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MODULATION OF TLR-MEDIATED IMMUNE RESPONSES USING ADAPTOR
OLIGONUCLEOTIDES
Related Applications
This application claims priority under 35 U.S.C. 119 from U.S. provisional
application serial number 60/720,981, filed September 27, 2005, entitled
"MODULATION
OF TLR-MEDIATED 1MMUNE RESPONSES USING ADAPTOR
OLIGONUCLEOTIDES", the entire contents of which are incorporated by reference
herein.
Field of the Invention
The invention relates to modulation of TLR-mediated immune responses using
particular nucleic acids.
Backlzround of the Invention
Reaction to certain motifs in bacterial DNA is an important function of
natural
immunity. Bacterial DNA has long been known to be mitogenic for mammalian B
lymphocytes (B cells), whereas mammalian DNA generally is not. The discovery
that this
immune recognition was directed to specific DNA sequences centered on a motif
containing
an unmethylated CpG dinucleotide opened the field to molecular immunologic
approaches.
Krieg AM et al. (1995) Nature 374:546-9. The immunostimulatory effects of so-
called CpG
DNA can be reproduced using synthetic oligodeoxynucleotides (ODN) containing
CpG
dinucleotides in the context of certain preferred flanking sequence, a CpG
motif. CpG-
containing ODN (CpG-ODN) have been reported to exert a number of effects on
various
types of cells of the.immune system, including protecting primary B cells from
apoptosis,
promotion of cell cycle entry, and skewing an immune response toward a Thl-
type immune
response, e.g., induction of interleukin 6 (IL-6), interleukin 12 (IL-12),
gamma interferon
(IFN-y), activation of antigen-specific cytolytic T lymphocytes (CTL), and
induction in the
mouse of IgG2a.
Recently it has been reported that the immunomodulatory effects of CpG DNA
involve signaling by Toll-like receptor 9 (TLR9). It is believed that CpG DNA
is internalized
into a cell via a sequence-nonspecific pathway and traffics to the endosomal
compartment,
where it interacts with TLR9 in a sequence-specific manner. TLR9 signaling
pathways lead
to induction of a number of immune-function related genes, including notably
NF-xB.
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The TLRs are a large family of receptors that recognize specific molecular
structures
that are present in pathogens (pathogen-associated molecular patterns or
PAMPs) and are also
termed pattern recognition receptors (PRRs). Immune cells expressing PRRs are
activated
upon recognition of PAMPs and trigger the generation of optimal adaptive
immune responses.
PRRs consisting of 10 different TLR subtypes, TLRI to TLR10, have been
described. Such
TLRs have been described to be involved in the recognition of double-stranded
RNA (TLR3),
lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), small anti-viral
compounds
(TLR7 and TLR8), and bacterial DNA or CpG ODN (TLR9). (See review by Uhlmann
et al.
(2003) Curr Opin Drug Discov Devel 6:204-17.) In addition, RNA molecules have
been
identified that are believed to interact with and signal through TLR7 and
TLR8. (See
International patent application PCT/US03/10406.) Such immunostinlulatory RNA
molecules are believed to have a base sequence that includes at least one
guanine and at least
one uracil. The immunostimulatory G,U-rich RNA does not require a CpG motif as
described
for TLR9. The corresponding class of RNA molecules found in nature is believed
to be
present in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA),
and
viral RNA (vRNA).
Following the discovery of immunostimulatory CpG DNA, a number of reports
appeared describing short DNA sequences with immunoinhibitory effects. It has
long been
known that poly-G sequences were immunoinhibitory. Published PCT patent
application WO
00/14217 describes ODN containing an inhibitory motif N1N2GN3G in which at
least any two
of Nl, N2, and N3 are G (guanosine). Krieg and colleagues described a group of
inhibitory 15-
mer ODN, having three or four consecutive G, that blocked apoptosis protection
and cell-
cycle entry induced by stimulatory ODN. Lenert P et al. (2001) Antisense
Nucleic Acid Drug
Dev 11:247-56; Stunz LL et al. (2002) Eur Jlmmunol 32:1212-22; Lenert P et al.
(2003)
Antisense Nucleic Acid Drug Dev 13:143-50. The immunoinhibitory effect of
these ODN was
reported to be specific for CpG-ODN and to involve a mechanism other than
simple
competition for cellular uptake. Stunz LL et al. (2002) Eur Jlmmunol 32:1212-
22.
Independently, Klinman and colleagues reported a single immunoinhibitory ODN.
Zeuner
RA et al. (2002) Arthritis Rheum 46:2219-24; Yamada H et al. (2002) Jlmmunol
169:5590-4.
Summary of the Invention
The invention provides methods and compositions for modulating TLR-mediated
immune responses including inhibiting some responses, potentiating other
responses, or some
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combination thereof. The invention is premised in part on the finding that
certain
oligonucleotides are apparently able to change the TLR signaling profile of
TLR ligands.
Thus, these "adaptor" oligonucleotides are able to essentially convert TLR7
ligands into
TLR8 ligands, according to one embodiment, and TLR7/8 ligands into TLR8
ligands,
according to another embodiment.
Thus, in one aspect, the invention provides a method for stimulating a TLR8-
mediated
immune response comprising administering to a subject in need thereof a TLR7/8
ligand and
an adaptor oligonucleotide in an amount effective to stiniulate a TLR8-
mediated immune
response.
In another aspect, the invention provides a method for redirecting a TLR7-
mediated
immune response to a TLR8-mediated immune response comprising administering to
a
subject experiencing a TLR7-mediated immune response an adaptor
oligonucleotide in an
amount effective to redirect a TLR7-mediated immune response to a TLR8-
mediated immune
response.
In yet another aspect, the invention provides a composition comprising a
TLR7/8
ligand and an adaptor oligonucleotide.
In one embodiment, the TLR7/8 ligand is a TLR7 ligand, such as a TLR7 specific
ligand. The TLR7 ligand may be a guanosine analogue such as but not limited to
a C8-
substituted guanosine. The TLR7 ligand may be 3M-001. The TLR7 ligand maybe an
adenosine-based compound such as but not limited to 6-amino-9-benzyl-2-(3-
hydroxy-
propoxy)-9H-purin-8-ol, or 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol. The TLR7
ligand
may be 7-deaza-guanosine.
Examples of C8-substituted guanosine include 7-allyl-7,8-dihydro-8-oxo-
guanosine
(loxoribine), 7-thia-8-oxoguanosine (immunosine, Isatoribine, ANA245, 7-thia-8-
oxo-7,8-
dihydroguanosine, 5-amino-3-(D-D-ribofuranosyl)-3H,6H-thiazol[4,5-d]pyrimidine-
2,7-
dione), 8-mercaptoguanosine, 8-bromoguanosine, 8-methylguanosine, 8-oxo-7,8-
dihydroguanosine, C8-arylamino-2'-deoxyguanosine, C8-propynyl-guanosine, C8-
and N7-
substituted guanine ribonucleosides, 7-methyl-8-oxoguanosine, 8-
aminoguanosine, 8-
hydroxy-2'-deoxyguanosine, 7-deaza-8-substituted guanosine, and 8-
hydroxyguanosine. In
some embodiments, the C8-substituted guanosine is loxoribine, immunosine, or 7-
deaza
guanosine.
In another embodiment, the TLR7/8 ligand is a TLR8 ligand including a TLR8
specific ligand. The TLR8 ligand may be 3M-002. In embodiments in which the
ligand is a
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TLR8 ligand, the TLR8-mediated immune response may be enhanced at least 2-
fold, 3-fold,
4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more over the level of immune
response achieved
in the absence of the adaptor oligonucleotide.
In another embodiment, the TLR7/8 ligand is a TLR7 ligand and a TLR8 ligand.
An
example of such a TLR 7/8 ligand is an imidazoquinoline. Examples of
imidazoquinolines
include imidazoquinoline amine, imidazopyridine amine, 6,7-fused
cycloalkylimidazopyridine amine, 1,2 bridged imidazoquinoline amine, R-848 (S-
28463 or
resiquimod), 4-amino-2ethoxymethyl-a,a-dimethyl-1H-imidazo[4,5-c]quinolines-1 -
ethanol,
1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (R-837 or Imiquimod), or
S-27609.
In some important embodiments, the imidazoquinoline is R848.
The TLR7/8 ligand may be 3M-003. In embodiments in which the ligand is a TLR7
and TLRB ligand, the TLR8-mediated immune response may be enhanced at least 2-
fold, 3-
fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more over the level of TLR8-
mediated
immune response achieved in the absence of the adaptor oligonucleotide.
In one embodiment, the adaptor oligonucleotide comprises the formula 5' X - N5
- X
3' or the formula 5' X - N4 3', wherein X can be any nucleotide and may be
present or absent
and N4 and N5 represent four or five contiguous T(thymidine), U (uracil) or A
(adenine) such
that every N in N4 or N5 is identical. Depending on the embodiment, the
oligonucleotide is 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or more nucleotides in length. In
important embodiments,
it comprises at least one phosphorothioate intemucleotide linkage (up to and
including a
completely phosphorothioated backbone).
In a related embodiment, the adaptor oligonucleotide comprises the formula
5'Xa -
TTTTT - Xb 3', wherein Xa and Xb can independently be anynucleotide and may be
present
or absent. Xa and Xb may be one or more nucleotides (e.g., 1-100 nucleotides).
In one
embodiment, the oligonucleotide comprises 6, 7 or more contiguous T. In one
important
embodiment, the adaptor oligonucleotide is a thymidine (dT) homopolymer, that
is optionally
17 nucleotides in length.
In another embodiment, the adaptor oligonucleotide comprises the formula 5' Xa
-
UUUUU - Xb 3' wherein Xa and Xb can independently be any nucleotide and may be
present
or absent. X. and Xb may be one or more nucleotides (e.g., 1-100 nucleotides).
In one
embodiment, the oligonucleotide may comprise 6, 7 or more contiguous U. In an
important
embodiment, the oligonucleotide is a uracil (dU) homopolymer, that is
optionally 17
nucleotides in length.
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In yet another embodiment, the adaptor oligonucleotide comprises the formula
5' Xa -
AAAAA- Xb 3' wherein Xa and Xb can independently be any nucleotide and may be
present
or absent. Xa and Xb may be one or more nucleotides (e.g., 1-100 nucleotides).
In one
embodiment, the oligonucleotide may comprise 6, 7 or more contiguous A. In an
important
embodiment, the oligonucleotide is an adenine (dA) homopolymer, that is
optionally 17
nucleotides in length.
In still another embodiment, the adaptor oligonucleotide comprises the formula
5' Cn-
Tm - Cp 3', wherein n is an integer ranging from 0-100, p is an integer
ranging from 0-100,
and m is an integer ranging from 0-100. In one embodiment, the sum of n and p
is equal to or
less than the value of m such that C content of the entire oligonucleotide is
50%, less than
50%, less than 45%, less than 40%, less than 35%, less than 30%, less than
25%, less than
20%, less than 15%, less than 10%, less than 5%, or less. In some embodiments,
n ranges
from 3-7, m ranges from 2-10 and p ranges from 4-8, provided the percentages
cited above
are satisfied. Examples include 5'C3TioC4 3'(SEQ ID NO: 1) and 5'C4T8C5 3'
(SEQ ID NO:
2).
The adaptor oligonucleotide may comprise a CpG motif, or it may lack such a
motif.
The motif may be an unmethylated CpG motif.
In one embodiment, the TLR 7/8 ligand and the adaptor oligonucleotide are
administered separately to a subject. In these and other embodiments, the
ligand and
oligonucleotide may still be administered substantially simultaneously with
each other. In
still another embodiment, the oligonucleotide and the TLR7/8 ligand are
conjugated to each
other. In one embodiment, the adaptor oligonucleotide is covalently attached
to the TLR 7/8
ligand.
In one embodiment, the adaptor oligonucleotide is a DNA while in another it is
an
RNA. In one embodiment, the adaptor oligonucleotide is not immunostimulatory
when used
alone. In one embodiment, the adaptor oligonucleotide alone does not stimulate
a TLR7- or
TLR8-mediated immune response.
In one embodiment, the composition further comprises an antigen and/or the
method
further comprises administering an antigen to the subject.
In one embodiment, the subject has an infection. In another embodiment, the
subject
has cancer. In yet another embodiment, the subject has allergy or asthma.
The composition may be a pharmaceutical preparation further comprising a
pharmaceutically acceptable carrier.
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In one aspect, the invention provides a use of the TLR ligand and adaptor
oligonucleotide combination, as described above, for the preparation of a
medicament for
vaccinating a subject.
In one aspect, the invention provides a method for preparing a vaccine. The
method
includes the step of placing the TLR ligand and adaptor oligonucleotide
combination, as
described above, in intimate association with an antigen and, optionally, a
pharmaceutically
acceptable carrier.
In one aspect, the invention provides a use of the TLR ligand and adaptor
oligonucleotide combination, as described above, for the preparation of a
medicament for
treating infection in a subject.
In one aspect, the invention provides a composition useful for the treatment
of
infection that includes the TLR ligand and adaptor oligonucleotide
combination, as described
above, and an anti-microbial agent or medicament.
In one aspect, the invention provides a use of the TLR ligand and adaptor
oligonucleotide combination, as described above, for the preparation of a
medicarnent for
treating cancer in a subject.
In one aspect, the invention provides a composition useful for the treatment
of cancer
that includes the TLR ligand and adaptor oligonucleotide combination, as
described above,
and a cancer medicament.
In one aspect, the invention provides a use of the TLR ligand and adaptor
oligonucleotide combination, as described above, for the preparation of a
medicament for
treating an allergic condition in a subject.
In one aspect, the invention provides a composition useful for the treatment
of an
allergic condition that includes the TLR ligand and adaptor oligonucleotide
combination, as
described above, and an allergy medicament.
In one aspect, the invention provides a use of the TLR ligand and adaptor
oligonucleotide combination, as described above, for the preparation of a
medicament for
treating asthma in a subject.
In one aspect, the invention provides a composition useful for the treatment
of asthma
that includes the TLR ligand and adaptor oligonucleotide combination, as
described above,
and an asthma medicament.
In other aspects, the invention provides screening methods for identifying
TLR7/8
ligands and adaptor oligonucleotides. Thus, in one aspect, the invention
provides a method
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for identifying a TLR8 ligand comprising contacting a TLR8-expressing cell
with a test ligand
in the presence and absence of an adaptor oligonucleotide, and measuring
stimulation of the
TLR8-expressing cell in response to the test ligand in the presence and
absence of the adaptor
oligonucleotide. A TLR8 ligand is identified by an increased stimulation in
the presence of
the adaptor oligonucleotide. The increased stimulation is preferably an
increase over anon-
zero stimulation level in the absence of the adaptor oligonucleotide.
In another aspect, the invention provides a method for identifying a TLR7
ligand
comprising contacting a TLR7-expressing cell and a TLR8-expressing cell with a
test ligand
in the presence and absence of an adaptor oligonucleotide, and measuring
stimulation of the
TLR7-expressing cell and the TLR8-expressing cell in response to the test
ligand in the
presence and absence of the adaptor oligonucleotide. A TLR7 ligand is
identified by a
decreased stimulation in the TLR7-expressing cell and an increased stimulation
in the TLR8-
expressing cell in the presence of the adaptor oligonucleotide. The increased
stimulation, in
this aspect, is preferably an increase over a zero stimulation level in the
absence of the adaptor
oligonucleotide.
In another aspect, the invention provides a method for identifying an adaptor
oligonucleotide comprising contacting a TLR8-expressing cell with a known TLR7
ligand in
the presence and absence of a test adaptor oligonucleotide, and measuring
stimulation of the
TLR8-expressing cell in response to the TLR7 ligand in the presence of the
test adaptor
oligonucleotide. The level of stimulation may be compared to the level in the
absence of the
test oligonucleotide, the level in the absence of the TLR7 ligand and the
presence of the test
oligonucleotide, andlor in the presence of the TLR7 ligand and a known adaptor
oligonucleotide. An adaptor oligonucleotide is identified by increased
stimulation of the
TLR8-expressing cell in its presence as compared to in its absence, in one
embodiment,
provided that the oligonucleotide itself is not mediating a TLR8 -mediated
immune response
(as determined by the control assays mentioned above).
The TLR7 and TLR8 ligands and the adaptor oligonucleotides can be any of those
recited in the proceeding aspects and embodiments. The TLR7- or TLR8-
expressing cells can
be cells that naturally express TLR7 or TLR8 or they may be cells that are
engineered to
express (e.g., ectopically) TLR7 or TLRB. Stimulation may be measured by
signaling through
TLR7 or TLR8 and downstream effects thereof including but not limited to
increased gene
expression (e.g., as visualized using a reporter construct that is conjugated
to the promoter
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elements of a downstream target of TLR7 or TLR8), increased growth factor
(cytokine)
expression, production or secretion, and the like.
These and other aspects and embodiments of the invention will be described in
greater
detail herein.
Brief Description of the Drawings
FIGs. lA-lB show the selective enhancement and inhibition of R-848-mediated
activity on human TLR8 and TLR7 by ODN. FIG. 1A shows the inhibition of R-848
activity
on human TLR7 by ODN 6056 (SEQ ID NO: 3) and 1982 (SEQ ID NO: 4) (for
sequences see
Tables 1 and 2). HEK293 cells stably expressing TLR7 and an NF-KB luciferase
reporter
construct were incubated with 2 p,M R-848 in the presence or absence of
indicated amounts of
the oligo(dT)17 homopolymer ODN 6056, or the heteropolymer ODN 1982. Activity
of R-
848 alone was set to 100%. All data points were assayed in triplicate and mean
(+/- SD) is
displayed. Results shown are one out of more than two independent experiments.
FIG. 1B
shows the sequence-selective enhancement of R-848 activity on human TLR8 by an
oligo(dT)17 ODN. HEK293 cells stably expressing hTLRB and a NF-xB-luciferase
reporter
construct were incubated with increasing amounts of R-848 in the absence or
presence of 0.1,
1 and 5 M of the oligo(dT)17 homopolymer ODN 6056, or the heteropolymer ODN
1982.
Stimulation of NF-xB activation was calculated in reference to medium
background. All data
points were assayed in triplicate and mean ( SD) is displayed. Results shown
are one out of
four independent experiments.
FIGs. 2A-C show the altered target specificity of the TLR7 ligands, loxoribine
and 7-
deaza-guanosine, when combined with an oligo(dT)17 ODN. In FIG. 2A, HEK293
cells
stably expressing hTLR7, hTLR8 or hTLR9 were incubated with increasing amounts
of
loxoribine. NF-xB activation was measured by assaying luciferase activity 16h
later and
displayed as fold stimulation above medium background. In FIG. 2B, HEK293
cells
expressing hTLR8 were incubated with increasing amounts of loxoribine in the
absence or
presence of the indicated concentrations of the homopolymer ODN 6056 or the
heteropolymer
ODN 1982. NF-xB activation is given as fold induction above medium background.
In FIG.
2C, increasing amounts of 7-deaza-guanosine (left panel) and inosine (right
panel) were
assayed on hTLR8 expressing HEK293 cells in the absence (filled circles) or
presence (open
circles) of 5 M ODN 6056 and NF-xB stimulation above medium background was
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calculated. All data points were assayed in triplicate. Shown are data from
one out of three
experiments.
FIGs. 3A-3B show that ODN 6056 inhibits TLR7-mediated NF-xB activation in
HEK293 cells. HEK293 cells stably expressing hTLR7 and a NF-xB-luciferase
reporter
construct were incubated for 16h with 2.5 mM loxoribine (FIG. 3A) or 2 M R-
848 (FIG. 3B)
in the presence or absence of indicated concentrations of ODN 6056.
Stimulation of NF-xB-
activation was calculated in reference to medium background. All data points
were assayed in
triplicate and mean ( SD) is displayed. Results shown are from one out of two
independent
experiments.
FIGs. 4A-4D show redirection of loxoribine-induced cytokine production by
specific
ODN. Human PBMC were incubated with 1 mM loxoribine in the absence or separate
presence of increasing amounts of ODN 6056 and ODN 1982. Supernatants were
collected
24h later and amounts of IFN-a (FIG. 4A), IL-12p40 (FIG. 4B), IFN-y (FIG. 4C)
and TNF-a
(FIG. 4D) were measured by ELISA. Values represent mean of 3 donors ( SEM).
The data
are from one representative experiment out of four experiments.
FIGs. 5A-5F show stimulation of human monocytes to produce IL-12p40 and TNF-a
upon coculture with loxoribine and oligo(dT)17 ODN. Human PBMC were incubated
with
indicated amounts of loxoribine and ODN. Intracellular staining was performed
using
anti-.IL-12p40/p70 (FIG. 5A) and anti-TNF-a (FIG. 5B) antibodies. Cells were
stained
simultaneously with anti-CD 14 and anti-CD 19 antibodies. Cells were gated for
CD 14-
positive cells and percentage of IL-12p40/p70- or TNF-a-positive cells was
calculated.
Results show mean of three donors ( - SEM). FIGs. 5C-5F display representative
flow
cytometry dot blots. Shown is one out of two independent experiments.
