
encapsulated within the aqueous interior and be delivered to cells
in a biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In addition to mammalian cells, liposomes have
been used for delivery of polynucleotides in plant, yeast and
bacterial cells. In order for a liposome to be an efficient gene
transfer vehicle, the following characteristics should be present:
(1) encapsulation of the genes encoding the antisense
polynucleotides at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6.682,
1988).
[0080] The composition of the liposome is usually a combination of
phospholipids, particularly high-phasetransition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0081] Examples of lip ids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are iacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0082] The targeting of liposomes can be classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0083] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various well known linking
groups can be used for joining the lipid chains to the targeting
ligand (see, e.g., Yanagawa, et al., Nuc. Acids Symp. Ser. 19:139
(1988); Grabarek, et al. Anal. Biochem., 185:131 (1990); Staros, et
al., Anal. Biochem., 156:220 (1986) and Boujrad, et al., Proc.
Natl. Acad. Sci USA, 90:5728 (1993), the disclosures of which are
incorporated herein by reference solely to illustrate the standard
level of knowledge in the art concerning conjugation of
oligonucleotides to lipids). Targeted delivery of ISS-ODN can also
be achieved by conjugation of the ISS-ODN to a the surface of viral
and non-viral recombinant expression vectors, to an antigen or
other ligand, to a monoclonal antibody or to any molecule which has
the desired binding specificity.
[0084] Examples of other useful conjugate partners include any
immunogenic antigen (including allergens, live and attenuated viral
particles and tumor antigens), targeting peptides (such as receptor
ligands, antibodies and antibody fragments, hormones and enzymes),
non-peptidic antigens (coupled via a peptide linkage, such as
lipids, polysaccharides, glycoproteins, gangliosides and the like)
and cytokines (including interleukins, interferons, erythropoietin,
tumor necrosis factor and colony stimulating factors). Such
conjugate partners can be prepared according to conventional
techniques (e.g., peptide synthesis) and many are commercially
available.
[0085] Those of ordinary skill in the art will also be familiar
with, or can readily determine, methods useful in preparing
oligonucleotide-peptide conjugates. Conjugation can be accomplished
at either termini of the ISS-ODN or at a suitably modified base in
an internal position (e.g., a cytosine or uracil). For reference,
methods for conjugating oligonucleotides to proteins and to
oligosaccharide moieties of Ig are known (se e.g., O'Shannessy, et
al., J. Applied Biochem., 7:347 (1985), the disclosure of which is
incorporated herein by reference solely to illustrate the standard
level of knowledge in the art concerning oligonucleotide
conjugation). Another useful reference is Kessler: "Nonradioactive
Labeling Methods for Nucleic Acids", in Kricka (ed.), Nonisotopic
DNA Probe Techniques (Acad. Press, 1992)).
[0086] Briefly, examples of known, suitable conjugation methods
include: conjuration through 3' attachment via solid support
chemistry see, e.g., Haralambidis, et al., Nuc. Acids Res., 18:493
(1990) and Haralambidis, et al., Nuc. Acids Res., 18:501 (1990)
[solid support synthesis of peptide partner]; Zuckermann, et al.,
Nuc. Acids Res., 15:5305 (1987), Core, et al., Science, 238:1401
(1987) and Nelson, et al., Nuc. Acids Res., 17:1781 (1989) [solid
support synthesis of oligonucleotide partner]).
[0087] Amino-amino group linkages may be performed as described in
Benoit. et al., Neuromethods. 6:43 (1987), while thiol-carboxyl
group linkages may be performed as described in Sinah, et al.,
Oligonucleotide Analogues: A Practical Approach (IRL Press, 1991).
In these latter methods, the oligonucleotide partner is synthesized
on a solid support and a linking group comprising a protected
amine, thiol or carboxyl group opposite a phosphoramidite is
covalently attached to the 5'-hydroxy (see, e.g., U.S. Pat. Nos.
4,849,513; 5,015,733; 5,118,800 and 5,118,802).
[0088] Linkage of the oligonucleotide partner to a peptide may also
be made via incorporation of a linker arm (e.g., amine or carboxyl
group) to a modified cytosine or uracil base (e.g., Ruth, 4th
Annual Congress for Recombinant DNA Research at 123). Affinity
linkages (e.g., biotin-streptavidin) may also be used (see e.g.,
Roget, et al., Nuc. Acids Res., 17:7643 (1989)).