FIGs. 6A-6L show stimulation of human NK cells to produce IFN-y upon co-
culture
with loxoribine and oligo(dT)17 ODN 6056. Human PBMC were incubated with
indicated
amounts of loxoribine and ODN. Intracellular staining was performed using anti-
IFN-y
antibodies. Cells were stained with anti-CD56 and anti-CD3 antibodies. Cells
were gated for
CD56-positive/ CD3 negative cells (NK cells). Percentage of IFN-y-positive
cells was
calculated and is displayed within the flow cytometry dot blots. Shown are two
representative
donors out of five.
FIGs. 7A and 7B show influence of co-incubation of loxoribine with ODN on
cytokine production from isolated cell populations. In FIG. 7A, monocytes were
isolated
from human PBMC (96% purity) using CD14 mAb and incubated with indicated
amounts of
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loxoribine in the presence or absence of ODN. The amount of IL-12p40 was
determined in
the supematants by ELISA. Data display results for two individual donors (gray
and black
bars). Shown is one out of 3 similar experiments. In FIG. 7B, PDC were
enriched (85%
purity) from human PBMC of two donors (gray and black bars) using BDCA-4 mAb
and
incubated with indicated combinations of loxoribine and ODN for 24h. IFN-a was
detected
in the supematants by ELISA. Shown are the data from one out of three similar
experiments.
FIG. 8A shows the effect on hTLR8-LUC-293 cells incubated with 50 M R-848 in
the presence of indicated amounts of ODN for 16h. NF-xB stimulation was
measured by
assaying luciferase activity. NF-xB activity induced by 50 M R-848 alone was
set to 100%.
FIG. 8B shows hTLR8-LUC-293 cells incubated with increasing concentrations of
R-848 in
the presence of 1 M of indicated ODN for 16h. NF-icB stimulation was measured
by
assaying luciferase activity.
FIGs. 9A and 9B show TLR7 and TLR8 signaling using a combination of the TLR7
specific ligand 6-amino-9 benzyl-2-butoxy-9H-purin-8-ol and ODN 6056. FIG. 9A
shows
the effects of the combination on TLR7 signaling. FIG. 9B shows the effects of
the
combination on TLR8 signaling.
FIG. 10 shows the effect on TLR8 signaling of two different RNAs. The poly(rU)
18
(SEQ ID NO: 5) stimulates TLR8 in the absence of a TLR7/8 ligand. The mixed
RNA oligo
(SEQ ID NO: 16) does not stimulate TLR8 alone.
FIG. 11 shows the effects of two RNAs on R-848 TLR8 signaling, in the presence
of a
TLR7/8 ligand.
FIG. 12 shows the TLR8-mediated effects of ODN 6056 on TLR7 ligands
(loxoribine,
immunosine), a ligand that is both a TLR7 and a TLR8 ligand (R-848), and a
compound that
is not a TLR7/8 ligand (ribavirin).
FIGs. 13A-D show the structures of immunosine (A), two immunosine
variants/monomers (B and C) that can be conjugated to an oligonucleotide at a
3' end or
internally (B) or the 5' end (C), and 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol
(D).
FIG. 14 shows the structure of an immunosine-oligonucleotide conjugate
comprising a
linker.
I FIGs. 15A-C show the structures of R-848 (A), CL-029 (B) and immunosine (C),
and
points at which linkers and/or oligonucleotides may be attached.
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Detailed Description of the Invention
The invention relates in part to the discovery that certain oligonucleotides
are able to
modulate TLR-mediated immune responses. As used herein, modulating a TLR-
mediated
immune response refers to the ability to manipulate signaling of one or more
TLRs. For
example, it is possible according to the invention to change the TLR profile
of particular TLR
ligands such that in the presence of certain oligonucleotides these ligands
signal through a
different TLR. As another example, in the presence of certain oligonucleotides
TLR ligands
known to signal through more than one TLR demonstrate signaling through only
one TLR,
and in many instances such signaling is enhanced. In still another example, in
the presence of
certain oligonucleotides the efficacy and potency of TLR signaling is
increased significantly.
Thus, the invention makes it possible to selectively inhibit and/or enhance
signaling by any
one of or by any combination of TLR7, TLR8, and TLR9.
The invention is premised in part on the observation that co-incubation of the
imidazoquinoline derivative Resiquimod (R-848), which by itself activates both
TLR7 and
TLR8, with an oligo(dT)17 homopolymer oligonucleotide (ODN 6056) increased
activity of R-
848 on TLR8-expressing HEK 293 cells significantly, whereas TLR7-mediated
signaling was
abolished. Similarly, the combination of the guanosine analogue loxoribine,
which by itself
activates TLR7, and the oligo(dT)17 ODN 6056 induced TLR8-mediated signaling
in a
sequence-selective fashion, and abolished TLR7-mediated signaling.
The cytokine profile induced by loxoribine in human immune cells was also
altered by
co-incubation with the oligo(dT)17. IL-12, TNF-a or IFN-y were highly
secreted, but IFN-a
production was abrogated. Although not intending to be bound by any particular
mechanism,
it is presumed that the observed alteration in induced cytokines is due to a
shift in the cell
types being activated from plasmacytoid DC by loxoribine alone, to monocytes
by its
combination with ODN and indicates that monocytes do not express functional
TLR7.
The invention is therefore useful for modulating a range of immune responses.
The
invention in a specific embodiment is useful in inhibiting TLR7 signaling (and
the associated
TLR7-mediated immune response), optionally while inducing TLR8 signaling (and
the
associated TLRB-mediated immune response). In another specific embodiment, the
invention
provides for induction (including enhancement) of TLR8 signaling (and the
associated TLR8-
mediated immune response). Depending on the nature of the adaptor
oligonucleotide, it is
further possible to induce TLR9 signaling at the same time (and the associated
TLR9-
mediated immune response). For example, a combination of an adaptor
oligonucleotide that
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comprises a CpG motif and a TLR7 ligand such as loxoribine would induce TLR8
and TLR9
signaling (and their associated immune responses).
The invention is also usef-ul for treating a range of conditions that would
benefit from
inhibition of TLR7-mediated immune responses and/or induction (including
enhancement) of
TLR8-mediated immune responses, optionally in the absence or presence of TLR9-
mediated
immune responses.
Conditions that would benefit from inhibition of TLR7-mediated immune
responses
include autoimmune conditions such as but not limited to lupus and rheumatoid
arthritis,
particularly those associated with infection by a DNA virus. Inhibition of a
TLR7-mediated
immune response with concomitant induction of a TLR8-mediated immune response
can be
used where it is desirable to enhance a T cell response that is being
suppressed by T
regulatory cells. Such conditions include, but are not limited to, some forms
of cancer, as
well as HCV, HBV and HIV infection. Combined TLR7 inhibition and TLR8
induction
could also be beneficial in treating autoimmune disease or where it is
desirable to induce an
immune response but avoid TLR7 associated toxicity. Induction (including
enhancement) of
a TLR8-mediated immune response, independent of modulation of a TLR7-mediated
immune
response, is useful in, inter alia, the treatment of cancer and infections, in
a vaccine or non-
vaccine setting.
The invention is also useful in the treatment of conditions that would benefit
from
shifts in cytokine production, or where a subject is or becomes refractory to
the effect of
certain cytokines (e.g., IFN-a, as is sometimes observed in the treatment of
viral infections).
TLRs
Toll-like receptors (TLRs) are a family of highly conserved polypeptides that
play a
critical role in innate immunity in mammals. Currently ten family members,
designated
TLR1 - TLR1O, have been identified. The cytoplasmic domains of the various
TLRs are
characterized by a Toll-interleukin 1(IL-1) receptor (TIR) domain. Medzhitov R
et al. (1998)
Mol Cell 2:253-8. Recognition of microbial invasion by TLRs triggers
activation of a
signaling cascade that is evolutionarily conserved in Drosophila and mammals.
The TIR
domain-containing adaptor protein MyD88 has been reported to associate with
TLRs and to
recruit IL-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF)
receptor-
associated factor 6 (TRAF6) to the TLRs. The MyD88-dependent signaling pathway
is
believed to lead to activation of NF-xB transcription factors and c-Jun NH2
terminal kinase
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(Jnk) mitogen-activated protein kinases (MAPKs), critical steps in immune
activation and
production of inflammatory cytokines. For a review, see Aderem A et al. (2000)
Nature
406:782-87.
TLRs are believed to be differentially expressed in various tissues and on
various
types of immune cells. For example, human TLR7 has been reported to be
expressed in
placenta, lung, spleen, lymph nodes, tonsil and on plasmacytoid precursor
dendritic cells
(pDCs). Chuang T-H et al. (2000) Eur Cytokine Netw 11:372-8); Kadowaki N et
al. (2001) J
Exp Med 194:863-9. Human TLR8 has been reported to be expressed in lung,
peripheral
blood leukocytes (PBL), placenta, spleen, lymph nodes, and on monocytes.
Kadowaki N et
al. (2001) JExp Med 194:863-9; Chuang T-H et al. (2000) Eur Cytokitae Netw
11:372-8.
Human TLR9 is reportedly expressed in spleen, lymph nodes, bone marrow, PBL,
and on
pDCs and B cells. Kadowaki N et al. (2001) JExp Med 194:863-9; Bauer S et al.
(2001)
Proc Natl Acad Sci USA 98:9237-42; Chuang T-H et al. (2000) Eur Cytokine Netw
11:372-8.
Nucleotide and amino acid sequences of human and murine TLR7 are known. See,
for example, GenBank Accession Nos. AF240467, AF245702, NM 016562, AF334942,
NM 133211; and AAF60188, AAF78035, NP 057646, AAL73191, and AAL73192, the
contents of all of which are incorporated herein by reference. Human TLR7 is
reported to be
1049 amino acids long. Murine TLR7 is reported to be 1050 amino acids long.
TLR7
polypeptides include an extracellular domain having a leucine-rich repeat
region, a
transmembrane domain, and an intracellular domain that includes a TIR domain.
Nucleotide and amino acid sequences of human and murine TLR8 are known. See,
for example, GenBank Accession Nos. AF246971, AF245703, NM 016610, XM 045706,
AY035890, NM 133212; and AAF64061, AAF78036, NP_057694, XP~045706, AAK62677,
and NP_573475, the contents of all of which are incorporated herein by
reference. Human
TLR8 is reported to exist in at least two isoforms, one that is 1041 amino
acids long and
another that is 1059 amino acids long. Murine TLR8 is 1032 amino acids long.
TLR8
polypeptides include an extracellular domain having a leucine-rich repeat
region, a
transmembrane domain, and an intracellular domain that includes a TIR domain.
Nucleotide and amino acid sequences of human and murine TLR9 are known. See,
for example, GenBank Accession Nos. NM_017442, AF259262, AB045180, AF245704,
AB045181, AF348140, AF314224, NM_031178; and NP_059138, AAF72189, BAB19259,
AAF78037, BAB 19260, AAK29625, AAK28488, and NP_112455, the contents of all of
which are incorporated herein by reference. Human TLR9 is reported to exist in
at least two
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isoforms, one that is 1032 amino acids long and another that is 1055 amino
acids. Murine
TLR9 is 1032 amino acids long. TLR9 polypeptides include an extracellular
domain having a
leucine-rich repeat region, a transmembrane domain, and an intracellular
domain that includes
a TIR domain.
TLR-mediated immune responses
As used herein, the term "TLR signaling" refers to any aspect of intracellular
signaling
associated with signaling through a TLR. As used herein, the term "TLR-
mediated immune
response" refers to the immune response that is associated with (e.g., is the
result of) TLR
signaling.
A TLR7-mediated immune response is a response associated with TLR7 signaling.
TLR7-mediated immune response is generally characterized by the induction of
IFN-a and
IFN-inducible cytokines such as IP-10 and I-TAC. The levels of cytokines IL-1
a/(3, IL-6, IL-
8, MIP-la/f3 and MIP-3a/0 induced in a TLR7-mediated immune response are less
than those
induced in a TLR8-mediated immune response.
A TLR8-mediated immune response is a response associated with TLR8 signaling.
This response is characterized by the induction of pro-inflammatory cytokines
such as IFN-y,
IL-12p40/70, TNF-a, IL-1a/(3, IL-6, IL-8, MIP-1 a/(3 and MIP-3 a/(3.
A TLR9-mediated immune response is a response associated with TLR9 signaling.
This response is characterized at least by the production and/or secretion of
IFN-y and IL-12,
albeit at levels lower than are achieved via a TLR8-mediated immune response.
TLR 7/8 ligand
As used herein, a "TLR7/8 ligand" collectively refers to any agent that is
capable of
increasing TLR7 and/or TLR8 signaling (i.e., an agonist of TLR7 and/or TLR8).
Thus, when
used in the absence of the adaptor oligonucleotides of the invention, some
TLR7/8 ligands
induce TLR7 signaling alone (e.g., TLR7 specific ligands), some induce TLR8
signaling
alone (e.g., TLR8 specific ligands), and others induce both TLR7 and TLR8
signaling. It has
been found according to the invention that when such ligands are combined with
adaptor
oligonucleotides, the TLR signaling profile of these ligands is altered. In
some instances,
TLR7 signaling is inhibited and TLR8 signaling is induced. In other instances,
TLR8
signaling is induced (i.e., increased from a background level) or enhanced
(i.e., increased
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from a non-background level), and in still other embodiments, both TLR8 and
TLR9 signaling
are induced or enhanced.
As used herein, the term "TLR7 ligand" refers to any agent that is capable of
increasing TLR7 signaling (i.e., an agonist of TLR7). In this respect, the
level of TLR7
signaling may be enhanced over a pre-existing level of signaling or it may be
induced over a
background level of signaling. TLR7 ligands include, without limitation,
guanosine
analogues such as C8-substituted guanosines, mixtures of ribonucleosides
consisting
essentially of G and U, guanosine ribonucleotides and RNA or RNA-like
molecules
(PCTlCTS03l10406), and adenosine-based compounds (e.g., 6-amino-9-benzyl-2-(3-
hydroxy-
propoxy)-9H-purin-8-ol (CL-029, Sumitomo), 6-amino-9-benzyl-2-butoxy-9H-purin-
8-ol
(shown in FIG. 13D), and other related compounds such as those described in US
6310070
B1). TLR7 ligands are also disclosed in Gorden et al. J. Itnmunol. 2005,
174:1259-1268 (e.g.,
3M-001, N-[4-(4-amino-2-ethyl-lH-imidazo[4,5-c]quinolin-1-yl)butyl-
]methanesulfonamide;
C17H23N502S; mw 361).
As used herein, the term "guanosine analogues" refers to a guanosine-like
nucleotides
(excluding guanosine) having a chemical modification involving the guanine
base, guanosine
nucleoside sugar, or both the guanine base and the guanosine nucleoside sugar.
Guanosine
analogues specifically include, without limitation, 7-deaza-guanosine.
Guanosine analogues further include C8-substituted guanosines such as 7-thia-8-
oxoguanosine (immunosine), 8-mercaptoguanosine, 8-bromoguanosine, 8-
methylguanosine,
8-oxo-7,8-dihydroguanosine, C8-arylamino-2'-deoxyguanosine, C8-propynyl-
guanosine, C8-
and N7- substituted guanine ribonucleosides such as 7-allyl-8-oxoguanosine
(loxoribine) and
7-methyl-8-oxoguanosine, 8-aminoguanosine, 8-hydroxy-2'-deoxyguanosine, 8-
hydroxyguanosine, and 7-deaza 8-substituted guanosine.
As used herein, the term "TLR8 ligand" refers to any agent that is capable of
increasing TLR8 signaling (i.e., an agonist of TLR8). In this respect, the
level of TLR8
signaling may be enhanced over a pre-existing level of signaling or it may be
induced over a
background level of signaling. TLR8 ligands include mixtures of
ribonucleosides consisting
essentially of G and U, guanosine ribonucleotides and RNA or RNA-like
molecules
(PCT/US03/10406). Additional TLR8 ligands are also disclosed in Gorden et al.
J. Immunol.
2005, 174:1259-1268 (e.g., 3M-002, 2-propylthiazolo[4,5-c]quinolin-4-amine;
C13H13N3S;
mw 243).
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Some TLR7/8 ligands are ligands of botll TLR7 and TLR8. These include
imidazoquinolines, mixtures of ribonucleosides consisting essentially of G and
U, guanosine
ribonucleotides, and RNA or RNA-like molecules (PCT/US03/10406). Additional
TLR7/8
ligands are also disclosed in Gorden et al. J. Immunol. 2005, 174:1259-1268
(e.g., 3M-003, 4-
amino-2-(ethoxymethyl)-a, a-dimethyl-6, 7, 8, 9-tetrahydro-1 H-imidazo [4, 5-
c] quino line-l-
ethanol hydrate; C17H26N402; mw 318).
Imidazoquinolines are immune response modifiers thought to induce expression
of
several cytokines including interferons (e.g., IFN-a), TNF-a and some
interleukins (e.g., IL-1,
IL-6 and IL-12). Imidazoquinolines are capable of stimulating a Thl immune
response, as
evidenced in part by their ability to induce increases in IgG2a levels.
Imidazoquinoline
agents reportedly are also capable of inhibiting production of Th2 cytokines
such as IL-4, IL-
5, and IL-13. Some of the cytokines induced by imidazoquinolines are produced
by
macrophages and dendritic cells. Some species of imidazoquinolines have been
reported to
increase NK cell lytic activity and to stimulate B cell proliferation and
differentiation, thereby
inducing antibody production and secretion.
As used herein, imidazoquinolines include imidazoquinoline amines,
imidazopyridine
amines, 6,7-fused cycloalkylimidazopyridine anlines, and 1,2 bridged
imidazoquinoline
amines. These compounds have been described in U.S. Patent Nos. 4689338,
4929624,
5238944, 5266575, 5268376, 5346905, 5352784, 5389640, 5395937, 5494916,
5482936,
5525612, 6039969 and 6110929. Particular species of iniidazoquinolines include
R-848 (S-
28463); 4-amino-2ethoxymethyl-a,a-dimethyl-lH-imidazo[4,5-c]quinolines-l-
ethanol; 1-(2-
methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (R-837 or Imiquimod), and S-
27609.
Imiquimod is currently used in the topical treatment of warts such as genital
and anal warts
and has also been tested in the topical treatment of basal cell carcinoma.
As used herein, the term "TLR9 ligand" refers to any agent that is capable of
increasing TLR9 signaling (i.e., an agonist of TLR9). TLR9 ligands
specifically include,
without limitation, immunostimulatory nucleic acids, and in particular CpG
immunostimulatory nucleic acids.
As used herein, the term "immunostimulatory CpG nucleic acids" refers to any
CpG-
containing nucleic acid that is capable of activating an immune cell. At least
the C of the
CpG dinucleotide is typically, but not necessarily, unmethylated.
Immunostimulatory CpG
nucleic acids are described in a number of issued patents and published patent
applications,
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includingU.S. Pat. Nos. 6,194,388; 6,207,646; 6,218,371; 6,239,116; 6,339,068;
6,406,705;
and 6,429,199.
In some embodiments, the TLR ligand is a specific ligand. As used herein, a
TLR7
specific ligand is one that, when used in the absence of the adaptor
oligonucleotides of the
invention, signals through TLR7 but not TLR8 or TLR9. Similarly, a TLR8
specific ligand is
one that, when used in the absence of the adaptor oligonucleotides of the
invention, signals
through TLR8 but not TLR7 or TLR9. Preferably, a TLR7 specific ligand signals
through
TLR7 and no other TLRs, when used in the absence of the adaptor
oligonucleotide, and a
TLR8 specific ligand signals through TLR8 and no other TLRs, when used in the
absence of
the adaptor oligonucleotide.
In some embodiments, the TLR ligand is not an RNA.
The use of TLR7/8 ligands with adaptor oligonucleotides may stimulate both a
TLRB
and a TLR9, resulting in downstream effects from both receptors concurrently.
For example,
stimulation of the TLR9 by the oligonucleotide may result in for example IFN-
alpha
production while stimulation of TLR8 by the oligonucleotide may result in for
example IL-12,
TNF-alpha and IFN-gamma production. The ligands and oligonucleotides may be
conjugated
to each other or may be physically separate from each other.
Adaptor oligonucleotides
According to the invention, TLR7/8 ligands are used in combination with
adaptor
oligonucleotides. An adaptor oligonucleotide, as used herein, is an
oligonucleotide which
when used with a TLR7/8 ligand modulates the activity of that ligand by
inhibiting TLR7
signaling by a TLR7 ligand, inducing TLRB signaling by a TLR7 ligand, and/or
enhancing
TLRB signaling by a ligand that is both a TLR7 and a TLR8 ligand.
As used herein, the term oligonucleotide is used interchangeably with the term
nucleic
acid. Preferably, the term excludes plasmids, vectors and/or anti-sense
nucleic acids. In some
embodiments, the oligonucleotide may be immunostimulatory while in other
embodiments it
may be non-immunostimulatory when used alone. The oligonucleotides may be any
length
but preferably are 7 or 8 up to 100 nucleotides in length.
One broad class of adaptor oligonucleotides comprises the formula 5' X - N4 -
X 3' or
5' X - N5 - X 3' wherein X can be any nucleotide and may be present or absent
and N4 and N5
represents four and five contiguous T (thymidine), U (uracil), or A (adenine)
such that every
N in N4 or N5 is identical. The oligonucleotide may be 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17
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or more nucleotides in length. It preferably comprises at least one
phosphorothioate
internucleotide linkage (up to and including a completely phosphorothioated
backbone).