[0089] Methods for linking oligonucleotides to lipids are also
known and include synthesis of oligophospholipid conjugates see,
e.g., Yanagawa, et al., Nuc. Acids Symp. Ser., 19:189 (1988)),
synthesis of oligo-fatty acids conjugates (see. e.g., Grabarek, et
al., Anal. Biochem., 185:131 (1990)) and oligo-sterol conjugates
(see, e.g., Boujrad, et al., Proc. Natl. Acad. Sci USA, 90:5728
(1993)).
[0090] Each of the foregoing references is incorporated herein by
reference for the sole purpose of illustrating the level of
knowledge and skill in the art with respect to oligonucleotide
conjugation methods.
[0091] Co-administration of a peptide drug with an ISS-ODN
according to the invention may also be achieved by incorporating
the ISS-ODN in cis or in trans into a recombinant expression vector
(plasmid, cosmid, virus or retrovirus) which codes for any
therapeutically beneficial protein deliverable by a recombinant
expression vector. If incorporation of an ISS-ODN into an
expression vector for use in practicing the invention is desired,
such incorporation may be accomplished using conventional
techniques which do not require detailed explanation to one of
ordinary skill in the art. For review, however, those of ordinary
skill may wish to consult Ausubel, Current Protocols in Molecular
Biology, supra.
[0092] Briefly, construction of recombinant expression vectors
(including those which do not code for any protein and are used as
carriers for ISS-ODN) employs standard ligation techniques. For
analysis to confirm correct sequences in vectors constructed, the
ligation mixtures may be used to transform a host cell and
successful transformants selected by antibiotic resistance where
appropriate. Vectors from the transformants are prepared, analyzed
by restriction and/or sequenced by, for example, the method of
Messing, et al., (Nucleic Acids Res., 9:309, 1981), the method of
Maxam, et al., (Methods in Enzymology, 65:499, 1980), or other
suitable methods which will be known to those skilled in the art.
Size separation of cleaved fragments is performed using
conventional gel electrophoresis as described, for example, by
Maniatis, et al., (Molecular Cloning, pp. 133-134, 1982).
[0093] Host cells may be transformed with expression vectors and
cultured in conventional nutrient media modified as is appropriate
for inducing promoters, selecting transformants or amplifying
genes. The culture conditions, such as temperature, pH and the
like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan.
[0094] If a recombinant expression vector is utilized as a carrier
for the ISS-ODN of the invention, plasmids and cosmids are
particularly preferred for their lack of pathogenicity. However,
plasmids and cosmids are subject to degradation in vivo more
quickly than viruses and therefore may not deliver an adequate
dosage of ISS-ODN to substantially inhibit ISS-ODN
immunostimulatory activity exerted by a systemically administered
gene therapy vector. Of the viral vector alternatives,
adenoassociated viruses would possess the advantage of low
pathogenicity. The relatively low capacity of adeno-associated
viruses for insertion of foreign genes would pose no problem in
this context due to the relatively small size in which ISS-ODN of
the invention can be synthesized.
[0095] Other viral vectors that can be utilized in the invention
include adenovirus, adeno-associated virus, herpes virus, vaccinia
or an RNA virus such as a retrovirus. Retroviral vectors are
preferably derivatives of a murine, avian or human HIV retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A
number of additional retroviral vectors can incorporate multiple
genes. All of these vectors can transfer or incorporate a gene for
a selectable marker so that transduced cells can be identified and
generated.
[0096] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence that enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines that have deletions of the
packaging signal include, but are not limited to, T2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
helper cells in which the packaging signal is intact, but the
structural genes are replaced by other genes of interest, the
vector can be packaged and vector virion can be produced.
[0097] By inserting one or more sequences of interest into the
viral vector, along with another gene which encodes the ligand for
a receptor on a specific target cell, for example, the vector can
be rendered target specific. Retroviral vectors can be made target
specific by inserting, or example, a polynucleotide encoding a
sugar, a glycolipid, or a protein. Preferred targeting is
accomplished by using an antibody to target the retroviral vector.
Those of skill in the art will know of, or can readily ascertain
without undue experimentation, specific polynucleotide sequences
which can be inserted into the retroviral genome to allow target
specific delivery of the retroviral vector containing ISS-ODN.
C. Pharmaceutical Compositions of ISS-ODN
[0098] If to be delivered without use of a vector or other delivery
system, ISS-ODN will be prepared in a pharmaceutically acceptable
composition. Pharmaceutically acceptable carriers preferred for use
with the ISS-ODN of the invention may include sterile aqueous of
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
antioxidants, chelating agents, and inert gases and the like. A
composition of ISS-ODN may also be lyophilized using means well
known in the art, for subsequent reconstitution and use according
to the invention.
[0099] Absorption promoters, detergents and chemical irritants
(e.g., keratinolytic agents) can enhance transmission of an ISS-ODN
composition into a target tissue. For reference concerning general
principles regarding absorption promoters and detergents which have
been used with success in mucosal delivery of organic and
peptide-based drugs, see Chien, Novel Drug Delivery Systems, Ch. 4
(Marcel Dekker, 1992).
[0100] Examples of suitable nasal absorption promoters in
particular are set forth at Chien, supra at Ch. 5, Tables 2 and 3;
milder agents are preferred. Suitable agents for use in the method
of this invention for mucosal/nasal delivery are also described in
Chang, et al., Nasal Drug Delivery, "Treatise on Controlled Drug
Delivery", Ch. 9 and Table 3-4B thereof, (Marcel Dekker, 1992).
Suitable agents which are known to enhance absorption of drugs
through skin are described in Slean, Use of Solubility Parameters
from Regular Solution Theory to Describe Partitioning-Driven
Processes, Ch. 5, "Prodrugs: Topical and Ocular Drug Delivery"
(Marcel Dekker, 1992), and at places elsewhere in the text. All of
these references are incorporated herein for the sole purpose of
illustrating the level of knowledge and skill in the art concerning
drug delivery techniques.
D. Kits for Use in Practicing the Methods of the Invention
[0101] For use in the methods described above, kits are also
provided by the invention. Such kits may include any or all of the
following: ISS-ODN (conjugated or unconjugated); a pharmaceutically
acceptable carrier (may be pre-mixed with the ISS-ODN) or
suspension base for reconstituting lyophilized ISS-ODN; additional
medicaments; a sterile vial for each ISS-ODN and additional
medicament, or a single vial for mixtures thereof; device(s) for
use in delivering ISS-ODN to a host; assay reagents for detecting
indicia that the anti-inflammatory and/or immunostimulatory effects
sought have been achieved in treated animals and a suitable assay
device.
[0102] Examples illustrating the practice of the invention are set
forth below. The examples are for purposes of reference only and
should not be construed to limit the invention, which is defined by
the appended claims. All abbreviations and terms used in the
examples have their expected and ordinary meaning unless otherwise
specified.
EXAMPLE I
Murine Model for the Airway Hyperreactivity of Allergic Asthma
[0103] Sensitizing-antigen challenged mice of different strains
model the airway hyperreactivity seen in allergic asthma. Suitable
murine strains for use in modeling the disease include Balb/c mice
(which are biased genetically toward the Th2 phenotype and produce
enhanced concentrations of IL-4 and IL-5 in response to antigen
challenge to CD4+ lymphocytes), C57BL/6 mice (which are IL-5
deficient, for detailed study of IL-5 induced tissue damage in
asthma) and W/W.sup.v mice (which are mast cell deficient, for
detailed study of mast cell activation in asthma).
[0104] Disease modeling mice are conveniently prepared by
intraperitoneal or subcutaneous injection of a model sensitizing
antigen, ovalbumin ("OVA") in carrier (e.g., sterile saline or a
carrier with adjuvant, such as alum), followed by antigen challenge
with aerosolized antigen. For example, mice may be immunized with
25 .mu.g OVA by subcutaneous injection (with or without adjuvant)
weekly for 4-6 weeks, then challenged with 2 or 3 weekly
aerosolizations of OVA at a concentration of 50 mg/ml in phosphate
buffered saline (PBS) delivered in 20 minute intervals or at a
concentration of 10 mg/ml 0.9% saline daily for about a week (in
three 30 minute intervals daily). Nebulizer devices for use in the
aerosolization available from Aerotech II, CIS-US, Bedford, Mass.,
with a nasal chamber adapted for murine nasal passages (e.g. a
nose-only chamber from Intox Products, Albuquerque, N. Mex.). When
driven by compressed air it a rate of 10 liters/min., the devices
described produce aerosol particles having a median aerodynamic
diameter of 1.4 .mu.m.
[0105] Control mice are preferably littermates which are
protein-antigen challenged without prior immunization. For further
details concentration this animal model, those of skill in the art
may wish to refer to Foster, et al., J. Exp. Med., 195-101, 1995;
and, Corry, et al., J. Exp. Med., 109-117, 1996.