Thus, one class of adaptor oligonucleotides comprises the formula 5'Xa - TTTTT
- Xb
3', wherein Xa and Xb can independently be any nucleotide and may be present
or absent. Xa
and Xb may represent one or more nucleotides (e.g., 1-100 nucleotides). The
oligonucleotide
may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more
nucleotides in
length. The oligonucleotide may comprise 6, 7 or more contiguous T.
Preferably, the adaptor
oligonucleotide is a dT homopolymer (i.e., oligo dT of a length recited
herein). Even more
preferably, the adaptor oligonucleotide is a thymidine (dT) homopolymer 17
nucleotides in
length. Most preferably, it comprises at least one phosphorothioated
internucleotide linkage
(up to and including a completely phosphorothioated backbone).
The adaptor oligonucleotide may be comprised of 100% T, 99% T, 98% T, 97% T,
96% T, 95% T, 94% T, 93% T, 92% T, 91% T, 90% T, 85% T, 80% T, 75% T, 70% T,
65%
T, 60% T, 55% T, 50% T, 45% T or less, depending on the embodiment.
Another class of adaptor oligonucleotides comprises the formula 5' Xa - UUUUU -
Xb
3' wherein Xa and Xb can independently be any nucleotide and may be present or
absent. Xa
and Xb may represent one or more nucleotides (e.g., 1-100 nucleotides). The
oligonucleotide
may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more
nucleotides in
length. The oligonucleotide may comprise 6, 7 or more contiguous U. In an
important
embodiment, the oligonucleotide is a dU homopolymer that is preferably 17
nucleotides in
length and having at least one phosphorothioated intemucleotide linkage (up to
and including
a completely phosphorothioated backbone).
The adaptor oligonucleotide may be comprised of 100% U, 99% U, 98% U, 97% U,
96% U, 95% U, 94% U, 93% U, 92% U, 91% U, 90% U, 85% U, 80% U, 75% U, 70% U,
65% U, 60% U, 55% U, 50% U, 45% U or less, depending on the embodiment.
Yet, another class of adaptor oligonucleotides comprises the formula 5' Xa -
AAAAA-
Xb 3' wherein Xa and Xb can independently be any nucleotide and may be present
or absent.
X. and Xb may represent one or more nucleotides (e.g., 1-100 nucleotides). The
oligonucleotide may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or more
nucleotides in length. The oligonucleotide may comprise 6, 7 or more
contiguous A. In an
important embodiment, the oligonucleotide is a dA homopolymer that is
preferably 17
nucleotides in length and having at least one phosphorothioated
internucleotide linkage (up to
and including a completely phosphorothioated backbone).
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The adaptor oligonucleotide may be comprised of 100% A, 99% A, 98% A, 97% A,
96% A, 95% A, 94% A, 93% A, 92% A, 91% A, 90% A, 85% A, 80% A, 75% A, 70% A,
65% A, 60% A, 55% A, 50% A, 45% A or less, depending on the embodiment.
Another class of adaptor oligonucleotides comprises the formula 5' Cn - Tm -
Cp 3'a
wherein n is an integer ranging from 0-100 (e.g., 3-7), p is an integer
ranging from 0-100
(e.g., 4-8), and m is an integer ranging from 0-100 (e.g., 2-10). Preferably,
the sum of n and p
is equal to or less than the value of m such that C content is 50%, less than
50%, less than
45%, less than 40%, less than 35%, less than 30%, less than 25%, less than
20%, less than
15%, less than 10%, less than 5%, or less. In some embodiments, n ranges from
3-7, m
ranges from 2-10 and p ranges from 4-8, provided the percentages cited above
are satisfied.
Some species of this formula are shown in FIG. 8. Examples include 5'C3T10C4
3' (SEQ ID
NO: 1), 5'C4T$C5 3' (SEQ ID NO: 2), 5' C5T6C6 3' (SEQ ID NO: 6), 5' C6T4C7 3'
(SEQ ID
NO: 7), and 5' C7T2C8 3' (SEQ ID NO: 8).
In some embodiments, the adaptor oligonucleotides comprise an
immunostimulatory
CpG motif. The motif may be methylated or unmethylated. In the latter
instance, the entire
oligonucleotide can be unmethylated or portions may be unmethylated but at
least the C of the
5' CG 3' must be unmethylated. The unmethylated motif and its effects on
immune
modulation have been described extensively in U.S. Patents such as US
6,194,388 B1; US
6,207,646 B l; US 6,239,116 B 1; and US 6,218,371 B 1; and published patent
applications,
such as PCT/tJS98/03678, PCT/US98/10408, PCT/US98/04703, and PCT/US99/09863.
The
entire contents of each of these patents and patent applications is hereby
incorporated by
reference.
As stated above, the terms "oligonucleotide" and "nucleic acid" are used
interchangeably to mean multiple nucleotides (i.e., molecules comprising a
sugar (e.g., ribose
or deoxyribose) linked to a phosphate group and to an exchangeable organic
base, which is
either a pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a
purine (e.g., adenine
(A) or guanine (G)). Thus, the term embraces both DNA and RNA
oligonucleotides. The
terms shall also include polynucleosides (i.e., a polynucleotide minus the
phosphate) and any
other organic base containing polymer. Oligonucleotides can be obtained from
existing
nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic
(e.g., produced by
a nucleic acid synthesizer).
The oligonucleotides can be double-stranded or single-stranded. In certain
embodiments, when the oligonucleotide is single stranded, it is capable of
forming secondary
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and tertiary structures (e.g., by folding back on itself, or by hybridizing
with itself either
throughout its entirety or at select segments along its length). Accordingly,
while the primary
structure of such an oligonucleotide may be single stranded, its higher order
structures may be
double or triple stranded.
The oligonucleotides may range in length but they are preferably equal to or
less than
100 nucleotides (or bases) in length. The adaptor oligonucleotides are
preferably not
plasmids or vectors. They are also preferably not capable of anti-sense
activity, or at least the
effects manifest according to the invention are not related to any anti-sense
activity.
The oligonucleotides may have modified backbones. For example, they may
comprise
at least one intemucleotide linkage which is not a phosphodiester linkage.
Such a linkage
may be a phosphorothioate linkage. In some embodiments, the oligonucleotides
may have
chiineric backbones (i.e., backbones comprised of at least two different
typesof
internucleotide linkages).
As used herein, the term "phosphorothioate backbone" refers to a stabilized
sugar
phosphate backbone of an oligonucleotide in which a non-bridging phosphate
oxygen is
replaced by sulfur at at least one internucleotide linkage. In one enibodiment
a non-bridging
phosphate oxygen is replaced by sulfur at each and every intemucleotide
linkage.
Source and Preparation of Oligonucleotides
The oligonucleotides of the instant invention can encompass various chemical
modifications and substitutions, in comparison to natural RNA and DNA,
involving a
phosphodiester intemucleoside bridge, a(3-D-ribose unit and/or a natural
nucleoside base
(adenine, guanine, cytosine, thymine, uracil). Examples of chemical
modifications are known
to the skilled person and are described, for example, in Uhlmann E et al.
(1990) Chem Rev
90:543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties
& Synthesis
and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;
Crooke ST
et al. (1996) Annu Rev Pharmacol Toxicol 36:107-29; and Hunziker J et al.
(1995) Mod Synth
Metlaods 7:331-417. An oligonucleotide according to the invention may have one
or more
modifications, wherein each modification is located at a particular
phosphodiester
intemucleoside bridge and/or at a particular (3-D-ribose unit and/or at a
particular natural
nucleoside base position in comparison to an oligonucleotide of the same
sequence which is
composed of natural DNA or RNA.
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For example, the oligonucleotides may include one or more modifications and
wherein
each modification is independently selected from:
a) the replacement of a phosphodiester internucleoside bridge located at the
3' and/or the
5' end of a nucleoside by a modified internucleoside bridge,
b) the replacement of phosphodiester bridge located at the 3' and/or the 5'
end of a
nucleoside by a dephospho bridge,
c) the replacement of a sugar phosphate unit from the sugar phosphate backbone
by
another unit,
d) the replacement of a(3-D-ribose unit by a modified sugar unit, and
e) the replacement of a natural nucleoside base by a modified nucleoside base.
More detailed examples for the chemical modification of an oligonucleotide
follow.
The oligonucleotides may include modified internucleotide linkages, such as
those
described in (a) or (b) above. These modified linkages may be partially
resistant to
degradation (e.g., are stabilized). A "stabilized oligonucleotide" shall mean
an
oligonucleotide that is relatively resistant to in vivo degradation (e.g., via
an exo- or endo-
nuclease) resulting from such modifications. Oligonucleotides having
phosphorothioate
linkages, in some embodiments, may provide maximal activity and protect the
oligonucleotide
from degradation by intracellular exo- and endo-nucleases.
A phosphodiester intemucleoside bridge located at the 3' and/or the 5' end of
a
nucleoside can be replaced by a modified intemucleoside bridge, wherein the
modified
intemucleoside bridge is for example selected from phosphorothioate,
phosphorodithioate,
NR1R2-phosphoramidate, boranophosphate, a-hydroxybenzyl phosphonate, phosphate-
(C1-
C21)-O-alkyl ester, phosphate-[(C6-C12)aryl-(Cl-C2l)-O-alkyl]ester, (C1-
C8)alkylphosphonate
and/or (C6-Cla)arylphosphonate bridges, (C7-C12)-a-hydroxymethyl-aryl (e.g.,
disclosed in
WO 95/01363), wherein (C6-C12)aryl, (C6-C20)aryl and (C6-C14)aryl are
optionally substituted
by halogen, alkyl, alkoxy, nitro, cyano, and where Rl and RZ are,
independently of each other,
hydrogen, (Ci-C18)-alkyl, (C6-C20)-aryl, (C6-C14)-aryl-(Cl-C$)-alkyl,
preferably hydrogen,
(Cl-C8)-alkyl, preferably (Cl-C4)-alkyl and/or methoxyethyl, or Rl and R2
form, together with
the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can
additionally
contain a further heteroatom from the group 0, S and N.
The phosphodiester bridge located at the 3' and/or the 5' end of a nucleoside
can be
replaced by a dephospho bridge (dephospho bridges are described, for example,
in Uhlmann E
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and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for
Oligonucleotides
and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. 355
ff), wherein
a dephospho bridge is for example selected from the dephospho bridges
formacetal, 3'-
thioformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo,
dimethylenesulfone and/or silyl groups.
A sugar phosphate unit (i.e., a(3-D-ribose and phosphodiester internucleoside
bridge
together forming a sugar phosphate unit) from the sugar phosphate backbone
(i.e., a sugar
phosphate backbone is composed of sugar phosphate units) can be replaced by
another unit,
wherein the other unit is for example suitable to build up a "morpholino-
derivative" oligomer
(as described, for example, in Stirchak EP et al. (1989) Nucleic Acids Res
17:6129-41), that is,
for example, the replacement with a morpholino-derivative unit, or to build up
a polyamide
nucleic acid ("PNA"; as described for example, in Nielsen PE et al. (1994)
Bioconjug Claem
5:3-7), that is, for example, the replacement with a PNA backbone unit, e.g.,
by 2-
aminoethylglycine. The oligonucleotide may have other carbohydrate backbone
modifications and replacements, such as peptide nucleic acids with phosphate
groups
(PHONA), locked nucleic acids (LNA), and oligonucleotides having backbone
sections with
alkyl linkers or amino linkers. The alkyl linker may be branched or
unbranched, substituted
or unsubstituted, and chirally pure or a racemic mixture.
The (3-ribose unit or a(3-D-2'-deoxyribose unit can be replaced by a modified
sugar
unit, wherein the modified sugar unit is for example selected from (3-D-
ribose, a-D-2'-
deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F-arabinose, 2'-O-(Cl-
C6)alkyl-ribose,
2'-O-methylribose, 2'-O-(CZ-C6)alkenyl-ribose, 2'-[O-(C1-C6)alkyl-O-(Cl-
C6)alkyl]-ribose, 2'-
NH2-2'-deoxyribose, (3-D-xylo- furanose, a-arabinofuranose, 2,4-dideoxy-(3-D-
erythro-hexo-
pyranose, and carbocyclic (described, for example, in Froehler (1992) JAm
Chena Soc
114:8320) and/or open-chain sugar analogs (described, for example, in
Vandendriessche et al.
(1993) Tetrahedron 49:7223) and/or bicyclosugar analogs (described, for
example, in Tarkov
M et al. (1993) Flelv Chirn Acta 76:481).
In some embodiments, the modified sugar is a 2' modified ribose. In some
embodiments the sugar is 2'-O-methylribose, particularly for one or both
nucleotides linked
by a phosphodiester or phosphodiester-like intemucleoside linkage.
Nucleic acids also include substituted purines and pyrimidines such as C-5
propyne
pyrimidine and 7-deaza-7-substituted purine modified bases. Wagner RW et al.
(1996) Nat
Biotechnol 14:840-4. Purines and pyrimidines include but are not limited to
adenine,
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cytosine, guanine, and thymine, and other naturally and non-naturally
occurring nucleobases,
substituted and unsubstituted aromatic moieties.
A modified base is any base which is chemically distinct from the naturally
occurring
bases typically found in DNA and RNA such as T, C, G, A, and U, but which
share basic
chemical structures with these naturally occurring bases. The modified
nucleoside base may
be, for example, selected from hypoxanthine, uracil, dihydrouracil,
pseudouracil, 2-thiouracil,
4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-(C2-C6)-alkenyluracil, 5-
(C2-C6)-
alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-
bromouracil,
5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-
C6)-
alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-
dimethylguanine,
2,4-diamino-purine, 8-azapurine, a substituted 7-deazapurine, preferably
7-deaza-7-substituted and/or 7-deaza-8-substituted purine, 5-
hydroxymethylcytosine, N4-
alkylcytosine, e.g., N4-ethylcytosin.e, 5-hydroxydeoxycytidine, 5-
hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g., N4-
ethyldeoxycytidine, 6-
thiodeoxyguanosine, and deoxyribonucleosides of nitropyrrole, C5-
propynylpyrimidine, and
diaminopurine e.g., 2,6-diaminopurine, inosine, 5-methylcytosine, 2-
aminopurine,
2-amino-6-chloropurine, hypoxanthine or other modifications of a natural
nucleoside bases.
This list is meant to be exemplary and is not to be interpreted to be
limiting.
In particular formulae described herein modified bases may be incorporated.
For
example, a cytosine may be replaced with a modified cytosine. A modified
cytosine as used
herein is a naturally occurring or non-naturally occurring pyrimidine base
analog of cytosine
which can replace this base without impairing the immunostimulatory activity
of the
oligonucleotide. Modified cytosines include but are not limited to 5-
substituted cytosines
(e.g., 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-
cytosine, 5-iodo-
cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-
cytosine, and
unsubstituted or substituted 5-alkynyl-cytosine), 6-substituted cytosines, N4-
substituted
cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine, 2-mercapto-cytosine,
isocytosine, pseudo-
isocytosine, cytosine analogs with condensed ring systems (e.g., N,N'-
propylene cytosine or
phenoxazine), and uracil and its derivatives (e.g., 5-fluoro-uracil, 5-bromo-
uracil, 5-
bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil). Some
of the preferred
cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-
hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodiment of the
invention, the
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cytosine base is substituted by a universal base (e.g., 3-nitropyrrole, P-
base), an aromatic ring
system (e.g., fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer).
As another example, a guanine may be replaced with a modified guanine base. A
modified guanine as used herein is a naturally occurring or non-naturally
occurring purine
base analog of guanine which can replace this base without impairing the
immunostimulatory
activity of the oligonucleotide. Modified guanines include but are not limited
to
7-deazaguanine, 7-deaza-7-substituted guanine (such as 7-deaza-7-(C2-
C6)alkynylguanine),
7-deaza-8-substituted guanine, hypoxanthine, N2-substituted guanines (e.g., N2-
methyl-
guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-
diaminopurine,
2-aminopurine, purine, indole, adenine, substituted adenines (e.g., N6-methyl-
adenine, 8-oxo-
adenine), 8-substituted guanine (e.g., 8-hydroxyguanine and 8-bromoguanine),
and
6-thioguanine. In another embodiment of the invention, the guanine base is
substituted by a
universal base (e.g., 4-methyl-indole, 5-nitro-indole, and K-base), an
aromatic ring system
(e.g., benzimidazole or dichloro- benzimidazole, 1-methyl-lH-[1,2,4]triazole-3-
carboxylic
acid amide) or a hydrogen atom (dSpacer).
For use in the instant invention, the oligonucleotides of the invention can be
synthesized de novo using any of a number of procedures well lmown in the art
including, for
example, the (3-cyanoethyl phosphoramidite method (Beaucage SL et al. (1981)
Tetrahedron
Lett 22:1859), or the nucleoside H-phosphonate method (Garegg et al. (1986)
Tetrahedron
Lett 27:4051-4; Froehler BC et al. (1986) Nucleic Acids Res 14:5399-407;
Garegg et al.
(1986) Tetrahedron Lett 27:4055-8; Gaffiiey et al. (1988) Tetrahedron Lett
29:2619-22).
These chemistries can be perfonned by a variety of commercially available
automated nucleic
acid synthesizers. These oligonucleotides are referred to as synthetic
oligonucleotides. An
isolated oligonucleotide generally refers to an oligonucleotide which is
separated from
components which it is normally associated with in nature. As an example, an
isolated
oligonucleotide may be one which is separated from a cell, from a nucleus,
from mitochondria
or from chromatin.
Modified backbones such as phosphorothioates may be synthesized using
automated
techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl-
and
alkyl-phosphonates can be made, for example, as described in U.S. Pat. No.
4,469,863; and
alkylphosphotriesters (in which the charged oxygen moiety is alkylated as
described in U.S.
Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by
automated solid
phase synthesis using commercially available reagents. Methods for making
other DNA
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backbone modifications and substitutions have been described (e.g., Uhlmann E
et al. (1990)
Claern Rev 90:544; Goodchild J(1990) Bioconjugate Claena 1:165).
Isolated
In certain embodiments the TLR7/8 ligands and/or the adaptor oligonucleotides
are
isolated. An isolated ligand or oligonucleotide is a ligand or oligonucleotide
that is
substantially pure or is free of other substances with which it is ordinarily
found in nature or
in in vivo systems to an extent practical and appropriate for its intended
use. In particular, the
ligand or oligonucleotide is sufficiently pure and is sufficiently free from
other biological
constituents of cells so as to be useful in, for example, producing
pharmaceutical preparations.
Because an isolated ligand or oligonucleotide may be admixed with a
pharmaceutically
acceptable carrier in a pharmaceutical preparation, the ligand or
oligonucleotide may
comprise only a small percentage by weight of the preparation. The ligand or
oligonucleotide
is nonetheless isolated in that it has been substantially separated from the
substances with
which it may be associated in living systems.
Conjugates
As used herein, the term "conjugate" refers to any combination of two or more
component parts that are linked together, directly or indirectly, via any
physicochemical
interaction. In one embodiment the conjugate is a combination of two or more
component
parts that are linked together, directly or indirectly, via covalent bonding.
In one aspect the invention provides a composition including a conjugate of a
TLR
ligand and an adaptor oligonucleotide. In one embodiment the conjugate is made
via a
covalent bond. In one embodiment the conjugate comprises a linker.
The conjugate can include one or more adaptor,oligonucleotides and one or more
TLR7/8 ligands of the invention. The conjugate can, alternatively or
additionally, include one
or more other molecules including but not limited to antigens or other
medicaments.
FIGs. 13-15 illustrate many features of such conjugates including points of
attachment
on both the ligands and the oligonucleotides, and the optional use of linkers
to facilitate such
conjugation.
In one embodiment, the conjugate is a conjugate of an adaptor oligonucleotide
and
certain TLR7 specific ligands such as but not limited to immunosine or
loxoribine. The
adaptor oligonucleotide may be but is not limited to a poly(dT)
oligonucleotide ranging in
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length from 8-20, preferably 10-20, and more preferably 14-18 nucleotides in
length. In some
embodiments, conjugates of adaptor oligonucleotides and certain TLR7/8 ligands
such as
imidazoquinolines and C8-substituted guanosines are excluded, particularly if
the adaptor
oligonucleotide is itself immunostimulatory or signals through TLR9.
FIGs. 13A-C illustrate the structure of immunosine as well as two monomer
variants
thereof that can be used in the conjugation process. The variant shown in FIG.
13B can be
conjugated to an oligonucleotide at an internal position or at a 3' end. Thus,
there may be one
or more of these variants attached to an oligonucleotide. The variant shown in
FIG. 13C can
be conjugated to an oligonucleotide at a 5' end. FIG. 14 illustrates the
conjugation of
immunosine to an oligonucleotide using a linker. FIG. 15 illustrates
attachment points in R-
848, CL-029 and immunosine to which linkers and/or oligonucleotides may be
conjugated.
The conjugates of the invention may comprise 1, 2, 3, 4, 5, or more linkers,
or alternatively,
they may lack linkers. An example of a conjugate is poly(dT)14-L2-IM (where L
represents a
linker and IM represents immunosine and subscripts denote the number of each
unit in the
conjugate) (SEQ ID NO: 9). Another example is IM-L2-(dT)14 (SEQ ID NO: 10).
The
difference between these examples is the placement of the immunosine and the
linkers at
either the 3' end or the 5' end.
The linkers may be attached to any reactive moiety on the oligonucleotide
including
but not limited to a backbone phosphate group or a sugar hydroxyl group. For
example, they
may be incorporated via phosphodiester, phosphorothioate, methylphosphonate
and/or amide
linkages.
The linkers may be non-nucleotide in nature. These include, for example,
abasic
residues (dSpacer), oligoethyleneglycol such as triethyleneglycol (spacer 9)
or
hexaethylenegylcol (spacer 18), or alkane-diol such as butanediol. Linkers may
be attached to
each other by phosphodiester or phosphorothioate bonds. Other linkers are
alkylamino
linkers, such as C3, C6, C12 amino linkers, and also alkylthiol linkers, such
as C3 or C6 thiol
linkers. Different types of linkers may also be used in one conjugate.
The oligonucleotides may be linked by aromatic residues which may be further
substituted by alkyl or substituted alkyl groups. The oligonucleotides may
also contain a
doubler or trebler unit, which allow conjugation of multiple ligands of one or
different types
to the oligonucleotide. The oligonucteotides may also contain linker resulting
from peptide
modifying reagents or oligonucleotide modifying reagents. Furthermore, they
may contain
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one or more natural or unnatural amino acid residues which are connected by
peptide (amide)
linkages.
The different oligonucleotides are synthesized by established methods and can
be
linked together on-line during solid-phase synthesis. Alternatively, they may
be linked
together following synthesis of the individual units.
Imnaune Responses
In one embodiment, the TLR-mediated immune response is Thl-like immune
response. As used herein, the term "Thl -like" refers to having a feature
characteristic of a
Thl immune response. A Thl immune response characteristically may include
induction of
certain cytokines such as IFN-y, secretion (in mice) of IgG2a immunoglobulins,
and
macrophage activation. The term"Thl -like" is to be contrasted with the term
"Th2-like",
which refers to having a feature characteristic of a Th2 immune response, as
discussed below.
A Thl-like immune response can include expression of any of certain cytokines
and
chemokines, including IFN-a, IFN-(3, IFN-y, TNF-a, IL-12, IL-18, IP-10, and
any
combination thereof, that are characteristically associated with a Thl immune
response. Thl
immune responses and Th2 immune responses are believed to be counter-
regulatory. In some
embodiments the Thl-like immune response can include suppression of certain
Th2-associated cytokines, including IL-4, IL-5, IL-10, and IL-13. The Thl-like
immune
response can include expression of certain antibody isotypes, including (in
the mouse) IgG2a,
with or without suppression of certain Th2-associated antibody isotypes,
including IgE and (in
the mouse) IgGI. In one embodiment a Thl-like immune response is a Thl
response.
A Th2-like immune response can include expression of any of certain cytokines
and
chemokines, including IL-4, IL-5, IL- 10, IL- 13, and any combination thereof,
that are
characteristically associated with a Th2 immune response. In some embodiments
the Th2-like
immune response can include suppression of certain Thl-associated cytokines.
The Th2-like
immune response can include expression of certain antibody isotypes, including
IgE and (in
the mouse) IgGl, with or without suppression of certain Thl-associated
antibody isotypes,
including (in the mouse) IgG2a. In one embodiment a Th2-like immune response
is a Th2
response.
Thus, in one embodiment the invention provides a method for inducing a Thi-
like
immune response in a subject. Inducing a Thl-like immune response includes
augmenting or
enhancing a Thl -like immune response. The method includes the step of
administering to a
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subject an effective amount of an TLR7/8 ligand and adaptor oligonucleotide of
the invention
to induce a Thl-like immune response in the subject.
In one embodiment the invention provides a method for suppressing a Th2-like
immune response in a subject. The method includes the step of administering to
a subject an
effective amount of an TLR7/8 ligand and adaptor oligonucleotide of the
invention to
suppress a Th2-like immune response in the subject. Such method may find
particular use in
the treatment of subjects having or at risk of having a condition
characterized by an immune
response with predominant Th2 character. Such conditions include, without
limitation,
allergy and asthma.
In one embodiment the immune response involves upregulation of cell surface
markers of immune cell activation, such as CD25, CD80, CD86, and CD154.
Methods for
measuring cell surface expression of such markers are well known in the art
and include
FACS analysis.
For measurement of immune response in a cell or population of cells, in one
embodiment the cell or population of cells expresses at least one and in some
instances
preferably only one of TLR7, TLR8, or TLR9. The cell can express the TLR
naturally, or it
can be manipulated to express the TLR though introduction into the cell of a
suitable
expression vector for the TLR. In one embodiment the cell or population of
cells is obtained
as peripheral blood mononuclear cells (PBMC). In one embodiment the cell or
population of
cells is obtained as a cell line expressing the TLR. In one embodiment the
cell or population
of cells is obtained as a cell line transiently transfected with a TLR. In one
embodiment the
cell or population of cells is obtained as a cell line stably transfected with
a TLR.
Also for use in measuring an immune response in a cell or population of cells,
it may
be convenient to introduce into the cell or population of cells a reporter
construct that is
responsive to (i.e., regulated by) intracellular signaling by a TLR. In one
embodiment such a
reporter is a gene placed under the control of an NF-xB promoter. In one
embodiment the
gene placed under control of the promoter is luciferase although other
reporter genes can be
used including green fluorescent protein (GFP), R-galactosidase, alkaline
phosphatase, and the
like. Under suitable conditions of activation, as an example, the luciferase
reporter construct
is expressed and emits a detectable light signal that may be measured
quantitatively using a
luminometer. These and other reporter constructs are commercially available.
In other
embodiments, TLR-mediated immune responses can be measured by the production
(mRNA
or protein) or secretion of cytokines such as IFN-alpha, TNF-alpha, IL-12, IFN-
gamma and
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the like. The examples demonstrate these types of assays. The invention also
contemplates
the use of cell-free methods of detecting TLR activation.
The invention also includes a method for inducing antigen specific immune
responses
(as described in greater detail herein) and non-specific innate immune
activation and broad
spectrum resistance to infectious challenge. The term antigen non-specific
innate immune
activation, as used herein, refers to the activation of immune cells other
than B cells including
NK cells, T cells or other immune cells that can respond in an antigen
independent fashion, or
some combination of these cells. A broad spectrum resistance to infectious
challenge is
induced because the inunune cells are in active form and are primed to respond
to any
invading compound or microorganism. The cells do not have to be specifically
primed
against a particular antigen. This is particularly useful in biowarfare, and
the other
circumstances described above such as travelers.
Subjects
As used herein, a subject refers to a human or non-human vertebrate. Non-human
vertebrates include livestock animals, companion animals, and laboratory
animals. Non-
human subjects also specifically include non-human primates as well as
rodents. Non-human
subjects also specifically include, without limitation, chickens, horses,
cows, pigs, goats,
dogs, cats, guinea pigs, hamsters, mink, and rabbits.
As used herein, a subject at risk of developing a condition refers to a
subject with a
known or suspected exposure to an agent known to cause or to be associated
with the
condition or a known or suspected predisposition to develop the condition
(e.g., a genetic
marker for or a family history of the condition).
In one aspect the invention provides a method of treating a subject having an
immune
system deficiency. The method according to this aspect of the invention
includes the step of
administering to the subject an effective amount of a composition of the
invention to treat the
subject. An immune system deficiency as used herein refers to an abnormally
depressed
ability of an imnlune system to mount an immune response to an antigen. In one
embodiment
an immune system deficiency is a disease or disorder in which the subject's
immune system is
not functioning in normal capacity or in which it would be useful to boost the
subject's
immune response, for example to eliminate a tumor or cancer or an infection in
the subject. A
subject having an immune deficiency, as used herein, refers to a subject in
whom there is a
depressed ability of the subject's immune system to mount an immune response
for example,
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to an antigen. Subjects having an immune deficiency include subjects having an
acquired
immune deficiency as well as subjects having a congenital immune system
deficiency.
Subjects having acquired immune deficiency include, without limitation,
subjects having a
chronic inflammatory condition, subjects having chronic renal insufficiency or
renal failure,
subjects having infection, subjects having cancer, subjects receiving
immunosuppressive
drugs, subjects receiving other immunosuppressive treatment, and subjects with
malnutrition.
In one embodiment the subject has a suppressed CD4+ T-cell population. In one
embodiment
the subject has an infection with human immunodeficiency virus (HIV) or has
acquired
immunodeficiency syndrome (AIDS). The method according to this aspect of the
invention
thus provides a method for boosting an immune response or boosting the ability
to mount an
immune response in a subject in need of a more vigorous immune response.
As used herein, inhibit shall mean reduce an outcome or effect compared to
normal.
As used herein, treat as used in reference to a disease or condition shall
mean to
intervene in such disease or condition so as to prevent or slow the
development of, prevent or
slow the progression of, halt the progression of, or eliminate the disease or
condition. For
example, treating cancer includes preventing the development of a cancer
(e.g., from a
precancerous state), reducing the symptoms of cancer, and/or inhibiting the
growth of an
established cancer.
Conditions
The invention intends to treat conditions which would benefit from inhibition
and/or
induction of certain TLR-mediated immune responses. As used herein, a
condition associated
with TLR7-mediated immunostimulation or immune response refers to any disease
or other
condition in a subject in which there is immune activation associated with
TLR7 signaling,
and such activation is detrimental. Such conditions typically involve
activation of TLR7
signaling in response to contact with a TLR7 ligand.
As used herein, a condition associated with TLRB-inediated immunostimulation
or
immune response refers to any disease or other condition in a subject in which
there is
immune activation associated with TLR8 signaling, and such activation is
detrimental. Such
conditions typically involve activation of TLR8 signaling in response to
contact with a TLRB
ligand.
As used herein, a condition associated with TLR9-mediated immunostimulation or
invmune response refers to any disease or other condition in a subject in
which there is
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immune activation associated with TLR9 signaling, and such activation is
detrimental. Such
conditions typically involve activation of TLR9 signaling in response to
contact with a TLR9
ligand.
Infection
The invention can be used to treat conditions such as infection. Infection
refers to any
condition in which there is an abnormal collection or population of viable
intracellular or
extracellular microbes in a subject. Such microbes may be endogenous to the
subject or they
may be foreign to the subject. A subject having an infection is a subject that
has a disorder
arising from the invasion of the subject, superficially, locally, or
systemically, by an
infectious microorganism or from abnormal upregulated growth of naturally
occurring
endogenous microbes in a subject. The infectious microorganism can be a virus,
bacterium,
fungus, or parasite, as described above. Various types of microbes can cause
infection,
including microbes that are bacteria, microbes that are viruses, microbes that
are fungi, and
microbes that are parasites.
Bacteria are unicellular organisms which multiply asexually by binary fission.
They
are classified and named based on their morphology, staining reactions,
nutrition and
metabolic requirements, antigenic structure, chemical composition, and genetic
homology.
Bacteria can be classified into three groups based on their morphological
forms, spherical
(coccus), straight-rod (bacillus) and curved or spiral rod (vibrio,
campylobacter, spirillum, and
spirochaete). Bacteria are also more commonly characterized based on their
staining
reactions into two classes of organisms, gram-positive and gram-negative. Gram
refers to the
method of staining which is commonly performed in microbiology labs. Gram-
positive
organisms retain the stain following the staining procedure and appear a deep
violet color.
Gram-negative organisms do not retain the stain but take up the counter-stain
and thus appear
pink.
Infectious bacteria include, but are not limited to, gram negative and gram
positive
bacteria. Gram positive bacteria include, but are not limited to Pasteurella
species,
Staphylococci species, and Streptococcus species. Gram negative bacteria
include, but are not
limited to, Escherichia coli, Pseudomonas species, and Salmonella species.
Specific
examples of infectious bacteria include but are not limited to:
Helicobacterpyloris, Borrelia
burgdozferi, Legionella pneumopliilia, Mycobacteria sps (e.g., M.
tuberculosis, M. avium, M.
intracellulare, M. kansasii, M. gordonae), Staphylococcus aureus, Neisseria
gonorrhoeae,
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Neisseria naeningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group
A
Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus (viridans
group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic
species),
Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus
influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacteriurn
sp.,
Erysipelothrix rhusiopatlaiae, Clostridium perfringens, Clostridium tetani,
Enterobacter
aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,
Fusobacterium
nucleatuna, Streptobacillus moniliformis, Treponema pallidum, Treponerna
pertenue,
Leptospira, Rickettsia, and Actinomyces israelli.
Bacteria include, but are not limited to, Pasteurella species, Staphylococci
species,
Streptococcus species, Escherichia coli, Pseudomonas species, and Salnzonella
species.
Specific examples of infectious bacteria include but are not limited to,
Helicobacter pyloris,
Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M.
tuberculosis, M.
avium, M. intracellulare, M. kansasii, M gordonae), Staphylococcus aureus,
Neisseria
gonorrizoeae, Neisseria nzeningitidis, Listeria monocytogenes, Streptococcus
pyogenes
(Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus),
Streptococcus
(viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus
(anaerobic sps.),
Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp.,
Haemophilus
influenzae, Bacillus antlaracis, Corynebacterium diphtheriae, Corynebacterium
sp.,
Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani,
Enterobacter
aerogenes, Klebsiella pneunzoniae, Pasturella multocida, Bacteroides sp.,
Fusobacterium
nucleatum, Streptobacillus moniliformis, Treponeina pallidum, Treponema
pertenue,
Leptospira, Rickettsia, and Actinomyces israelii.
Viruses are small infectious agents which generally contain a nucleic acid
core and a
protein coat, but are not independently living organisms. Viruses can also
take the form of
infectious nucleic acids lacking a protein. A virus cannot survive in the
absence of a living
cell within which it can replicate. Viruses enter specific living cells either
by endocytosis or
direct injection of DNA (phage) and multiply, causing disease. The multiplied
virus can then
be released and infect additional cells. Some viruses are DNA-containing
viruses and others
are RNA-containing viruses. In some aspects, the invention also intends to
treat diseases in
which prions are implicated in disease progression such as for example bovine
spongiform
encephalopathy (i.e., mad cow disease, BSE) or scrapie infection in animals,
or Creutzfeldt-
Jakob disease in humans.
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Viruses include, but are not limited to, enteroviruses (including, but not
limited to,
viruses that the family picornaviridae, such as polio virus, coxsackie virus,
echo virus),
rotaviruses, adenovirus, hepatitis virus. Specific examples of viruses that
have been found in
humans include but are not limited to: Retroviridae (e.g., human
immunodeficiency viruses,
such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III;
and other
isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A
virus; enteroviruses,
human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,
strains that cause
gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella
viruses); Flaviviridae
(e.g., dengue viruses, encephalitis viruses, yellow fever viruses);
Coronaviridae (e.g.,
coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies
viruses); Filoviridae
(e.g., ebola viruses); Pararnyxoviridae (e.g., parainfluenza viruses, mumps
virus, measles
viras, respiratory syncytial virus); Ortlzomyxoviridae (e.g., influenza
viruses); Bunyaviridae
(e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses);
Arenaviridae
(hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and
rotaviruses);
Birnaviridae=, Hepadnaviridae (Hepatitis B virus); Parvoviridae
(parvoviruses);
Papovaviridae (papillomaviruses, polyoma viruses); Adenoviridae (most
adenoviruses);
Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus,
cytomegalovirus
(CMV)); Poxviridae (variola viruses, vaccinia viruses, pox viruses);
Iridoviridae (e.g.,
African swine fever virus); and unclassified viruses (e.g., the etiological
agents of spongiform
encephalopathies, the agent of delta hepatitis (thought to be a defective
satellite of hepatitis B
virus), the agents of non-A, non-B hepatitis (class 1= internally transmitted;
class 2=
parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and
astroviruses).
Examples of viruses that have been found in humans include but are not limited
to:
Retroviridae (e.g., human immunodeficiency viruses, such as HIV-l (also
referred to as
HTLV-IIl, LAV or HTLV-IIULAV, or HIV-III; and other isolates, such as HIV-LP;
Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human
Coxsackie viruses,
rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause
gastroenteritis); Togaviridae
(e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g.,
dengue viruses,
encephalitis viruses, yellow fever viruses); Coronaviridae (e.g.,
coronaviruses);
Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses);
Filoviridae (e.g., ebola
viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles
virus,
respiratory syncytial virus); Orthoinyxoviridae (e.g., influenza viruses);
Bunyaviridae (e.g.,
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae
(hemorrhagic
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fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);
Bornaviridae;
Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae
(papilloma
viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae
(herpes simplex
virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes
virus; Poxviridae
(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g.,
African swine fever
virus); and unclassified viruses (e.g., the agent of delta hepatitis (thought
to be a defective
satellite of hepatitis B virus), Hepatitis C; Norwalk and related viruses, and
astroviruses).
Fungi are eukaryotic organisms, only a few of which cause infection in
vertebrate
mammals. Because fungi are eukaryotic organisms, they differ significantly
from prokaryotic
bacteria in size, structural organization, life cycle and mechanism of
multiplication. Fungi are
classified generally based on morphological features, modes of reproduction
and culture
characteristics. Although fungi can cause different types of disease in
subjects, such as
respiratory allergies following inhalation of fungal antigens, fi.tngal
intoxication due to
ingestion of toxic substances, such as Arnanita phalloides toxin and
phallotoxin produced by
poisonous inushrooms and aflatoxins, produced by aspergillus species, not all
fungi cause
infectious disease.
Infectious fungi can cause systemic or superficial infections. Primary
systemic
infection can occur in normal healthy subjects, and opportunistic infections
are most
frequently found in irnmunocompromised subjects. The most common fungal agents
causing
primary systemic infection include Blastonayces, Coccidioides, and
Histoplasma. Common
fungi causing opportunistic infection in immunocompromised or immunosuppressed
subjects
include, but are not limited to, Candida albicans, Cryptococcus neoforrnans,
and various
Aspergillus species. Systemic fungal infections are invasive infections of the
internal organs.
The organism usually enters the body through the lungs, gastrointestinal
tract, or intravenous
catheters. These types of infections can be caused by primary pathogenic fungi
or
opportunistic fungi.
Superficial fungal infections involve growth of fungi on an external surface
without
invasion of internal tissues. Typical superficial fungal infections include
cutaneous fungal
infections involving skin, hair, or nails.
Diseases associated with fungal infection include aspergillosis,
blastomycosis,
candidiasis, chromoblastomycosis, coccidioidomycosis, cryptococcosis, fungal
eye infections,
fungal hair, nail, and skin infections, histoplasmosis, lobomycosis, mycetoma,
otomycosis,
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paracoccidioidomycosis, disseminated Penicillium inarneffei,
phaeohyphomycosis,
rhinosporidioisis, sporotrichosis, and zygomycosis.
Fungi include yeasts and molds. Examples of fungi include without limitation
Aspergillus spp including Aspergillus furnigatus, Blastomyces derinatitidis,
Candida spp
including Candida albicans, Coccidioides immitis, Cryptococcus neoformans,
Histoplasrna
capsulatum, Pneurnocystis carinii, Rhizornucor spp, and Rhizopus spp.
Parasites are organisms which depend upon other organisms in order to survive
and
thus must enter, or infect, another organism to continue their life cycle. The
infected
organism, i.e., the host, provides both nutrition and habitat to the parasite.
Although in its
broadest sense the term parasite can include all infectious agents (i.e.,
bacteria, viruses, fungi,
protozoa and helminths), generally speaking, the term is used to refer solely
to protozoa,
helminths, and ectoparasitic arthropods (e.g., ticks, mites, etc.). Protozoa
are single-celled
organisms which can replicate both intracellularly and extracellularly,
particularly in the
blood, intestinal tract or the extracellular matrix of tissues. Helminths are
multicellular
organisms which almost always are extracellular (an exception being
Trichinella spp.).
Helminths normally require exit from a primary host and transmission into a
secondary host
in order to replicate. In contrast to these aforementioned classes,
ectoparasitic arthropods
form a parasitic relationship with the external surface of the host body.
Parasites include intracellular parasites and obligate intracellular
parasites. Examples
of parasites include but are not limited to Plasmodium falciparum, Plasmodium
ovale,
Plasmodium malariae, Plasmdodium vivax, Plasmodium ktzowlesi, Babesia microti,
Babesia
divergens, Trypanosoina cruzi, Toxoplasma gondii, Trichinella spiralis,
Leishmania rnajor,
Leishmania donovani, Leishmania braziliensis, Leishmania tropica, Trypanosoma
gambiense,
Trypanosoma rhodesiense and Schistosoma mansoni.
Other infectious organisms (i.e., protists) include Plasmodium spp. such as
Plasmodiurn falciparum, Plasmodium malariae, Plasmodiurn ovale, and Plasmodium
vivax
and Toxoplasma gondii. Blood-borne and/or tissue parasites include Plasmodium
spp.,
Babesia naicroti, Babesia divergens, Chlarnydia trachonaatis, Leishmania
tropica, Leishmania
spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and
Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi
(Chagas' disease),
and Toxoplasma gondii.