EXAMPLE II
Reduction of Eosinophil Accumulation in Lung Tissue in a Murine
Asthma Model by Administration of ISS-ODN
[0106] BALB/c mice, 6-10 weeks of age, were prepared as models of
allergic asthma as described in Example I (subcutaneous injection
of OVA followed by antigen challenge at a concentration of 50 mg
OVA/ml PBS). Prior to each inhalation with OVA according to this
scheme, sets of 8 mice each were treated as described in the Table
below. Control mice were antigen challenged but untreated and naive
mice were not challenged with antigen. All ISS doses were 100 .mu.g
per administration. Dexamethasone (a conventional steroidal
anti-inflammatory used in the treatment of asthma) doses were 5
mg/kg/mouse. Priming doses of antigen were 25 .mu.g OVA adsorbed to
alum in 0.2 ml phosphate buffered saline (PBS). Challenge doses of
antigen were 10 ml of 50 mg OVA/ml PBS. 1N=intranasal;
IP=intraperitoneal; SC=subcutaneous and N/A=not applicable.
TABLE-US-00002 Set # Material Received Route and Timing 1 Naive
mice (no antigen) N/A 2 DY1018 (ISS-ODN) IN, 1 day before the first
inhalation 3 DY1018 IN, 1 day before the second inhalation 4 DY1018
IN, with the second inhalation 5 DY1018 IN, 2 days after the second
inhalation 6 DY1018 IP, 1 day before the first inhalation 7 DY1018
IP, 1 day before the second inhalation 8 DY1018 IP, with the second
inhalation 9 DY1018 IP, 2 days after the second inhalation 10
DY1018 IT, 2 days after the second inhalation 11 DY1019 (M-ISS-ODN)
IN, 2 days after inhalation 12 DY1019 IP, 2 days after the second
inhalation 13 DY1019 IT, 2 days after the second inhalation 14
Dexamethansone SC, 2 days after the second inhalation 15
Dexamethansone SC, 7 days after the second inhalation 16 control
mice (antigen only) N/A
[0107] DY1018 has the nucleotide sequence:
5'-TGACTGTGAACGTTCGAGATGA-3' (SEQ. ID. No. 19) with a
phosphothioate backbone and DY1019 has the nucleotide sequence:
[0108] 5'-TGACTGTGAAGGTTGGAGATGA-3' (SEQ. ID. No. 20) with a
phosphothioate backbone.
[0109] On day 32, each mouse was bled by fail snip (approximately
50 .mu.l volume) into a 0.1 mM solution of PBS and EDTA. Red blood
cells in solution were lysed with 150 mM NH.sub.4Cl and 10 mM
KHCO.sub.3 in dH.sub.2O then stained (Wright-Giesma stain). Lung
lavage from each mouse was obtained sacrifice by canalization of
the trachea and lavage with 300 microliters PBS, then the lavage
product was stained. Bone marrow samples from each mouse were
obtained by flushing of extended femur marrow with PBS.
Histological specimens of lung and trachea tissue were obtained
from the right lower lobe of the lung and trachea. Specimens were
frozen, sectioned to a 5 micron width and stained with DAB
peroxidase.
[0110] Results are expressed in the Table below as percent
eosinophils compared to total leukocytes (inflammatory infiltrate)
in each sample, except for the "lung" results, which represent the
number of eosinophils per microscopic field (5 randomly selected
fields were evaluated for each sample). In summary, the control
mice had an average of 67% eosinophils in the lung/trachea tissue
samples, while mice who received the mutant ISS-ODN (M-ISS-ODN;
DY1019) had 52% and 88% (.+-.12%) average accumulation of
eosinophils in lung tissue after IP and IN administration,
respectively. The higher values for the mice treated with
MI-ISS-ODN after antigen challenge is most likely owing to the
immunoinhibitory properties of DY1019 (see, the co-pending,
commonly owned U.S. patent application entitled "Inhibitors of DNA
Immunostimulatory Sequence Activity"; Eyal Raz, inventor; filed
Jun. 6, 1997 (Ser. No. 60/048,793)). Mouse sets 7 and 8 therefore
model an partially immune incompetent host with allergic
asthma.
[0111] In startling contrast, the mice pre-treated with the DY1018
ISS-ODN delivered intranasally had less than about 10% eosinophil
accumulation in the lung and trachea when treated after antigen
challenge and only about 19% eosinophil accumulation when treated
before antigen challenge. These values represent up to an 80%
reduction in eosinophil accumulation compared to the control mice
and more than a 90% reduction in comparison to M-ISS-ODN (IN)
treated mice.
[0112] The IP ISS-ODN treated mice fared even better, with a 6%
eosinophil accumulation in the lung and trachea on treatment before
and after antigen challenge. This value represents an 86% reduction
in eosinophil accumulation as compared to the control mice and an
91% reduction as compared to M-ISS-ODN (IP) treated mice.
[0113] These data indicate that the IL-5 stimulated eosinophil
accumulation in lung tissue which characterizes the late phase of
allergic asthma is inhibited by the ISS-ODN therapeutic methods of
the invention.
TABLE-US-00003 Broncheo- Lung and alveolar Tracheal Set # Bone
Marrow Lavage Blood Tissue 1 (naive) 3 .+-. 2 0 2 .+-. 1 2 .+-. 1 2
(ISS) 5 .+-. 1 10 .+-. 2 3 .+-. 1 8 .+-. 1 3 (ISS) 12 .+-. 1 17
.+-. 4 6 .+-. 2 19 .+-. 5 4 (ISS) 5 .+-. 1 3 .+-. 1 2 .+-. 1 6 .+-.
1 5 (ISS) 8 .+-. 2 4 .+-. 3 3 .+-. 1 6 .+-. 4 6 (ISS) 10 .+-. 1 10
.+-. 1 4 .+-. 1 16 .+-. 4 7 (M-ISS) 13 .+-. 1 51 .+-. 3 10 .+-. 1
88 .+-. 12 8 (M-ISS) 13 .+-. 1 43 .+-. 3 10 .+-. 1 52 .+-. 14 9
(control) 3 .+-. 2 42 .+-. 4 14 .+-. 3 67 .+-. 5
EXAMPLE III
Antigen Dependent Reduction of Eosinophil Accumulation in Lung
Tissue
[0114] To evaluate whether the eosinophil suppression demonstrated
by the data in Example II is dependent upon immune stimulation by
the ISS-ODN, mice were sensitized to OVA using a conventional, Th2
stimulatory adjuvant (alum), treated with ISS-ODN or a control, and
measured for eosinophil suppression before ISS-ODN stimulation of
the mouse immune system would be expected to occur.
[0115] More specifically, groups of four mice were immunized with
25 .mu.g OVA in 1 mg alum by subcutaneous injection on days 1, 7,
14 and 21. This immunization protocol is known to stimulate a Th2
type response to the antigen in mice. On day 27, one group of
animals received 100 .mu.g of the DY1018 ISS-ODN described in
Example I by intraperitoneal administration. A control group
received the mutant DY1019 M-ISS-ODN described in Example I by the
same route.
[0116] On day 28, the animals in each group received 10 mg OVA/ml
phosphate buffered saline by inhalation for 30 minutes. On day 30,
some of the animals in each group received a second injection of
ISS-ODN- or M-ISS-ODN and the animals who had not been treated on
day 27 were treated with ISS-ODIN or M-ISS-ODN. The inhalation
challenge with OVA was repeated on day 31 and the animals were
sacrificed for eosinophil counting within 24 hours.
[0117] The results of this experiment are set forth in the Table
below. Animals that received two treatments with ISS-ODN on days 27
and 30 had only 5.8% eosinophils in the broncheo-alveolar fluid
(BALF) lavage on Day 32, even though immune stimulation by the
ISS-ODN would be minimal so shortly after treatment. Even after
only one treatment with ISS-ODN (on day 30), eosinophil
accumulation in the BALF the treated animals was limited to 10.3%.
In contrast, the control animals twice treated with M-ISS-ODN had
42.3% eosinophils in extracted BALF.
TABLE-US-00004 Treated on Day Animals 28 Blood Bone Marrow BALF
ISS-ODN Yes 1.9% .+-. 0.8 5.8% .+-. 2.5 5.8% .+-. 2.8 M-ISS-ODN Yes
9.8% .+-. 2.1 13.0% .+-. 0.9 42.3% .+-. 3.5 ISS-ODN No 3.5% .+-.
0.6 10.5% .+-. 1.4 10.3% .+-. 1.3
[0118] These data establish that practice of the invention can
inhibit allergic inflammation in animals and that the inhibition
can occur as quickly as one day after treatment.
EXAMPLE IV
Selective induction of a Th1 response in a host After
Administration of an ISS-ODN Containing Plasmid
[0119] In mice, IgG 2A antibodies are serological markers for a Th1
type immune response, whereas IgG 1 antibodies are indicative of a
Th2 type immune response. Th2 responses include the
allergy-associated IgE antibody class; soluble protein antigens
tend to stimulate relatively strong Th2 responses. In contrast, Th1
responses are induced by antigen binding to macrophages and
dendritic cells.