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Other medically relevant microorganisms have been described extensively in the
literature, e.g., see C.G.A Thomas, Medical Microbiology, Bailliere Tindall,
Great Britain
1983, the entire contents of which is hereby incorporated by reference.
Cancer
In one aspect the invention provides a method of treating a subject having a
cancer.
A subject having a cancer is a subject that has detectable cancerous cells.
The cancer
may be a malignant or non-malignant cancer. "Cancer" as used herein refers to
an
uncontrolled growth of cells which interferes with the normal functioning of
the bodily organs
and systems. Cancers which migrate from their original location and seed vital
organs can
eventually lead to the death of the subject through the functional
deterioration of the affected
organs. Hemopoietic cancers, such as leukemia, are able to outcompete the
normal
hemopoietic compartments in a subject, thereby leading to hemopoietic failure
(in the form of
anemia, thrombocytopenia and neutropenia) ultimately causing death.
A metastasis is a region of cancer cells, distinct from the primary tumor
location,
resulting from the dissemination of cancer cells from the primary tumor to
other parts of the
body. At the time of diagnosis of the primary tumor mass, the subject may be
monitored for
the presence of metastases. Metastases are most often detected through the
sole or combined
use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans,
blood
and platelet counts, liver function studies, chest X-rays and bone scans in
addition to the
monitoring of specific symptoms.
Cancers include, but are not limited to, basal cell carcinoma, biliary tract
cancer;
bladder cancer; bone cancer; brain and central nervous system (CNS) cancer;
breast cancer;
cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue
cancer; cancer
of the digestive system; endometrial cancer; esophageal cancer; eye cancer;
cancer of the head
and neck; intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia;
liver cancer;
lung cancer (e.g., small cell and non-small cell); lymphoma including
Hodgkin's and Non-
Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer
(e.g., lip,
tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate
cancer;
retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory
system; sarcoma;
skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine
cancer; cancer of the
urinary system, as well as other carcinomas, adenocarcinomas, and sarcomas.
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Autoitnmune Conditions
Conditions that involve an innate immune response or a Thl-like immune
response,
include inflammation, acute and chronic allograft rejection, graft-versus-host
disease (GvHD),
certain autoimmune diseases, and sepsis. The invention can be used to treat
such conditions
in view of the selective inhibition of TLR signaling that can be achieved
according to the
invention.
Autoimmune diseases can be generally classified as antibody-mediated, T-cell
mediated, or a combination of antibody-mediated and T-cell mediated. The
adaptor ODN and
TLR ligand combinations of the invention are believed to be useful for
treating various types
of autoimmunity involving antibody-mediated or T-cell mediated immunity,
including
insulin-dependent (type I) diabetes mellitus, rheumatoid arthritis, multiple
sclerosis, systemic
lupus erythematosus (SLE), and inflammatory bowel disease (i.e., Carohn's
disease and
ulcerative colitis). Animal models for these autoimmune diseases are available
and are useful
for assessing the efficacy of the combinations of the invention in these
diseases. Other
autoimmune diseases include, without limitation, alopecia areata, acquired
hemophilia,
ankylosing spondylitis, antiphospholipid syndrome, autoimmune hepatitis,
autoimmune
hemolytic anemia, Behget's syndrome, cardiomyopathy, celiac sprue dermatitis,
chronic
fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory
demyelinating
polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST
syndrome, cold
agglutinin disease, discoid lupus, essential mixed cryoglobulinemia,
fibromyalgia,
fibromyositis, Guillain-Barre syndrome, idiopathic pulmonary fibrosis,
idiopathic
thrombocytopenic purpura, IgA nephropathy, juvenile arthritis, lichen planus,
myasthenia
gravis, polyarteritis nodosa, polychondritis, polyglandular syndromes,
dermatomyositis,
primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's
phenomena,
Reiter's syndrome, sarcoidosis, stiff-man syndrome, Takayasu arthritis,
temporal
arteritis/giant cell arteritis, uveitis, vasculitis, and vitiligo.
In several autoimmune diseases antibodies to self antigens are frequently
observed.
For example for systemic lupus erythematosus autoantibodies have been
described to single-
stranded and double-stranded DNA or RNA. Vallin H et al. (1999) Jlnamunol
163:6306-13;
Hoet RM et al. (1999) Jbnnaunol 163:3304-12; ven Venrooij (1990) J Clin Invest
86:2154-
60. The levels of autoantibodies found in the serum of autoimmune patients
very often are
found to correlate with disease severity. The pattern of autoantibodies that
arise, e.g., in
human SLE, suggest that intact macromolecular particles, such as RNA- or DNA-
containing
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complexes, could themselves be immunogenic and anti-nucleic acid antibodies
could
therefore arise. Lotz M et al. (1992) Mol Biol Rep 16:127; Mohan C et al.
(1993) JExp Med
177:1367-81. Such DNA or RNA released from, e.g., apoptotic cells or DNA- or
RNA-
containing microbes present in serum of autoimmune patients, could be
responsible for
inflammation that contributes to the autoimmune disease. Fatenejad S (1994)
Jlmmunol
152:5523-31; Malmegrim KC et al. (2002) Isr Med Assoc J4:706-12; Newkirk MM et
al.
(2001) Artlzr-itis Res 3:253-8. Indeed CpG-containing sequences could be
identified from SLE
serum that induces an efficient immune response dominated by IFN-a secretion
that is
thought to contribute to the development of autoimmune diseases. Magnusson M
et al. (2001)
Scand Jlmrnunol 54:543-50; Ronnblom L et al. (2001) JExp Med 194:F59-63. In
addition,
the epitopes for anti-RNA antibodies could be identified and are composed of
G,U-rich
sequences. Tsai DE et al. (1992) Proc Natl Acad Sci USA 89:8864-8; Tsai DE et
al. (1993) J
Immunol 150:1137-45.
Allergy
An "allergic condition" or "allergy" refers to acquired hypersensitivity to a
substance
(allergen). A "subject having an allergic condition" shall refer to a subject
that is currently
experiencing or has previously experienced an allergic reaction in response to
an allergen.
Allergic conditions include but are not limited to eczema, allergic rhinitis
or coryza, hay
fever, allergic conjunctivitis, bronchial asthma, urticaria (hives) and food
allergies, other
atopic conditions including atopic dermatitis; anaphylaxis; drug allergy; and
angioedema.
Allergy is typically an episodic condition associated with the production of
antibodies
from a particular class of irnmunoglobulin, IgE, against allergens. The
development of an
IgE-mediated response to common aeroallergens is also a factor which indicates
predisposition towards the development of asthma. If an allergen encounters a
specific IgE
which is bound to an IgE Fc receptor (FcsR) on the surface of a basophil
(circulating in the
blood) or mast cell (dispersed throughout solid tissue), the cell becomes
activated, resulting in
the production and release of mediators such as histamine, serotonin, and
lipid mediators.
An allergic reaction occurs when tissue-sensitizing immunoglobulin of the IgE
type
reacts with foreign allergen. The IgE antibody is bound to mast cells and/or
basophils, and
these specialized cells release chemical mediators (vasoactive amines) of the
allergic reaction
when stimulated to do so by allergens bridging the ends of the antibody
molecule. Histamine,
platelet activating factor, arachidonic acid metabolites, and serotonin are
among the best
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known mediators of allergic reactions in man. Histamine and the other
vasoactive amines are
normally stored in mast cells and basophil leukocytes. The mast cells are
dispersed
throughout animal tissue and the basophils circulate within the vascular
system. These cells
manufacture and store histamine within the cell unless the specialized
sequence of events
involving IgE binding occurs to trigger its release.
Symptoms of an allergic reaction vary, depending on the location within the
body
where the IgE reacts with the antigen. If the reaction occurs along the
respiratory epithelium,
the symptoms generally are sneezing, coughing and asthmatic reactions. If the
interaction
occurs in the digestive tract, as in the case of food allergies, abdominal
pain and diarrhea are
common. Systemic allergic reactions, for example following a bee sting or
administration of
penicillin to an allergic subject, can be severe and often life-threatening.
Allergy is associated with a Th2-type of immune response, which is
characterized at
least in part by Th2 cytokines IL-4 and IL-5, as well as antibody isotype
switching to IgE.
Thl and Th2 immune responses are mutually counter-regulatory, so that skewing
of the
immune response toward a Thl-type of immune response can prevent or ameliorate
a Th2-
type of immune response, including allergy.
Asthma
"Asthma" as used herein refers to a disorder of the respiratory system
characterized by
inflammation and narrowing of the airways, and increased reactivity of the
airways to inhaled
agents. Asthma is frequently, although not exclusively, associated with an
atopic or allergic
condition. Symptoms of asthma include recurrent episodes of wheezing,
breathlessness, chest
tightness, and coughing, resulting from airflow obstruction. Airway
inflammation associated
with asthma can be detected through observation of a number of physiological
changes, such
as, denudation of airway epithelium, collagen deposition beneath basement
membrane,
edema, mast cell activation, inflammatory cell infiltration, including
neutrophils, eosinophils,
and lymphocytes. As a result of the airway inflammation, asthma patients often
experience
airway hyper-responsiveness, airflow limitation, respiratory symptoms, and
disease
chronicity. Airflow limitations include acute bronchoconstriction, airway
edema, mucous
plug formation, and airway remodeling, features which often lead to bronchial
obstruction. In
some cases of asthma, sub-basement membrane fibrosis may occur, leading to
persistent
abnormalities in lung function.
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Research over the past several years has revealed that asthma likely results
from
complex interactions among inflammatory cells, mediators, and other cells and
tissues
resident in the airways. Mast cells, eosinophils, epithelial cells,
macrophage, and activated T
cells all play an important role in the inflammatory process associated with
asthma.
Djukanovic R et al. (1990) Ana Rev Respir Dis 142:434-457. It is believed that
these cells can
influence airway function through secretion of preformed and newly synthesized
mediators
which can act directly or indirectly on the local tissue. It has also been
recognized that
subpopulations of T lymphocytes (Th2) play an important role in regulating
allergic
inflammation in the airway by releasing selective cytokines and establishing
disease
chronicity. Robinson DS et al. (1992) NEnglJMed 326:298-304.
Asthma is a complex disorder which arises at different stages in development
and can
be classified based on the degree of symptoms as acute, subacute, or chronic.
An acute
inflammatory response is associated with an early recruitment of cells into
the airway. The
subacute inflammatory response involves the recruitment of cells as well as
the activation of
resident cells causing a more persistent pattern of inflammation. Chronic
inflammatory
response is characterized by a persistent level of cell damage and an ongoing
repair process,
which may result in permanent abnormalities in the airway.
A "subject having asthma" is a subject that has a disorder of the respiratory
system
characterized by inflammation and narrowing of the airways and increased
reactivity of the
airways to inhaled agents. Factors associated with initiation of asthma
include, but are not
limited to, allergens, cold temperature, exercise, viral infections, and S02.
As mentioned herein, asthma may be associated with a Th2-type of immune
response,
which is characterized at least in part by Th2 cytokines IL-4 and IL-5, as
well as antibody
isotype switching to IgE. Thl and Th2 immune responses are mutually counter-
regulatory, so
that skewing of the immune response toward a Thl-type of immune response can
prevent or
ameliorate a Th2-type of immune response, including allergy.
The TLR7/8 ligand and adaptor oligonucleotide combination of the invention is
also
useful for improving survival, differentiation, activation and maturation of
dendritic cells.
In certain aspects the invention provides a method for enhancing epitope
spreading.
"Epitope spreading" as used herein refers to the diversification of epitope
specificity from an
initial focused, dominant epitope-specific immune response, directed against a
self or foreign
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protein, to subdominant and/or cryptic epitopes on that protein
(intramolecular spreading) or
other proteins (intermolecular spreading). Epitope spreading results in
multiple epitope-
specific immune responses.
Anti-naicrobial agents
The TLR7/8 ligand and adaptor oligonucleotide combination can be used together
with one or more anti-microbial agents. Anti-microbial agents or medicaments
include but
are not limited to anti-bacterial agents, anti-viral agents, anti-fungal
agents and anti-parasitic
agents. Phrases such as "anti-infective agent", "antibiotic", "anti-bacterial
agent", "anti-viral
agent", "anti-fungal agent", "anti-parasitic agent" and "parasiticide" have
well-established
meanings to those of ordinary skill in the art and are defined in standard
medical texts.
Briefly, anti-bacterial agents kill or inhibit bacteria, and include
antibiotics as well as other
synthetic or natural compounds having similar functions. Anti-viral agents can
be isolated
from natural sources or synthesized and are useful for killing or inhibiting
viruses. Anti-
fungal agents are used to treat superficial fungal infections as well as
opportunistic and
primary systemic fungal infections. Anti-parasite agents kill or inhibit
parasites. Many
antibiotics are low molecular weight molecules which are produced as secondary
metabolites
by cells, such as microorganisms. In general, antibiotics interfere with one
or more functions
or structures which are specific for the microorganism and which are not
present in host cells.
One of the problems with anti-infective therapies is the side effects
occurring in the
host that is treated with the anti-infective agent. For instance, many anti-
infectious agents can
kill or inhibit a broad spectrum of microorganisms and are not specific for a
particular type of
species. Treatment with these types of anti-infectious agents results in the
killing of the
normal microbial flora living in the host, as well as the infectious
microorganism. The loss of
the microbial flora can lead to disease complications and predispose the host
to infection by
other pathogens, since the microbial flora compete with and function as
barriers to infectious
pathogens. Other side effects may arise as a result of specific or non-
specific effects of these
chemical entities on non-microbial cells or tissues of the host.
Another problem with widespread use of anti-infectants is the development of
antibiotic-resistant strains of microorganisms. Already, vancomycin-resistant
enterococci,
penicillin-resistantpneumococci, multi-resistant S. aureus, and multi-
resistant tuberculosis
strains have developed and are becoming major clinical problems. Widespread
use of anti-
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infectants will likely produce many antibiotic-resistant strains of bacteria.
As a result, new
anti-infective strategies will be required to combat these microorganisms.
Anti-bacterial antibiotics which are effective for killing or inhibiting a
wide range of
bacteria are referred to as broad-spectrum antibiotics. Other types of
antibacterial antibiotics
are predominantly, effective against the bacteria of the class gram-positive
or gram-negative.
These types of antibiotics are referred to as narrow-spectrum antibiotics.
Other antibiotics
which are effective against a single organism or disease and not against other
types of
bacteria, are referred to as limited-spectrum antibiotics.
Anti-bacterial agents are sometimes classified based on their primary mode of
action.
In general, anti-bacterial agents are cell wall synthesis inhibitors, cell
membrane inhibitors,
protein synthesis inhibitors, nucleic acid synthesis or functional inhibitors,
and competitive
inhibitors. Cell wall synthesis inhibitors inhibit a step in the process of
cell wall synthesis,
and in general in the synthesis of bacterial peptidoglycan. Cell wall
synthesis inhibitors
include (3-lactam antibiotics, natural penicillins, semi-synthetic
penicillins, ampicillin,
clavulanic acid, cephalolsporins, and bacitracin.
The (3-lactams are antibiotics containing a four-membered (3-lactam ring which
inhibits the last step of peptidoglycan synthesis. (3-lactam antibiotics can
be synthesized or
natural. The (3-lactam antibiotics produced by penicillium are the natural
penicillins, such as
penicillin G or penicillin V. These are produced by fermentation of
Fenicillium chrysogenum.
The natural penicillins have a narrow spectrum of activity and are generally
effective against
Streptococcus, Gonococcus, and Staphylococcus. Other types of natural
penicillins, which are
also effective against gram-positive bacteria, include penicillins F, X, K,
and O.
Semi-synthetic penicillins are generally modifications of the molecule 6-
aminopenicillanic acid produced by a mold. The 6-aminopenicillanic acid can be
modified by
addition of side chains which produce penicillins having broader spectrums of
activity than
natural penicillins or various other advantageous properties. Some types of
semi-synthetic
penicillins have broad spectrums against gram-positive and gram-negative
bacteria, but are
inactivated by penicillinase. These semi-synthetic penicillins include
ampicillin,
carbenicillin, oxacillin, azlocillin, mezlocillin, and piperacillin. Other
types of semi-synthetic
penicillins have narrower activities against gram-positive bacteria, but have
developed
properties such that they are not inactivated by penicillinase. These include,
for instance,
methicillin, dicloxacillin, and nafcillin. Some of the broad spectrum semi-
synthetic
penicillins can be used in combination with (3-lactamase inhibitors, such as
clavulanic acids
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and sulbactam. The (3-lactamase inhibitors do not have anti-microbial action
but they
function to inhibit penicillinase, thus protecting the semi-synthetic
penicillin from
degradation.
Another type of (3-lactam antibiotic is the cephalolsporins. They are
sensitive to
degradation by bacterial (3-lactamases, and thus, are not always effective
alone.
Cephalolsporins, however, are resistant to penicillinase. They are effective
against a variety
of gram-positive and gram-negative bacteria. Cephalolsporins include, but are
not limited to,
cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin,
cefuroxine, cefoxitin,
cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone,
ceftazidine, and
moxalactam.
Bacitracin is another class of antibiotics which inhibit cell wall synthesis,
by inhibiting
the release of muropeptide subunits or peptidoglycan from the molecule that
delivers the
subunit to the outside of the membrane. Although bacitracin is effective
against gram-
positive bacteria, its use is limited in general to topical administration
because of its high
toxicity.
Carbapenems are another broad-spectrum (3-lactam antibiotic, which is capable
of
inhibiting cell wall synthesis. Examples of carbapenems include, but are not
limited to,
imipenems. Monobactams are also broad-spectrum (3-lactam antibiotics, and
include,
euztreonam. An antibiotic produced by Streptomyces, vancomycin, is also
effective against
gram-positive bacteria by inhibiting cell membrane synthesis.
Another class of anti-bacterial agents is the anti-bacterial agents that are
cell
membrane inhibitors. These compounds disorganize the structure or inhibit the
function of
bacterial membranes. One problem with anti-bacterial agents that are cell
membrane
inhibitors is that they can produce effects in eukaryotic cells as well as
bacteria because of the
similarities in phospholipids in bacterial and eukaryotic membranes. Thus
these compounds
are rarely specific enough to permit these compounds to be used systemically
and prevent the
use of high doses for local administration.
One clinically useful cell nlembrane inhibitor is Polymyxin. Polymyxins
interfere
with membrane function by binding to membrane phospholipids. Polyniyxin is
effective
mainly against Gram-negative bacteria and is generally used in severe
Pseudomonas
infections or Pseudomonas infections that are resistant to less toxic
antibiotics. The severe
side effects associated with systemic administration of this compound include
damage to the
kidney and other organs.
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Other cell membrane inhibitors include Amphotericin B and Nystatin which are
anti-
fungal agents used predominantly in the treatment of systemic fungal
infections and Candida
yeast infections. Imidazoles are another class of antibiotic that is a cell
membrane inhibitor.
Imidazoles are used as anti-bacterial agents as well as anti-fungal agents,
e.g., used for
treatment of yeast infections, dermatophytic infections, and systemic fungal
infections.
Imidazoles include but are not limited to clotrimazole, miconazole,
ketoconazole,
itraconazole, and fluconazole.
Many anti-bacterial agents are protein synthesis inhibitors. These compounds
prevent
bacteria from synthesizing structural proteins and enzymes and thus cause
inhibition of
bacterial cell growth or function or cell death. In general these compounds
interfere with the
processes of transcription or translation. Anti-bacterial agents that block
transcription include
but are not limited to Rifampins and Ethambutol. Rifampins, which inhibit the
enzyme RNA
polymerase, have a broad spectrum activity and are effective against gram-
positive and gram-
negative bacteria as well as Mycobacterium tuberculosis. Ethambutol is
effective against
Mycobacterium tuberculosis.
Anti-bacterial agents which block translation interfere with bacterial
ribosomes to
prevent mRNA from being translated into proteins. In general this class of
compounds
includes but is not limited to tetracyclines, chloramphenicol, the macrolides
(e.g.,
erythromycin) and the aminoglycosides (e.g., streptomycin).
The aminoglycosides are a class of antibiotics which are produced by the
bacterium
Streptoinyces, such as, for instance streptomycin, kanamycin, tobramycin,
amikacin, and
gentamicin. Aminoglycosides have been used against a wide variety of bacterial
infections
caused by Gram-positive and Gram-negative bacteria. Streptomycin has been used
extensively as a primary drug in the treatment of tuberculosis. Gentamicin is
used against
many strains of Gram-positive and Gram-negative bacteria, including
Pseudomonas
infections, especially in combination with Tobramycin. Kanamycin is used
against many
Gram-positive bacteria, including penicillin-resistant Staplzylococci. One
side effect of
aminoglycosides that has limited their use clinically is that at dosages which
are essential for
efficacy, prolonged use has been shown to impair kidney function and cause
damage to the
auditory nerves leading to deafiiess.
Another type of translation inhibitor anti-bacterial agent is the
tetracyclines. The
tetracyclines are a class of antibiotics that are broad-spectrum and are
effective against a
variety of gram-positive and gram-negative bacteria. Examples of tetracyclines
include
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tetracycline, minocycline, doxycycline, and chlortetracycline. They are
important for the
treatment of many types of bacteria but are particularly important in the
treatment of Lyme
disease. As a result of their low toxicity and minimal direct side effects,
the tetracyclines
have been overused and misused by the medical community,leading to problems.
For
instance, their overuse has led to widespread development of resistance.
Anti-bacterial agents such as the macrolides bind reversibly to the 50 S
ribosomal
subunit and inhibit elongation of the protein by peptidyl transferase or
prevent the release of
uncharged tRNA from the bacterial ribosome or both. These compounds include
erythromycin, roxithromycin, clarithromycin, oleandomycin, and azithromycin.
Erythromycin is active against most Gram-positive bacteria, Neisseria,
Legionella and
Haemophilus, but not against the Enterobacteriaceae. Lincomycin and
clindamycin, which
block peptide bond formation during protein synthesis, are used against gram-
positive
bacteria.
Another type of translation inhibitor is chloramphenicol. Chloramphenicol
binds the
70 S ribosome inhibiting the bacterial enzyme peptidyl transferase thereby
preventing the
growth of the polypeptide chain during protein synthesis. One serious side
effect associated
with chloramphenicol is aplastic anemia. Aplastic anemia develops at doses of
chloramphenicol which are effective for treating bacteria in a small
proportion (1/50,000) of
patients. Chloramphenicol which was once a highly prescribed antibiotic is now
seldom uses
as a result of the deaths from anemia. Because of its effectiveness it is
still used in life-
threatening situations (e.g., typhoid fever).
Some anti-bacterial agents disrupt nucleic acid synthesis or function, e.g.,
bind to
DNA or RNA so that their messages cannot be read. These include but are not
limited to
quinolones and co-trimoxazole, both synthetic chemicals and rifamycins, a
natural or semi-
synthetic chemical. The quinolones block bacterial DNA replication by
inhibiting the DNA
gyrase, the enzyme needed by bacteria to produce their circular DNA. They are
broad
spectrum and examples include norfloxacin, ciprofloxacin, enoxacin, nalidixic
acid and
temafloxacin. Nalidixic acid is a bactericidal agent that binds to the DNA
gyrase enzyme
(topoisomerase) which is essential for DNA replication and allows supercoils
to be relaxed
and reformed, inhibiting DNA gyrase activity. The main use of nalidixic acid
is in treatment
of lower urinary tract infections (UTI) because it is effective against
several types of Gram-
negative bacteria such as E. coli, Enterobacter aerogenes, K. pneumoniae and
Proteus
species which are common causes of UTI. Co-trimoxazole is a combination of
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sulfamethoxazole and trimethoprim, which blocks the bacterial synthesis of
folic acid needed
to make DNA nucleotides. Rifampicin is a derivative of rifamycin that is
active against
Gram-positive bacteria (including Mycobacterium tuberculosis and meningitis
caused by
Neisseria rneningitidis) and some Gram-negative bacteria. Rifampicin binds to
the beta
subunit of the polymerase and blocks the addition of the first nucleotide
which is necessary to
activate the polymerase, thereby blocking mRNA synthesis.
Another class of anti-bacterial agents is compounds that function as
competitive
inhibitors of bacterial enzymes. The competitive inhibitors are mostly all
structurally similar
to a bacterial growth factor and compete for binding but do not perform the
metabolic
function in the cell. These compounds include sulfonamides and chemically
modified forms
of sulfanilamide which have even higher and broader antibacterial activity.
The sulfonamides
(e.g., gantrisin and trimethoprim) are useful for the treatment of
Streptococcus pneumoniae,
beta-hemolytic streptococci and E. coli, and have been used in the treatment
of
uncomplicated UTI caused by E. coli, and in the treatment of ineningococcal
meningitis.
Anti-viral agents are compounds which prevent infection of cells by viruses or
replication of the virus within the cell. There are many fewer antiviral drugs
than
antibacterial drugs because the process of viral replication is so closely
related to DNA
replication within the host cell, that non-specific antiviral agents would
often be toxic to the
host. There are several stages within the process of viral infection which can
be blocked or
inhibited by antiviral agents. These stages include, attachment of the virus
to the host cell
(immunoglobulin or binding peptides), uncoating of the virus (e.g.,
amantadine), synthesis or
translation of viral mRNA (e.g., interferon), replication of viral RNA or DNA
(e.g.,
nucleoside analogues), maturation of new virus proteins (e.g., protease
inhibitors), and
budding and release of the virus.
Another category of anti-viral agents are nucleoside analogues. Nucleoside
analogues
are synthetic compounds which are similar to nucleosides, but which have an
incomplete or
abnormal deoxyribose or ribose group. Once the nucleoside analogues are in the
cell, they are
phosphorylated, producing the triphosphate form which competes with normal
nucleotides for
incorporation into the viral DNA or RNA. Once the triphosphate form of the
nucleoside
analogue is incorporated into the growing nucleic acid chain, it causes
irreversible association
with the viral polymerase and thus chain termination. Nucleoside analogues
include, but are
not limited to, acyclovir (used for the treatment of herpes simplex virus and
varicella-zoster
virus), gancyclovir (useful for the treatment of cytomegalovirus),
idoxuridine, ribavirin
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(useful for the treatment of respiratory syncitial virus), dideoxyinosine,
dideoxycytidine, and
zidovudine (azidothymidine).
Another class of anti-viral agents includes cytokines such as interferons. The
interferons are cytokines which are secreted by virus-infected cells as well
as immune cells.
The interferons function by binding to specific receptors on cells adjacent to
the infected cells,
causing the change in the cell which protects it from infection by the virus.
a and (3-
interferon also induce the expression of Class I and Class II MHC molecules on
the surface of
infected cells, resulting in increased antigen presentation for host immune
cell recognition. a
and (3-interferons are available as recombinant forms and have been used for
the treatment of
chronic hepatitis B and C infection. At the dosages which are effective for
anti-viral therapy,
interferons have severe side effects such as fever, malaise and weight loss.
Immunoglobulin therapy is used for the prevention of viral infection.
Immunoglobulin therapy for viral infections is different from bacterial
infections, because
rather than being antigen-specific, the immunoglobulin therapy functions by
binding to
extracellular virions and preventing them from attaching to and entering cells
which are
susceptible to the viral infection. The therapy is useful for the prevention
of viral infection for
the period of time that the antibodies are present in the host. In general
there are two types of
immunoglobulin therapies, normal immune globulin therapy and hyper-immune
globulin
therapy. Normal immune globulin therapy utilizes a antibody product which is
prepared from
the serum of normal blood donors and pooled. This pooled product contains low
titers of
antibody to a wide range of human viruses, such as hepatitis A, parvovirus,
enterovirus
(especially in neonates). Hyper-immune globulin therapy utilizes antibodies
which are
prepared from the serum of individuals who have high titers of an antibody to
a particular
virus. Those antibodies are then used against a specific virus. Examples of
hyper-immune
globulins include zoster immune globulin (useful for the prevention of
varicella in
immunocompromised children and neonates), human rabies immune globulin (useful
in the
post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis B
immune globulin
(useful in the prevention of hepatitis B virus, especially in a subject
exposed to the virus), and
RSV immune globulin (useful in the treatment of respiratory syncitial virus
infections).
Anti-fungal agents are useful for the treatment and prevention of infective
fungi.
Anti-fungal agents are sometimes classified by their mechanism of action. Some
anti-fungal
agents function as cell wall inhibitors by inhibiting glucose synthase. These
include, but are
not limited to, basiungin/ECB. Other anti-fungal agents function by
destabilizing membrane
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integrity. These include, but are not limited to, imidazoles, such as
clotrimazole,
sertaconzole, fluconazole, itraconazole, ketoconazole, miconazole, and
voriconacole, as well
as FK 463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,
butenafine, and
terbinafine. Other anti-fungal agents fun.ction by breaking down chitin (e.g.,
chitinase) or
immunosuppression (501 cream).
Parasiticides are agents that kill parasites directly. Such compounds are
known in the
art and are generally commercially available. Examples of parasiticides useful
for human
administration include but are not limited to albendazole, amphotericin B,
benznidazole,
bithionol, chloroquine HCI, chloroquine phosphate, clindamycin,
dehydroemetine,
diethylcarbamazine, diloxanide furoate, eflornithine, furazolidaone,
glucocorticoids,
halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine
antimoniate,
melarsoprol, metrifonate, metronidazole, niclosarnide, nifurtimox,
oxamniquine,
paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine
phosphate,
proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-
sulfadoxine,
quinacrine HCI, quinine sulfate, quinidine gluconate, spiramycin,
stibogluconate sodium
(sodium antimony gluconate), suramin, tetracycline, doxycycline,
thiabendazole, tinidazole,
trimethroprim-sulfamethoxazole, and tryparsamide.
Anti-cancer tlierapy
The TLR7/8 ligand and adaptor oligonucleotide combination of the invention can
be
used in conjunction with anti-cancer therapies.
Anti-cancer therapies include cancer medicaments, radiation, and surgical
procedures.
As used herein, a "cancer medicament" refers to an agent which is administered
to a subject
for the purpose of treating a cancer. Various types of medicaments for the
treatment of cancer
are described herein. For the purpose of this specification, cancer
medicaments are classified
as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone
therapy,
and biological response modifiers.
The chemotherapeutic agent may be selected from the group consisting of
methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing
chloroethylnitrosoureas,
5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol,
fragyline,
Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566,
RAS
famesyl transferase inhibitor, famesyl transferase inhibitor, MMP,
MTA/LY231514, Alimta,
LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/Topotecan, PKC412,
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Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Surarnin, Batimastat,
E7070, BCH-
4556, CS-682, 9-AC, AG3340, AG3433, InceUVX-710, VX-853, ZD0101, ISI641, ODN
698,
TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,
Lemonal
DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium
derivative,
Temodal/Temozolomide, EvacetJliposomal doxorubicin, Yewtaxan/Paclitaxel,
Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral
paclitaxel, Oral
Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609
(754)/RAS oncogene inhibitor, BMS- 18275 1/oral platinum, UFT(Tegafur/Uracil),
Ergamisol/Levaxn.isole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole,
Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine,
Paxex/Paclitaxel,
Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,
Fludara/Fludarabine,
Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU
103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD
0473/Anormed,
YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide,
Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin,
Plantinol/cisplatin,
Vepeside/Etoposide, ZD 9331, Taxotere/Docetaxel, prodrug of guanine
arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide,
Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,
Cytarabine HCI,
Dactinomycin, Daunorubicin HCI, Estramustine phosphate sodium, Etoposide (VP16-
213),
Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide),
Ifosfamide,
Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor
analogue), Lomustine
(CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna,
Mitotane (o.p'-
DDD), Mitoxantrone HCI, Octreotide, Plicamycin, Procarbazine HCI,
Streptozocin,
Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-
AMSA),
Azacitidine, Erthropoietin, Hexamethyhnelamine (HMM), Interleukin 2,
Mitoguazone
(methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin
(2'deoxycoformycin), Semustine (methyl-CCNU}, Teniposide (VM-26) and Vindesine
sulfate, but it is not so limited.
The immunotherapeutic agent may be selected from the group consisting of
3622W94,
4B5, ANA Ab, anti-FLK-2, anti-VEGF, ATRAGEN, AVASTIN (bevacizumab; Genentech),
BABS, BEC2, BEXXAR (tositumomab; GlaxoSmithKline), C225, CAMPATH
(alemtuzumab; Genzyme Corp.), CEACIDE, CMA 676, EMD-72000, ERBITUX (cetuximab;
ImClone Systems, Inc.), Gliomab-H, GNI-250, HERCEPTIN (trastuzumab;
Genentech),
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IDEC-Y2B8, ImmuRAIT-CEA, ior c5, ior egf.r3, ior t6, LDP-03, LymphoCide, MDX-
11,
MDX-22, MDX-210, MDX-220, MDX-260, MDX-447, MELIIVIMUNE-1, MELIMMUNE-
2, Monopharm-C, NovoMAb-G2, Oncolym, OV103, Ovarex, Panorex, Pretarget,
Quadramet,
Ributaxin, RITUXAN (rituximab; Genentech), SMART 1D10 Ab, SMART ABL 364 Ab,,
SMART M195, TNT, and ZENAPAX (daclizunlab; Roche), but it is not so limited.
The cancer vaccine may be selected from the group consisting of EGF, Anti-
idiotypic
cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate
vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-Vax, STn-KHL theratope, BLP25 (MUC-
1),
liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen
vaccines,
MVA-based vaccine, PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and
ImmuCyst/TheraCys, but it is not so limited.
Anti-allergy naedicaments
The TLR7/8 ligand and adaptor oligonucleotide of the invention can be used in
conjunction with anti-allergy medicaments.
Conventional methods for treating or preventing allergy have involved the use
of
allergy medicaments or desensitization therapies. Some evolving therapies for
treating or
preventing allergy include the use of neutralizing anti-IgE antibodies. Anti-
histamines and
other drugs which block the effects of chemical mediators of the allergic
reaction help to
regulate the severity of the allergic symptoms but do not prevent the allergic
reaction and
have no effect on subsequent allergic responses. Desensitization therapies are
performed by
giving small doses of an allergen, usually by injection under the skin, in
order to induce an
IgG-type response against the allergen. The presence of IgG antibody helps to
neutralize the
production of mediators resulting from the induction of IgE antibodies, it is
believed.
Initially, the subject is treated with a very low dose of the allergen to
avoid inducing a severe
reaction and the dose is slowly increased. This type of therapy is dangerous
because the
subject is actually administered the compounds which cause the allergic
response and severe
allergic reactions can result.
Allergy medicaments include, but are not limited to, anti-histamines,
corticosteroids,
and prostaglandin inducers. Anti-histamines are compounds which counteract
histamine
released by mast cells or basophils. These compounds are well known in the art
and
commonly used for the treatment of allergy. Anti-histamines include, but are
not limited to,
acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine,
buclizine,
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cetirizine, cetirizine analogues, chlorpheniramine, clemastine, CS 560,
cyproheptadine,
desloratadine, dexchlorphenirarnine, ebastine, epinastine, fexofenadine, HSR
609,
hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine,
norastemizole,
phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.
Corticosteroids include, but are not limited to, methylprednisolone,
prednisolone,
prednisone, beclomethasone, budesonide, dexamethasone, flunisolide,
fluticasone propionate,
and triamcinolone. Although dexamethasone is a corticosteroid having anti-
inflammatory
action, it is not regularly used for the treatment of allergy or asthma in an
inhaled form
because it is highly absorbed and it has long-term suppressive side effects at
an effective dose.
Dexamethasone, however, can be used according to the invention for treating
allergy or
asthma because when administered in combination with a composition of the
invention it can
be administered at a low dose to reduce the side effects. Some of the side
effects associated
with corticosteroid use include cough, dysphonia, oral thrush (candidiasis),
and in higher
doses, systemic effects, such as adrenal suppression, glucose intolerance,
osteoporosis, aseptic
necrosis of bone, cataract formation, growth suppression, hypertension, muscle
weakness,
skin thinning, and easy bruising. Barnes & Peterson (1993) Am Rev Respir Dis
148:S 1-S26;
and Kamada AK et al. (1996) Am JRespir Crit Care Med 153:1739-48.
Anti-astlima medicaments
The TLR 7/8 ligand and adaptor oligonucleotide combination of the invention
can be
used in conjunction with anti-asthma medicaments.
Medications for the treatment of asthma (i.e., anti-asthma medicaments) are
generally
separated into two categories, quick-relief medications and long-term control
medications.
Asthma patients take the long-term control medications on a daily basis to
achieve and
maintain control of persistent asthma. Long-term control medications include
anti-
inflammatory agents such as corticosteroids, chromolyn sodium and nedocromil;
long-acting
bronchodilators, such as long-acting (32-agonists and methylxanthines; and
leukotriene
modifiers. The quick-relief medications include short-acting (32 agonists,
anti-cholinergics,
and systemic corticosteroids. There are many side effects associated with each
of these drugs
and none of the drugs alone or in combination is capable of preventing or
completely treating
asthma.
Asthma medicaments include, but are not limited, PDE-4 inhibitors,
bronchodilatorlbeta-2 agonists, K+ channel openers, VLA-4 antagonists,
neurokin
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antagonists, thromboxane A2 (TXA2) synthesis inhibitors, xanthines,
arachidonic acid
antagonists, 5 lipoxygenase inhibitors, TXA2 receptor antagonists, TXA2
antagonists,
inhibitor of 5-lipox activation proteins, and protease inhibitors.
Bronchodilator/Pa agonists are a class of compounds which cause
bronchodilation or
smooth muscle relaxation. Bronchodilator/(32 agonists include, but are not
limited to,
salmeterol, salbutamol, albuterol, terbutaline, D2522/formoterol, fenoterol,
bitolterol,
pirbuerol methylxanthines and orciprenaline. Long-acting (32 agonists and
bronchodilators are
compounds which are used for long-term prevention of symptoms in addition to
the anti-
inflanunatory therapies. Long-acting (32 agonists include, but are not limited
to, salmeterol
and albuterol. These compounds are usually used in combination with
corticosteroids and
generally are not used without any inflanunatory therapy. They have been
associated with
side effects such as tachycardia, skeletal muscle tremor, hypokalemia, and
prolongation of
QTc interval in overdose.
Methylxanthines, including for instance theophylline, have been used for long-
term
control and prevention of symptoms. These compounds cause bronchodilation
resulting from
phosphodiesterase inhibition and likely adenosine antagonism. Dose-related
acute toxicities
are a particular problem with these types of compounds. As a result, routine
serum
concentration must be monitored in order to account for the toxicity and
narrow therapeutic
range arising from individual differences in metabolic clearance. Side effects
include
tachycardia, tachyarrhythmias, nausea and vomiting, central nervous system
stimulation,
headache, seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting
(32 agonists
include, but are not limited to, albuterol, bitolterol, pirbuterol, and
terbutaline. Some of the
adverse effects associated with the administration of short-acting (32
agonists include
tachycardia, skeletal muscle tremor, hypokalemia, increased lactic acid,
headache, and
hyperglycemia.
Chromolyn sodium and nedocromil are used as long-term control medications for
preventing primarily asthma symptoms arising from exercise or allergic
symptoms arising
from allergens. These compounds are believed to block early and late reactions
to allergens
by interfering with chloride channel function. They also stabilize mast cell
membranes and
inhibit activation and release of mediators from inosineophils and epithelial
cells. A four to
six week period of administration is generally required to achieve a maximum
benefit.
Anticholinergics are generally used for the relief of acute bronchospasm.
These
compounds are believed to function by competitive inhibition of muscarinic
cholinergic
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receptors. Anticholinergics include, but are not limited to, ipratropium
bromide. These
compounds reverse only cholinerigically-mediated bronchospasm and do not
modify any
reaction to antigen. Side effects include drying of the mouth and respiratory
secretions,
increased wheezing in some individuals, and blurred vision if sprayed in the
eyes.
The compositions of the invention may also be useful for treating airway
remodeling.
Airway remodeling results from smooth muscle cell proliferation and/or
submucosal
thickening in the airways, and ultimately causes narrowing of the airways
leading to restricted
airflow. The compositions of the invention may prevent further remodeling and
possibly even
reduce tissue build-up resulting from the remodeling process.
Adjuvants
The TLR7/8 ligands and adaptor oligonucleotides of the invention may be used
in
combination with other.agents, such as adjuvants. An adjuvant, as used herein,
refers to a
substance other than an antigen, a TLR7/8 ligand and an adaptor
oligonucleotide that
enhances immune cell activation in response to an antigen, e.g., a humoral
and/or cellular
immune response. Adjuvants promote the accumulation and/or activation of
accessory cells
to enhance antigen-specific immune responses. Adjuvants are used to enhance
the efficacy of
vaccines, i.e., antigen-containing compositions used to induce protective
immunity against the
antigen.
Adjuvants in general include adjuvants that create a depot effect, immune-
stimulating
adjuvants, and adjuvants that create a depot effect and stimulate the immune
system. An
adjuvant that creates a depot effect, as used herein, is an adjuvant that
causes the antigen to be
slowly released in the body, thus prolonging the exposure of immune cells to
the antigen.
This class of adjuvants includes but is not limited to alum (e.g., aluminum
hydroxide,
aluminum phosphate); emulsion-based formulations including mineral oil, non-
mineral oil,
water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as
Seppic ISA series
of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France);
MF-59 (a
squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron
Corporation,
Emeryville, Calif.); and PROVAX (an oil-in-water emulsion containing a
stabilizing
detergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation, San
Diego,
Calif.).
An immune-stimulating adjuvant is an adjuvant that causes activation of a cell
of the
immune system. It may, for instance, cause an immune cell to produce and
secrete cytokines.
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This class of adjuvants includes but is not limited to saponins purified from
the bark of the Q.
saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with
HPLC
fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);
poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research
Institute, USA);
derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi
ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and
threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide
related to
lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor
(a purified
Leishmania protein; Corixa Corporation, Seattle, Wash.). This class of
adjuvants also
includes CpG DNA.