[0120] To determine which response, if any, would be produced by
mice who received ISS-ODN according to the invention, nine groups
of Balb/c mice were immunized with 10 .mu.g .beta.-galactosidase
protein (conjugated to avidin; Sigma, St. Louis, Mo.) to produce a
model allergic phenotype and treated as follows:
TABLE-US-00005 Mouse Group ISS-ODN Treatment 1 None (.beta.-gal) 2
DY1018 (ISS-ODN) injected with the antigen 3 DY1018 injected 72
hrs. after the antigen (same site) 4 DY1019 (M-ISS-ODN) injected
with the antigen 5 DY1019 injected 72 hrs. after the antigen (same
site)
[0121] At 2 week intervals, any IgG 2a and IgG 1 to
(.beta.-galactosidase present in the serum of each mouse were
measured by enzyme-linked immunoabsorbent assay (using antibodies
specific for the IgG 1 and IgG 2A subclasses) on microtiter plates
coated with the enzyme.
[0122] As shown in FIG. 2, only the mice who received the ISS-ODN
produced high titers of IgG 2A antibodies, which increased in
number over a period of 12 weeks. As shown in FIG. 3, immunization
of the mice with the antigen itself or with the mutant ISS-ODN
induced production of relatively high titers of IgG 1 antibodies.
The data shown in the FIGURES comprise averages of the values
obtained from each group of mice.
[0123] These data indicate that a selective Th1 response is induced
by administration of an ISS-ODN according to the invention to an
antigen-challenged host. Further, the data indicate that ISS-ODN
administration according to the invention biases the immune system
toward the Th1 phenotype on antigen challenge, even when the
ISS-ODN are administered before antigen challenge (in this
instance, 72 hours before challenge).
EXAMPLE V
Suppression of IgE Antibody Response to Antigen by Immunization
with Antigen-Encoding Polynucleotides
[0124] To demonstrate the IgE suppression achieved through
stimulation of a Th1 type cellular immune response in preference to
a Th2 type cellular immune response, five to eight week old Balb/c
mice were immunized with one of two recombinant expression vectors:
ISS-ODN containing pCMV-Lac-Z (which contains two copies of
nucleotide sequences similar to the DY1018 ISS-ODN) or a control
plasmid, pCMV-BL. A third group of the mice received injections of
antigen (.beta.-galactosidase). Plasmid DNA was purified and its
endotoxin content reduced to 0.5-5 ng/l mg DNA by extraction with
TRITON X-114 (Sigma, St. Louis, Mich.). Before inoculation, pDNA
was precipitated in ethanol, washed with 70% ethanol and dissolved
in pyrogen free normal saline. Immunization was intradermal
injection of plasmid DNA loaded onto separate tynes of a
MONOVACC.RTM. multiple tyne devices (Connaught Lab, Inc.,
Swiftwater, Pa.). Briefly, the tyne devices were prepared after
extensive washing in DDW and overnight soaking in 0.5% SDS
(sulfated dodecyl saline), washed again in DDW, soaped overnight in
0.1N NaOH, washed again in DDW and dried at 37.degree. C. for 8
hours. Six .mu.l of plasmid DORA dissolved in normal saline were
pipetted onto the tynes of the tyne device just prior to each
inoculation described below. The total amount of pDNA leaded on the
device per inoculation was 25 .mu.g each of pCMV-Lac-Z and pCMV-BL.
For purposes of estimating actual doses, it was assumed that less
than 10% or the pDNA solution loaded onto the tyne device was
actually introduced on injection of the tynes into intradermal
tissue.
[0125] Each mouse was treated 3 times with 2 inoculations of each
plasmid in a one week interval injected intradermally at the base
of the tail. Another group of mice received a single intradermal
injection in the base of the tail of 10 .mu.g of .beta.
galactosidase protein (dissolved in 50 .mu.l of normal saline) in
lieu of pDNA.
[0126] Toward inducing an IgE antibody response to subsequent
sensitizing-antigen challenge, each group of mice was injected once
intraperitoneally with 0.1 ml of phosphate buffered saline (PBS)
solution containing 1 .mu.g of antigen (.beta. galactosidase;
Calbiochem, San Diego, Calif.) and 3 mg of ALUM aluminum hydroxide
as adjuvant (Pierce Chemical, Rockford, Ill.) 14 weeks after the
initial immunization. Total IgE was assayed in sera from the mice 4
times over the subsequent 4 consecutive weeks.
[0127] IgE was detected using a solid phase radioimmunoassay (RAST)
in a 96 well polyvinyl plate (a radioisotopic modification of the
ELISA procedure described in Coligan, "Current Protocols In
Immunology", Unit 7.12.4, Vol. 1, Wiley & Sons, 1994), except
that purified polyclonal goat antibodies specific for mouse
.epsilon. chains were used in lieu of antibodies specific for human
Fab. To detect anti-Lac-Z IgE, the plates were coated with
.beta.-galactosidase (10 .mu.g/ml). The lowest IgE concentration
measurable by the assay employed was 0.4 ng of IgE/ml.