Adjuvants that create a depot effect and stimulate the immune system are those
compounds which have both of the above-identified functions. This class of
adjuvants
includes but is not limited to ISCOMS (immunostimulating complexes which
contain mixed
saponins, lipids and form virus-sized particles with pores that can hold
antigen; CSL,
Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is
an oil-in-
water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB],
Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which
contains alum
and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as
CRL 1005
(these contain a linear chain of hydrophobic polyoxypropylene flanked by
chains of
polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation
(SAF, an
oil-in-water emulsion containing Tween 80 and a nonionic block copolymer;
Syntex
Chemicals, Inc., Boulder, Colo.).
The adjuvant may also be a lipopeptide such as Pam3Cys, a cationic
polysaccharide
such as chitosan, or a cationic peptide such as protamine.
Cytokines
The TLR7/8 ligand and adaptor oligonucleotide combination of the invention may
also
be used with cytokines. Cytokines are soluble proteins and glycoproteins
produced by many
types of cells that mediate inflammatory and immune reactions. Cytokines
mediate
communication between cells of the immune system, acting locally as well as
systemically to
recruit cells and to regulate their function and proliferation. Categories of
cytokines include
mediators and regulators of innate immunity, mediators and regulators of
adaptive immunity,
and stimulators of hematopoiesis. Included among cytokines are interleukins
(e.g., IL-1, IL-2,
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IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, IL-16, IL-
17, IL-18, and interleukins 19-32 (IL-19 - IL-32), among others), chemokines
(e.g., IP-10,
RANTES, MIP-la, MIP-1(3, M]P-3(x, MCP-1, MCP-2, MCP-3, MCP-4, eotaxin, I-TAC,
and
BCA-1, among others), as well as other cytokines including type 1 interferons
(e.g., IFN-a
and IFN-(3), type 2 interferon (e.g., IFN-y), tumor necrosis factor-alpha (TNF-
a),
transforming growth factor-beta (TGF-(3), and various colony stimulating
factors (CSFs),
including GM-CSF, G-CSF, and M-CSF.
Antigens
The TLR7/8 ligand and adaptor oligonucleotide combination of the invention can
be
used with an antigen, optionally in a vaccine formulation. An "antigen" as
used herein refers
to any molecule capable of being recognized by a T-cell antigen receptor or B-
cell antigen
receptor. The term broadly includes any type of molecule which is recognized
by a host
immune system as being foreign. Antigens generally include but are not limited
to cells, cell
extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide
conjugates, peptide
and non-peptide mimics of polysaccharides and other molecules, small
molecules, lipids,
lipoproteins, glycolipids, polysaccharides, carbohydrates, viruses and viral
extracts, and
multicellular organisms such as parasites, and allergens. With respect to
antigens that are
proteins, polypeptides, or peptides, such antigens can include nucleic acid
molecules encoding
such antigens. Antigens more specifically include, but are not limited to,
cancer antigens,
which include cancer cells and molecules expressed in or on cancer cells;
microbial antigens,
which include microbes and molecules expressed in or on microbes; and
allergens.
Accordingly, the invention in certain embodiments provides vaccines for
cancers, infectious
agents, and allergens.
In various embodiments the antigen is a microbial antigen, a cancer antigen,
or an
allergen. A "microbial antigen" as used herein is an antigen of a
microorganism and includes
but is not limited to viruses, bacteria, parasites, and fungi. Such antigens
include the intact
microorganism as well as natural isolates and fragments or derivatives thereof
and also
synthetic compounds which are identical to or similar to natural microorganism
antigens and
induce an immune response specific for that microorganism. A compound is
similar to a
natural microorganism antigen if it induces an immune response (humoral and/or
cellular) to a
natural microorganism antigen. Such antigens are used routinely in the art and
are well
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known to those of ordinary skill in the art. The invention intends to embrace
antigens derived
from any of the infectious agents described herein.
As used herein, the terms "cancer antigen" and "tumor antigen" are used
interchangeably to refer to a compound, such as a peptide, protein,
lipoprotein or
glycoprotein, which is associated with a tumor or cancer cell and which is
capable of
provoking an immune response when expressed on the surface of an antigen-
presenting cell in
the context of a major histocompatibility complex (MHC) molecule. Cancer
antigens which
are differentially expressed by cancer cells and can thereby be exploited in
order to target
cancer cells. Cancer antigens are antigens which can potentially stimulate
apparently
tumor-specific immune responses. Some of these antigens are encoded, although
not
necessarily expressed, by normal cells. These antigens can be characterized as
those which
are normally silent (i.e., not expressed) in normal cells, those that are
expressed only at certain
stages of differentiation, and those that are temporally expressed such as
embryonic and fetal
antigens. Other cancer antigens are encoded by mutant cellular genes, such as
oncogenes
(e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion
proteins resulting
from internal deletions or chromosomal translocations. Still other cancer
antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor viruses.
Cancer antigens can be prepared from cancer cells either by preparing crude
extracts
of cancer cells, for example, as described in Cohen PA et al. (1994) Cancer
Res 54:1055-8, by
partially purifying the antigens, by recombinant technology, or by de novo
synthesis of known
antigens. Cancer antigens include but are not limited to antigens that are
recombinantly
expressed, an immunogenic portion of, or a whole tumor or cancer or cell
thereof. Such
antigens can be isolated or prepared recombinantly or by any other means known
in the art.
Examples of tunlor antigens include MAGE, MART-1/Melan-A, gplOO, dipeptidyl
peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin
b,
colorectal associated antigen (CRC)--C017-1A/GA733, carcinoembryonic antigen
(CEA) and
its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, prostate specific
antigen (PSA) and
its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane
antigen
(PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g.,
MAGE-Al,
MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8,
MAGE-A9, MAGE-A10, MAGE-Al l, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4,
MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,
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GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,
1VIUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p2lras, RCAS1, a-
fetoprotein,
E-cadherin, a-catenin, (3-catenin and y-catenin, p120ctn, gpl00Pmel117, PRAME,
NY-ESO-1,
cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-
idiotype, p15,
gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus
proteins,
Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-
1, brain
glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1
and
CT-7, and c-erbB-2. This list is not meant to be limiting.
An "allergen" as used herein is a molecule capable of provoking an immune
response
characterized by production of IgE. An allergen is also a substance that can
induce an allergic
or asthmatic response in a susceptible subject. Thus, in the context of this
invention, the term
allergen means a specific type of antigen which can trigger an allergic
response which is
mediated by IgE antibody.
The list of allergens is enormous and can include pollens, insect venoms,
animal
dander dust, fungal spores and drugs (e.g., penicillin). Examples of natural
animal and plant
allergens include proteins specific to the following genuses: Canis
(Canisfamiliaris);
Dermatophagoides (e.g., Dernaatophagoides farinae); Felis (Felis domesticus);
Ambrosia
(Ambrosia artemisiifolia); Lolium (e.g., Lolium perenne and Loliunz
multiflorum);
Cryptomeria (Cryptomeriajaponica); Alternaria (Alternaria alterrzata); Alder;
Alnus (Alnus
gultinosa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea
europa);
Artemisia (Artemisia vulgaris); Plantago (e.g., Plantago lanceolata);
Parietaria (e.g.,
Parietaria officinalis and Parietariajudaica); Blattella (e.g., Blattella
germanica); Apis (e.g.,
Apis multiflorum); Cupressus (e.g., Cupressus semperviretzs, Cupressus
arizonica and
Cupressus macrocarpa); Juniperus (e.g., Juniperus sabinoides, Juniperus
virginiana,
Juniperus communis, and Juniperus ashei); Thuya (e.g., Thuya orientalis);
Chamaecyparis
(e.g., Chamaecyparis obtusa); Periplaneta (e.g., Periplaneta americana);
Agropyron (e.g.,
Agropyron repens); Secale (e.g., Secale cereale); Triticum (e.g., Triticum
aestivum); Dactylis
(e.g., Dactylis glomerata); Festuca (e.g., Festuca elatior); Poa (e.g.,
Poapratensis and Poa
compressa); Avena (e.g., Avena sativa); Holcus (e.g., Holcus lanatus);
Anthoxanthum (e.g.,
Antlzoxanthum odoratum); Arrhenatherum (e.g., Arrhenatherum elatius); Agrostis
(e.g.,
Agrostis alba); Phleum (e.g., Plaleum pratense); Phalaris (e.g., Phalaris
arundinacea);
Paspalum (e.g., Paspalunz notatum); Sorghum (e.g., Sorglaum halepensis); and
Bromus (e.g.,
Bronzus inernzis).
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Antibodies and ADCC
The TLR7/8 ligand and adaptor oligonucleotide combination of the invention
also
increases natural killer cell lytic activity and antibody-dependent cellular
cytotoxicity
(ADCC). ADCC can be performed using the combination with an antibody specific
for a
cellular target, such as a cancer cell, leading the subject's immune system to
kill the tumor
cell. The antibodies useful in the ADCC procedure include antibodies which
interact with a
cell in the body. Many such antibodies specific for cellular targets have been
described in the
art and many are commercially available. In one embodiment the antibody is an
IgG
antibody.
Therapeutic antibodies useful in the invention may be specific for microbial
antigens
(e.g., bacterial, viral, parasitic or fungal antigens), cancer or tumor-
associated antigens and
self antigens. Preferred antibodies are those that recognize and bind to
antigens present on or
in a cell. Examples of suitable antibodies include but are not limited to
RituxanTM (rituximab,
anti-CD20 antibody), Herceptin (trastuzumab), Quadramet, Panorex, IDEC-Y2B8,
BEC2,
C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210,
MDX-1 1, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447,
MELIMMUNE-2, MELI]VIlVIUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-
H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior
c5,
BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab, CC49
(mAb B72.3), ImmuRAIT-CEA, anti-IL-4 antibody, an anti-IL-5 antibody, an anti-
IL-9
antibody, an anti-Ig antibody, an anti-IgE antibody, serum-derived hepatitis B
antibodies,
recombinant hepatitis B antibodies, and the like.
Other antibodies similarly useful for the invention include alemtuzumab (B
cell
chronic lymphocytic leukemia), gemtuzumab ozogamicin (CD33+ acute myeloid
leukemia),
hP67.6 (CD33+ acute myeloid leukemia), infliximab (inflammatory bowel disease
and
rheumatoid arthritis), etanercept (rheumatoid arthritis), tositumomab, MDX-
210,
oregovomab, anti-EGF receptor mAb, MDX-447, anti-tissue factor protein (TF),
(Sunol); ior-
c5, c5, edrecolomab, ibritumomab tiuxetan, anti-idiotypic mAb mimic of
ganglioside GD3
epitope, anti-HLA-Dr10 mAb, anti-CD33 humanized mAb, anti-CD52 humAb, anti-CD1
mAb (ior t6), MDX-22, celogovab, anti-17-1A mAb, bevacizumab, daclizumab, anti-
TAG-72
(MDX-220), anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-
Mel-1),
anti-idiotypic mAb mimic of high molecular weight proteoglycan (I-Mel-2), anti-
CEA Ab,
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hmAbH11, anti-DNA or DNA-associated proteins (histones) mAb, Gliomab-H mAb,
GNI-
250 mAb, anti-CD22, CMA 676), anti-idiotypic human mAb to GD2 ganglioside, ior
egf/r3,
anti-ior c2 glycoprotein mAb, ior c5, anti-FLK-2/FLT-3 mAb, anti-GD-2
bispecific mAb,
antinuclear autoantibodies, anti-HLA-DR Ab, anti-CEA mAb, palivizumab,
bevacizumab,
alemtuzumab, BLyS-mAb, anti-VEGF2, anti-Trail receptor; B3 mAb, mAb BR96,
breast
cancer; and Abx-Cbl mAb.
Immunoglobulin Therapy
The agents of the invention can also be used with normal and hyper-immune
globulin
therapy. Normal immune globulin therapy utilizes an antibody product which is
prepared
from the serum of normal blood donors and pooled. This pooled product contains
low titers
of antibody to a wide range of antigens such as those of infectious pathogens
(e.g., bacteria,
viruses such as hepatitis A, parvovirus, enterovirus, fungi and parasites).
Hyper-immune
globulin therapy utilizes antibodies which are prepared from the serum of
individuals who
have high titers of an antibody to a particular antigen. Examples of hyper-
immune globulins
include zoster immune globulin (useful for the prevention of varicella in
immunocompromised children and neonates), human rabies immunoglobulin (useful
in the
post-exposure prophylaxis of a subject bitten by a rabid animal), hepatitis B
immune globulin
(useful in the prevention of hepatitis B virus, especially in a subject
exposed to the virus), and
RSV immune globulin (useful in the treatment of respiratory syncytial virus
infections).
Dosing and Administration
The ligand and oligonucleotide can be formulated in separate coinpositions
that are
used together to achieve a desired effect. For example, an adaptor
oligonucleotide and a
TLR7/8 ligand can be mixed together and administered to a subject or placed in
contact with a
cell substantially simultaneously as a combination. As another example, an
adaptor
oligonucleotide and a TLR7/8 ligand can be administered to a subject or placed
in contact
with a cell at different times. As yet another example, an adaptor
oligonucleotide and a
TLR7/8 ligand can be administered to a subject at different sites.
As mentioned above, the term "effective amount" refers generally to the amount
necessary or sufficient to realize a desired biologic effect. Combined with
the teachings
provided herein, by choosing among the various active compounds and weighing
factors such
as potency, relative bioavailability, patient body weight, severity of adverse
side-effects and
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preferred mode of administration, an effective prophylactic or therapeutic
treatment regimen
can be planned which does not cause substantial toxicity and yet is entirely
effective to treat
the particular subject. The effective amount for any particular application
can vary depending
on such factors as the disease or condition being treated, the particular
oligonucleotide being
administered, the size of the subject, or the severity of the disease or
condition. One of
ordinary skill in the art can empirically determine the effective amount of a
particular adaptor
oligonucleotide and TLR7/8 ligand and/or antigen and/or other therapeutic
agent witliout
necessitating undue experimentation.
Subject doses of the compounds described herein for systemic or local delivery
typically range from about 10 ng to 10 mg per administration, which degending
on the
application could be given daily, weekly, or monthly and any other amount of
time
therebetween or as otherwise required. More typically systemic or local doses
range from
about 1 gg to 1 mg per administration, and most typically from about 10 g to
100 g, with 2
- 4 administrations being spaced days or weeks apart. Higher doses may be
required for
parenteral administration. In some embodiments, however, parenteral doses for
these
purposes may be used in a range of 5 to 10,000 times higher than the typical
doses described
above.
For any compound described herein the therapeutically effective amount can be
initially determined from animal models. The applied dose can be adjusted
based on the
relative bioavailability and potency of the administered compound. Adjusting
the dose to
achieve maximal efficacy based on the methods described above and other
methods as are
well-known in the art is well within the capabilities of the ordinarily
skilled artisan.
Route ofAdministration
For clinical use the TLR7/8 ligand and adaptor oligonucleotide combination of
the
invention can be administered alone or formulated as a delivery complex via
any suitable
route of administration that is effective to achieve the desired therapeutic
result. Routes of
administration include enteral and parenteral routes of administration.
Examples of enteral
routes of administration include oral, gastric, intestinal, and rectal.
Nonlimiting examples of
parenteral routes of administration include intravenous, intramuscular,
subcutaneous,
intraperitoneal, intrathecal, local injection, topical, nasal, mucosal, and
pulmonary.
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Delivery Vehicles
The adaptor oligonucleotide and a TLR7/8 ligand and/or the antigen and/or
other
therapeutics may be administered alone (e.g., in saline or buffer) or using
any delivery vehicle
known in the art.
For example, the TLR7/8 ligand and adaptor oligonucleotide combination of the
invention may be directly administered to the subject or may be administered
in conjunction
with a nucleic acid delivery complex. A nucleic acid delivery complex shall
mean a nucleic
acid molecule associated with (e.g., ionically or covalently bound to; or
encapsulated within)
a targeting means (e.g., a molecule that results in higher affinity binding to
target cell.
Examples of nucleic acid delivery complexes include nucleic acids associated
with a sterol
(e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome),
or a target cell specific
binding agent (e.g., a ligand recognized by target cell specific receptor).
Preferred complexes
may be sufficiently stable in vivo to prevent significant uncoupling prior to
internalization by
the target cell. However, the complex can be cleavable under appropriate
conditions within
the cell so that the oligonucleotide is released in a functional form.
Other delivery vehicles that have been described and can be used according to
the
invention include cochleates (Gould-Fogerite et al., 1994, 1996); emulsomes
(Vancott et al.,
1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991,
Hu et., 1998,
Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989,
1992, de Haan
1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli,
bacillus Calmette-
Guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998,
Chatfield et al.,
1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g.,
Vaccinia, adenovirus,
Herpes simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et
al., 1998, Flexner
et al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et
al., 1996, Maloy
et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al.,
1989); nucleic acid
vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada
et al., 1997, Ishii
et al., 1997); polymers (e.g., carboxymethylcellulose, chitosan) (Hamajima et
al., 1998,
Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes
(Vancott et al.,
1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998);
transgenic plants
(Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); Virosomes (Gluck
et al., 1992,
Mengiardi et al., 1995, Cryz et al., 1998); virus-like particles (Jiang et
al., 1999, Leibl et al.,
1998). Other delivery vehicles are known in the art.
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Pharrnaceutical Conapositions
The TLR7/8 ligand and adaptor oligonucleotide combinations of the invention
can be
formulated as pharmaceutical compositions additionally comprising a
pharmaceutically
acceptable carrier. A pharmaceutically-acceptable carrier means one or more
compatible
solid or liquid filler, diluents or encapsulating substances which are
suitable for administration
to a human or other vertebrate animal. A carrier is an organic or inorganic
ingredient, natural
or synthetic, witli which the active ingredient(s) are combined to facilitate
the desired effect.
The components of a pharmaceutical composition are co-mingled in a manner such
that there
is no interaction which would substantially impair the desired pharmaceutical
efficiency.
For oral administration, the compounds (i.e., adaptor oligonucleotide and a
TLR7/8
ligand, antigens and/or other therapeutic agents) can be formulated readily by
combining the
active compound(s) with pharmaceutically acceptable carriers well known in the
art. Such
carriers enable the compounds of the invention to be formulated as tablets,
pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral
ingestion by a
subject to be treated. Pharmaceutical preparations for oral use can be
obtained as solid
excipient, optionally grinding a resulting mixture, and processing the mixture
of granules,
after adding suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable
excipients are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize starch, wheat
starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-
cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate. Optionally the oral
formulations may
also be formulated in saline or buffers for neutralizing internal acid
conditions or may be
adrninistered without any carriers.
Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar
solutions may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable
organic solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or
dragee coatings for identification or to characterize different combinations
of active
compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules
made
of gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol
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or sorbitol. The push-fit capsules can contain the active ingredients in
admixture with filler
such as lactose, binders such as starches, and/or lubricants such as talc or
magnesium stearate
and, optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or
suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycols.
In addition, stabilizers may be added. Microspheres formulated for oral
administration may
also be used. Such microspheres have been well defined in the art. All
formulations for oral
administration should be in dosages suitable for such administration.
For buccal administration, the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
The compounds may be administered by inhalation to pulmonary tract, especially
the
bronchi and more particularly into the alveoli of the deep lung, using
standard inhalation
devices. The compounds may be delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide,
or other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined
by providing a valve to deliver a metered amount. An inhalation apparatus may
be used to
deliver the compounds to a subject. An inhalation apparatus, as used herein,
is any device for
administering an aerosol, such as dry powdered form of the compounds. This
type of
equipment is well known in the art and has been described in detail, such as
that description
found in Remington: The Science and Practice of Pharmacy, 19th Edition, 1995,
Mac
Publishing Company, Easton, Pennsylvania, pages 1676-1692. Many U.S. patents
also
describe inhalation devices, such as U.S. Pat. No. 6,116,237.
"Powder" as used herein refers to a composition that consists of finely
dispersed solid
particles. Preferably the compounds are relatively free flowing and capable of
being
dispersed in an inhalation device and subsequently inhaled by a subject so
that the compounds
reach the lungs to permit penetration into the alveoli. A "dry powder" refers
to a powder
composition that has a moisture content such that the particles are readily
dispersible in an
inhalation device to form an aerosol. The moisture content is generally below
about 10% by
weight (% w) water, and in some embodiments is below about 5% w and preferably
less than
about 3% w. The powder may be formulated with polymers or optionally may be
formulated
with other materials such as liposomes, albumin and/or other carriers.
Aerosol dosage and delivery systems may be selected for a particular
therapeutic
application by one of skill in the art, such as described, for example in
Gonda, I. "Aerosols
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for delivery of therapeutic and diagnostic agents to the respiratory tract,"
in Critical Reviews
in Therapeutic Drug Carrier Systems, 6:273-313 (1990), and in Moren, "Aerosol
dosage
forms and formulations," in Aerosols in Medicine. Principles, Diagnosis and
Therapy,
Moren, et al., Eds., Elsevier, Amsterdam, 1985.
The compounds, when it is desirable to deliver them systemically, may be
formulated
for parenteral administration by injection, e.g., by bolus injection or
continuous infusion.