[0128] Measuring specifically the anti-antigen response by each
group of mice, as shown in FIG. 4, anti-Lac-Z IgE levels in the
ISS-ODN containing plasmid injected mice were consistently low both
before and after boosting (averaging about 250 CPM in RAST), while
the protein injected mice developed high levels of anti-Lac-Z,
particularly after the first antigen booster infection, when
anti-Lac-Z levels in the mice rose to an average of about 3000 CPM.
Consistent with acquisition of tolerance, anti-Lac-Z IgE levels in
the protein injected mice declined over time, but continued to rise
in the control mice who had not received an immunization to
.beta.-galactosidase.
[0129] These data show that the ISS-ODN containing plasmid injected
mice developed an antigen specific Th1 response to the plasmid
expression product with concomitant suppression of IgE production,
while tolerance was acquired in the protein injected mice only
after development of substantially higher levels of antigen
specific IgE antibodies.
EXAMPLE VI
IL-4, IL-5, IL-10 and INF.gamma. Levels, And CD4+ Lymphocyte
Proliferation, in Mice after Delivery on ISS-ODN
[0130] BALB/c mice were injected intravenously with 100 .mu.g of
DY1018, DY1019 or a random sequence control (DY1043) then
sacrificed 24 hrs later. Splenocytes were harvested from each
mouse.
[0131] 96 well microtiter plates were coated with anti-CD3 antibody
(Pharmingen, La Jolla, Calif.) at a concentration of 1 .mu.g/ml of
saline. The anti-CD3 antibody stimulates T cells by delivering a
chemical signal which mimics the effects of binding to the T cell
receptor (TCR) complex. The plates were washed and splenocytes
added to each well (4.times.10.sup.5/well) in a medium of RPMI 1640
with 10% fetal calf serum. Supernatants were obtained at days 1, 2
and 3.
[0132] Th2 cytokine (IL-4, IL-5 and IL-10) levels were assayed in
the supernatants using a commercial kit; Th1 cytokine (INF.gamma.)
levels were assayed with an anti-TNF, murine antibody assay (see,
e.g., Coligan, "Current Protocols in Immunology", Unit 6.9.5., Vol.
1, Wiley & Sons, 1994). Relatively high levels of IL-4 and
IL-10 with low levels of INF-.gamma. would be expected in mice with
a Th2 phenotype, while relatively low levels of IL-4 and IL-10 with
high levels of INF-.gamma. would be expected in mice with a Th1
phenotype. Relatively high levels of IL-5 characterize a
proinflammatory milieu, while the converse is true of relatively
low levels of IL-5.
[0133] As shown in FIGS. 5 and 6, levels of anti-CD3 stimulated
IL-4 and IL-10 secretion in DY1018 treated mice were substantially
lower than in the control mice. Levels in the DY1019 mice were
intermediate. Levels of pro-inflammatory IL-5 were reduced in
DY1018 treated mice to a comparable extent (FIG. 7).
[0134] Levels of T cell proliferation in response to antigen
challenge were greatly reduced in DY1018 (ISS-ODN) treated mice as
compared to DY1019 (mutant ISS-ODN) treated and control mice. This
suppression of T cell proliferation was reversible on
administration of IL-2, demonstrating that the suppression was due
to Th2 anergy in the ISS-ODN treated mice (see, Table below).
TABLE-US-00006 Treatment Control (CPM) ISS-ODN (CPM) M-ODN (CPM)
OVA (50 .mu.g/ml) 40680 .+-. 5495 15901 .+-. 4324 42187 .+-. 13012
OVA + IL-2 (1.5 65654 .+-. 17681 42687 .+-. 6329 79546 .+-. 10016
ng/ml) OVA-IL-2 (15 60805 .+-. 19181 57002 .+-. 10658 60293 .+-.
5442 ng/ml)
[0135] Levels of Th1 stimulated IFN-.gamma. secretion were greatly
increased in the DY1018 treated mice, but substantially reduced in
the DY1019 treated mice (as compared to the control), indicating
stimulation of a Th2-type milieu in the latter mice (FIG. 8).
Additional data demonstrating these results are shown in the Table
below. "b/f" in the Table refers to before; "1st", "2nd" and "each"
refer to administration of the compound before the 1st or 2nd
antigen challenge.
[0136] Importantly, treatment of mice before antigen challenge is
even more effective in shifting the immune response on antigen
challenge to a Th1 phenotype than is post-challenge treatment. As
shown in FIGS. 9 and 10, antigen primed (but unchallenged) animals
injected with ISS-ODN DY1019 72 hours before antigen challenge
(with .beta. galactosidase) mounted a more robust Th1-type immune
response to the antigen than did their post-challenge treated
littermates or littermates treated pre-challenge with a mutant,
inactive oligonucleotide (DY1019), as measured by increased
IFN.gamma. secretion (FIG. 9) and CD4+ lymphocyte proliferation
(FIG. 10).
TABLE-US-00007 Set # IL-5(pg/ml) IFN-.gamma.(pg/ml) 1 (naive)
<20 <20 2 (ISS) in b/f 1st 466 .+-. 40 246 .+-. 86 3 (ISS) in
b/f 2nd 531 .+-. 109 168 .+-. 22 4 (ISS) in with 2nd 575 .+-. 90 98
.+-. 44 5 (ISS) in b/f each 200 .+-. 66 443 .+-. 128 6 (ISS) ip;
b/f 1st 190 .+-. 52 664 .+-. 61 7 (ISS) ip; b/f 2nd 421 .+-. 102
252 .+-. 24 8 (ISS) ip; with 2nd 629 .+-. 110 104 .+-. 15 9 (ISS)
ip; b/f each 121 .+-. 18 730 .+-. 99 10 (ISS) it; b/f each 191 .+-.
49 610 .+-. 108 11 (M-ISS) in; b/f each 795 .+-. 138 31 .+-. 22 12
(M-ISS) ip; b/f each 820 .+-. 122 33 .+-. 33 13 (M-ISS) it; b/f
each 657 .+-. 52 102 .+-. 57 14 (steroid) sc; b/f each 424 .+-. 90
<20 15 (steroid) sc; daily 252 .+-. 96 <20 16 (control) not
treated 750 .+-. 124 24 .+-. 21
[0137] Further, ISS-ODN administered according to the invention
suppress Th2 cytokine release from Th2 sensitized mouse cells
(splenocytes harvested from OVA-primed mice, then incubated for 72
hours with 100 .mu.g/ml OVA in vitro). ISS-ODN treatment took place
either 1 (-1) or 3 (-3) days before sacrifice. These data are shown
below:
TABLE-US-00008 Group IL-3 (pg/ml) IL-5 (pg/ml) IFN-.gamma. (pg/ml)
Control 1299 .+-. 89 657 .+-. 52 <20 ISS-ODN (-1) 309 .+-. 26
112 .+-. 18 <20 ISS-ODN (-3) 463 .+-. 48 144 .+-. 27 <20
ISS-ODN (-1) 964 .+-. 81 508 .+-. 77 <20
Sequence CWU 1
1
2016DNAArtificial SequenceRecombinant or Synthetic Sequence 1aacgtt
626DNAArtificial SequenceRecombinant or Synthetic Sequence 2agcgtc
636DNAArtificial SequenceRecombinant or Synthetic Sequence 3gacgtt
646DNAArtificial SequenceRecombinant or Synthetic Sequence 4ggcgtt
656DNAArtificial SequenceRecombinant or Synthetic Sequence 5aacgtc
666DNAArtificial SequenceRecombinant or Synthetic Sequence 6agcgtc
676DNAArtificial SequenceRecombinant or Synthetic Sequence 7gacgtc
686DNAArtificial SequenceRecombinant or Synthetic Sequence 8ggcgtc
696DNAArtificial SequenceRecombinant or Synthetic Sequence 9aacgcc
6106DNAArtificial SequenceRecombinant or Synthetic Sequence
10agcgcc 6116DNAArtificial SequenceRecombinant or Synthetic
Sequence 11gacgcc 6126DNAArtificial SequenceRecombinant or
Synthetic Sequence 12ggcgcc 6136DNAArtificial SequenceRecombinant
or Synthetic Sequence 13agcgct 6146DNAArtificial
SequenceRecombinant or Synthetic Sequence 14gacgct
6156DNAArtificial SequenceRecombinant or Synthetic Sequence
15ggcgct 6166DNAArtificial SequenceRecombinant or Synthetic
Sequence 16ttcgaa 6176DNAArtificial SequenceRecombinant or
Synthetic Sequence 17ggcgtt 6186DNAArtificial SequenceRecombinant
or Synthetic Sequence 18aacgcc 61922DNAArtificial
SequenceRecombinant or Synthetic Sequence with a phosphothioate
backbone 19tgactgtgaa cgttcgagat ga 222022DNAArtificial
SequenceRecombinant or Synthetic Sequence with a phosphothioate
backbone 20tgactgtgaa ggttggagat ga 22
* * * * *