Formulations for injection maybe presented in unit dosage form, e.g., in
ampoules or in
multi-dose containers, with an added preservative. The compositions may take
such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles, and may
contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutica.l formulations for parenteral administration include aqueous
solutions
of the active compounds in water-soluble form. Additionally, suspensions of
the active
compounds may be prepared as appropriate oily injection suspensions. Suitable
lipophilic
solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may
contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Optionally, the suspension may also contain
suitable stabilizers
or agents which increase the solubility of the compounds to allow for the
preparation of
highly concentrated solutions.
Alternatively, the active compounds may be in powder form for constitution
with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal or vaginal compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such as
cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may also
be
formulated as a depot preparation. Such long acting formulations may be
formulated with
suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil)
or ion exchange resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble
salt.
The pharmaceutical compositions also may include suitable solid or gel phase
carriers
or excipients. Examples of such carriers or excipients include but are not
limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose derivatives,
gelatin, and
polymers such as polyethylene glycols.
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Suitable liquid or solid pharmaceutical preparation forms are, for example,
aqueous or
saline solutions for inhalation, microencapsulated, encochleated, coated onto
microscopic
gold particles, contained in liposomes, nebulized, aerosols, pellets for
iinplantation into the
skin, or dried onto a sharp object to be scratched into the skin. The
pharmaceutical
compositions also include granules, powders, tablets, coated tablets,
(micro)capsules,
suppositories, syrups, emulsions, suspensions, creams, drops or preparations
with protracted
release of active compounds, in whose preparation excipients and additives
and/or auxiliaries
such as disintegrants, binders, coating agents, swelling agents, lubricants,
flavorings,
sweeteners or solubilizers are customarily used as described above. The
pharmaceutical
compositions are suitable for use in a variety of drug delivery systems. For a
brief review of
methods for drug delivery, see Langer R (1990) Science 249:1527-33, which is
incorporated
herein by reference.
The agents of the invention and optionally other therapeutics and/or antigens
may be
administered per se (neat) or in the form of a pharmaceutically acceptable
salt. When used in
medicine the salts should be pharmaceutically acceptable, but non-
phamlaceutically
acceptable salts may conveniently be used to prepare pharmaceutically
acceptable salts
thereof. Such salts include, but are not limited to, those prepared from the
following acids:
hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic,
salicylic, p-toluene
sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic,
naphthalene-2-
sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline
metal or
alkaline earth salts, such as sodium, potassium or calcium salts of the
carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric
acid and a
salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and
a salt (0.8-2%
w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v);
chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-
0.02% w/v).
The pharmaceutical compositions of the invention contain an effective amount
of the
agents of the invention and optionally antigens and/or other therapeutic
agents optionally
included in a pharmaceutically acceptable carrier. The term pharmaceutically
acceptable
carrier means one or more compatible solid or liquid filler, diluents ox
encapsulating
substances which are suitable for administration to a human or other
vertebrate animal. The
term carrier denotes an organic or inorganic ingredient, natural or synthetic,
with which the
active ingredient is combined to facilitate the application. The components of
the
pharmaceutical compositions also are capable of being commingled with the
compounds of
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the present invention, and with each other, in a manner such that there is no
interaction which
would substantially impair the desired pharmaceutical efficiency.
Screening Metliods
The invention provides in yet other aspects methods for identifying TLR7/8
ligands
and/or adaptor oligonucleotides. The method steps vary depending on the agent
being
identified (i.e., ligand versus oligonucleotide), whether the ligand is a
TLR7, a TLR8 or a
TLR7/8 ligand, the type of cells used in the method, and the like.
Based on the teachings set forth herein, a TLR7 ligand may be identified by
its ability
to stimulate TLR7 signaling alone, to stimulate TL8 signaling (starting from a
background
level) in the presence of an adaptor oligonucleotide, andlor by inhibition of
TLR7 stimulation
in the presence of the adaptor oligonucleotide. A TLR8 ligand may be
identified by enhanced
TLR8 signaling (starting from an above background level) when used in
combination with an
adaptor oligonucleotide. A ligand that stimulates both TLR7 and 8 (when used
alone) can be
identified by any combination of these activities. An adaptor oligonucleotide
may be
identified by its ability to inhibit TLR7 stimulation by a TLR7 ligand, induce
TLR8
stimulation by a TLR7 ligand, and/or enhance TLR8 stimulation from a TLR8
ligand.
The methods may be performed in vivo or in vitro. In vitro assays are
documented in
the Examples. Suitable readouts include but are not limited to IL-12, TNF-
alpha and/or IFN-
gamma for TLR8 stimulation, and IFN-alpha for TLR7 stimulation. The assays may
alternatively employ reporter constructs having reporter genes linked to
transcriptional
regulation elements that are responsive to TLR7 and/or TLRB signaling (e.g.,
NF-xB
responsive elements). These constructs are described in the Examples. The
assays may
measure transcriptional up- or down-regulation, translational up- or down-
regulation, protein
expression and/or secretion, and the like.
As an example, a TLR8 ligand assay may involve contacting a TLR8-expressing
cell
(or cell population) with a test ligand in the presence and absence of an
adaptor
oligonucleotide. The cell preferably is one that expresses TLR8 but not TLR7
or TLR9.
TLR8 signaling is measured from the cell in response to the test ligand in the
presence and
absence of the oligonucleotide. A test ligand having a TLR8 signaling profile
that is
enhanced in the presence of the oligonucleotide, as compared to in the absence
of the
oligonucleotide, is then identified as a TLR8 ligand. The assay may also
include an analysis
of TLR7 signaling using a TLR7-expressing cell that does not express TLR8 or
TLR9. This
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latter analysis can be used to exclude the possibility that the ligand is a
TLR7 ligand that
switches to TLR8 signaling in the presence of the oligonucleotide.
Results from a similar assay using known ligands is shown in FIG. 12. Both
loxoribine and immunosine (TLR7 specific ligands) stimulate TLR8 signaling in
the presence
of ODN 6056. Putative TLR7 specific ligands would be expected to have a
qualitatively
similar profile. TLR8 signaling by R-848 (a TLR7 and TLR8 ligand) is enhanced
in the
presence of ODN 6056. Putative TLR8 ligands would be expected to have a
qualitatively
similar profile. Ribavirin, which is neither a TLR7 nor a TLR8 ligand, does
not stimulate
TLR8 signaling in the presence or absence of ODN 6056, indicating the
specificity of the
assay for TLR7/8 ligands.
An adaptor oligonucleotide assay may involve contacting a TLR8-expressing cell
(or
cell population) with a test oligonucleotide in the presence and absence of a
TLR7, TLR8 or
TLR7/8 ligand. If the ligand is a TLR7 ligand (when used alone), the assay may
measure
TLR7 signaling inhibition and/or induced TLR8 signaling when the
oligonucleotide is used
with the ligand. If the ligand is a TLR8 ligand (when used alone), the assay
may measure
enhanced TLR8 signaling when the oligonucleotide is used with the ligand as
compared to the
effect of the ligand alone. If the ligand stimulates both TLR7 and TLR8 (when
used alone),
the assay may measure one or more of the above-noted readouts: The assay
results may be
compared to positive control assays (e.g. assays using a known adaptor
oligonucleotide), and
the like. The method may also include an assay for determining if the
oligonucleotide is a
TLR7 and/or TLR8 ligand itself.
These methods may be used to identify ligands which when used alone are weakly
stimulatory, but which when combined with oligonucleotides have new and/or
enhanced
signaling profiles.
The present invention is further illustrated by the following Examples, which
in no
way should be construed as further limiting.
Examples
MATERIALS & METHODS
Oligodeo.xynucleotides and reagents
All oligonucleotides were purchased from Biospring (Frankfurt, Germany) or
provided by Coley Pharmaceutical GmbH (Langenfeld, Germany), controlled for
identity and
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purity by Coley Pharmaceutical GmbH and had undetectable endotoxin levels
(<0.1EU/ml)
measured by the Limulus assay (BioWhittaker, Verviers, Belgium).
Oligonucleotides were
suspended in sterile, endotoxin-free Tris-EDTA (Sigma, Deisenhofen, Germany),
and stored
and handled under aseptic conditions to prevent both microbial and endotoxin
contamination.
All dilutions were carried out using endotoxin-free Tris-EDTA. Sequences of
oligonucleotides are listed in Tables 1 and 2. Loxoribine (7-allyl-7,8-dihydo-
8-oxo-
guanosine) (Sigma) was dissolved first in 1N NaOH, diluted in RPMI-medium and
pH was
adjusted to 7.4 with 1N HCl (19). R-848 (1-(2-hydroxy2-methylpropyl)-2-methyl-
lH-
imidazo[4,5-c]quinolin-4-amine) was commercially synthesized by GLSynthesis
(Worcester,
MA, USA) and dissolved in 10% DMSO. 7-Deaza-guanosine (ChemGenes, Wilmington,
MA, USA) was dissolved at 1M in 1N NaOH. Inosine (Sigma) was dissolved in H20.
All
dilutions were done in endotoxin-free Tris-EDTA.
TLR assays
Stably transfected HEK293 cells expressing the human TLR9, TLR8 or TLR7 were
described before (16,20). Briefly, HEK293 cells were transfected by
electroporation with
vectors expressing the respective human TLR and a 6xNF-kappaB-luciferase
reporter
plasmid. Stable transfectants (3x104 cells/well) were incubated with
loxoribine or R-848 in
the absence or presence of ODN for 16h at 37 C in a humidified incubator. Each
data point
was done in triplicate. Cells were lysed and assayed for luciferase gene
activity (using the
BriteLite kit from Perkin-Elmer, Zaventem, Belgium). Stimulation indices were
calculated in
reference to reporter gene activity of medium without addition of
oligonucleotide.
Cell purification
Peripheral blood buffy coat preparations from healthy human donors were
obtained
from the Blood Bank of the University of Dusseldorf (Germany) and PBMC were
purified by
centrifugation over Ficoll-Hypaque (Sigma). Cells were cultured in a
humidified incubator at
37 C in RPMI 1640 medium supplemented with 5% (v/v) heat inactivated human AB
serum
(BioWhittaker) or 10% (v/v) heat inactivated FCS, 2mM L-glutamine, 100U/ml
penicillin and
100 g/mi streptomycin (all from Sigma).
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Cytokine detection and flow cytometric analysis
PBMC were resuspended at a concentration of 5x106 cells/ml and added to 96
well
round-bottomed plates (250 1/well). PBMC were incubated with various ODN
and/or
loxoribine concentrations and culture supematants (SN) were collected after
the indicated
time points. If not used immediately, SN were stored at -20 C until required.
Amounts of
cytokines in the SN were assessed using a commercially available ELISA Kit
(for IL-12p40,
from BD Biosciences, Heidelberg, Germany), IFN-y and TNF-a (from Diaclone,
Besangon,
France) or an in-house ELISA for IFN-y developed using commercially available
antibodies
(PBL, New Brunswick, NJ, USA).
For intracellular staining, PBMC were incubated at 5x106 cells/ml in 96 well
round-
bottomed plates (250 1/well) with indicated amounts of oligonucleotides and/or
loxoribine
concentrations, and Brefeldin A solution (BD Biosciences) was added. PBMC were
incubated for 6h. For intracellular IFN-y staining, cells were incubated with
oligonucleotides
and/or loxoribine for 16h before addition of Brefeldin A solution. Cells were
harvested and
intracellular staining performed using Intraprep reagent according to the
manufacturer
protocol (Beckman-Coulter, Neuss, Germany). Cells were stained with
appropriate antibodies
for identification of monocytes (CD 14), B cells (CD 19), and NK cells (CD56+,
CD3-).
Flow cytometric data were acquired on a FACSCalibur and were analyzed using
the computer
program Ce1lQuest (both from BD Biosciences). All monoclonal antibodies (niAb)
for flow
cytometric analysis were purchased from BD Biosciences, except CD11c from
Diaclone,
CD14 from Innlrnunotech (Marseille, France) and CD123 from Miltenyi (Bergisch
Gladbach,
Germany). Human monocytes were isolated from whole PBMC using the CD14 cell
isolation
kit as described by the manufacturer (Miltenyi). To determine purity, cells
were stained with
mAb to CD11c and CD14 and identified by flow cytometry. In all experiments,
monocytes
were more than 95% pure. Purified monocytes (4x106 cells/ml) were incubated
with
increasing concentrations of ODN for 24h. PDC were enriched using the BDCA-4
pDC
isolation kit as described by the manufacturer (Miltenyi). PDC purification
was confirmed by
staining with mAb to CD123 (from Miltenyi), HLA-DR and CD11c (from BD
Biosciences).
Purity was approximately 85%. Cells (5x105 cells/ml, 250 Uwell) were cultured
for 24h
with or without oligonucleotides and loxoribine. IFN-a or IL-12p40 in the SN
was measured
as described above.
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RESULTS
Sequence-selective enhancement of TLRB-mediated signaling by co-incubation
with
homopolyiner oligonucleotide
HEK293 cells stably expressing hTLR8 and a NF-icB-luciferase reporter
construct
were incubated with R-848 (1-(2-hydroxy-2-methylpropyl)-2-methyl-lH-
imidazo[4,5-
c]quinolin-4amine) in the presence or absence of oligonucleotide. Influence of
co-incubation
with different oligonucleotides on TLR8-mediated NF-icB activation was then
analyzed.
An oligo(dT)17 hoinopolymer ODN with a phosphorothioate backbone, ODN 6056,
was identified that markedly increased the level of NF-xB activation
stimulated by R-848
(FIG. 1B). ODN 6056 alone did not stimulate considerable NF-xB activation (not
more than
2.5 fold above background) at concentrations up to 25 M in TLR8-expressing
cells
(unpublished data). The effect appeared to be oligonucleotide-dependent as not
all of the
oligonucleotides tested enhanced TLR8 signaling equally well (e.g., an
unrelated
heteropolymer (ODN 1982) did not strongly influence NF-xB activation by R-
848).
Co-incubation of R-848 with ODN 6056 not only increased efficacy but also the
potency of TLRB activation significantly. The EC50 of R-848 was already
strongly decreased
in the presence of 0.1 M ODN 6056 (0.1 M ODN 6056: EC50 (R-848) = 4.9 M,
mean
from three experiments; EC50 (R-848) alone > 30 M). However, higher
concentrations of
ODN 6056 decreased the EC50 even more (1 M ODN 6056: EC50 (R-848) = 1.4 M; 5
M
ODN: EC50 (R-848) = 0.4 M). This effect was specific for ODN 6056 as the
decrease of
EC50 was considerably less for ODN 1982 (5 M 1982: EC50 (R-848) = 19.0 M).
In
contrast to the stimulatory effects on TLR8, co-incubation of ODN 6056 (or ODN
1982) with
the TLR9 ligand CpG ODN 2006 on hTLR9 expressing HEK293 cells did not affect
CpG-
mediated NF-xB stimulation (unpublished data).
The potential sequence requirements for the observed enhancement effect was
investigated and several different phosphorothioate homopolymers were tested
(Table 1). The
oligo(dT)17 (ODN 6056) had the strongest enhancing properties, followed by an
oligo(dU)17
(SEQ ID NO: 11) and oligo(dA)17 (SEQ ID NO: 12). Significantly less
enhancement was
detected for an oligo(dC)17 (SEQ ID NO: 13) and a randomised oligo(dN)15. The
heteropolymer ODN 1982 showed only minor enhancing activity and an oligo(dG)24
(SEQ ID
NO: 14) did not enhance activity, but rather inhibited R-848-mediated NF-xB
stimulation.
The sequence of ODN 6056 with a phosphodiester backbone did not influence TLR8-
mediated stimulation. Length of the oligonucleotide also seemed to have an
influence: an
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oligo(dA)24 (SEQ ID NO: 15) showed less synergistic effects with R-848 than
the shorter
oligo(dA)17 (SEQ ID NO: 12).
Stimulatioia of TM-expressing cells by a TLR7-specific ligand in the presence
of a
hornopolymer T oligonucleotide
R-848 is a ligand for hTLR7 as well as hTLR8 (16), whereas loxoribine (7-allyl-
7,8-
dihydo-8oxo-guanosine) was described as a TLR7-specific ligand (18).
Concentrations of up
to 10 mM loxoribine did not activate NF-xB signaling in HEK293 cells
transfected with either
hTLR8 or hTLR9, but activated hTLR7-mediated signaling (FIG. 2A). Although
being a
TLR7 ligand, co-incubation of hTLR8-expressing HEK293 cells with loxoribine
and
oligonucleotide activated NF-xB signaling (FIG. 2B). This concentration-
dependent effect
was observed exclusively in the presence of ODN 6056 but not ODN 1982 (FIG.
2B).
Similar observations were made with another TLR7 ligand, 7-deaza-guanosine
(18). 7-
Deaza-guanosine alone was inactive on hTLR8-expressing cells at concentrations
of up to 5
mM in the absence of ODN 6056 but stimulated TLR8-mediated NF-xB activation in
the
presence of ODN 6056. Inosine, a compound that appeared to activate NF-xB
signaling in
HEK293 cells nonspecifically (unpublished data), was also co-incubated with
ODN 6056 on
hTLR8-expressing HEK293 cells (FIG. 2C). NF-xB activation by inosine in the
presence of
ODN 6056 was not altered compared to nonspecific activation of inosine at
concentrations of
up to 10 mM.
A similar effect is observed with 6-amino-9-benzyl-2-butoxy-9H-purin-8-ol, an
adenosine-based compound. When used.alone, this compound is a TLR7 ligand but
not a
TLRB ligand (FIGs. 9A and 9B). When used in the presence of a poly(dT)17
adaptor
oligonucleotide, it is able to stimulate TLR8 signaling (FIG. 9B).
Sequence-independent inhibition of TLR 7 signaling
These data show that small molecule TLR7 ligands induce TLRS-dependent NF-xB
signaling in the presence of certain oligonucleotides. Nevertheless, in TLR7-
expressing
HEK293 cells the effects were converse. Oligonucleotides actually inhibited
TLR7-mediated
NF-icB activation by TLR7 specific ligands and TLR7/8 ligands as shown in
FIGs. 1A, 3 and
9A. The effect appeared to be sequence-unspecific as all phosphorothioate
oligonucleotides
tested so far inhibited hTLR7 activation (unpublished data).
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Enhancement of TLR8 ligand signaling using an RNA adaptor oligonucleotide
Similar experiments were carried out using R-848 as the TLR7/8 ligand and RNA-
based adaptor oligonucleotides. FIG. 10 shows the effect on NF-xB stimulation
in hTLR8-
LUC-293 cells by a mixed sequence oligonucleotide and a poly(rU)18
oligonucleotide. The
poly(rU)18 oligonucleotide
(rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU*rU; SEQ ID NO: 5) when
used alone signals through TLR8, as shown by the slight activation of NF-xB
with this
oligonucleotide. (FIG. 10) The mixed sequence RNA oligonucleotide
(rG*rC*rC*rA*rC*rC*rG*rA*rG*rC*rC*rG*rA*rA*rG*rG*rC*rA*rC*rC; SEQ ID NO:
16), when used alone, does not signal through TLR8.
In FIG. 11, hTLR8-LUC-HEK293 cells were incubated with increasing amounts of R-
848 in the presence of either (a) TE and 50 g/ml DOTAP, or (b) 5 M of
indicated ORN in
the presence of 50 g/ml DOTAP. Stimulation of NF-xB was measured 16 hours
later by
determining luciferase activity. Stimulation indices were calculated in
reference to
background in the presence of inedium alone. The Figure shows that the effect
of the
poly(rU)18 oligonucleotide on TLR8 signaling is dramatically enhanced when the
oligonucleotide is combined with R-848 (FIG. 11). The mixed sequence RNA
oligonucleotide, which is neither a TLR7 nor TLR8 ligand, also enhances TLR8
signaling by
R-848 as shown in FIG. 11. The data show that RNA-based oligonucleotides can
also
function as adaptors.
Co-incubation of loxoribine with oligo(dT)17 ODN changes the cytokine profile
produced by
human PBMC
The effect of co-incubation of loxoribine with ODN 6056 and 1982 on human
immune
cells was investigated. Incubation with loxoribine alone stimulated human PBMC
to produce
IFN-a (FIG. 4A). However, in the presence of increasing concentrations of ODN,
loxoribine-
induced IFN-a was reduced to background levels. Although the effect was
observed with
both ODN, ODN 6056 seemed to have a stronger suppressive effect.
The production of other cytokines was also studied: Loxoribine alone induced
only
minor amounts of IL-12p40 secretion from human PBMC (FIG. 4B). In contrast, in
the
presence of ODN 6056 IL-12p40 was produced in significant amounts, whereas the
same
concentration of ODN 1982 together with loxoribine did not induce IL-12p40
above the
background with media alone. Similar results were obtained for IL-12p7O
(unpublished data)
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- 73 -
and IFN-y (FIG. 4C). TNF-a production was also stimulated when loxoribine and
ODN 6056
were co-incubated. However, TNF-a production was also observed when ODN 1982
was co-
incubated with loxoribine, although considerably less than achieved by co-
incubation with
ODN 6056. None of the ODN nor loxoribine alone induced significant levels of
TNF-a
secretion (FIG. 4D).
IL-12p40 and TNF-a are produced by monocytes when co-stimulated with
loxoribine and
oligo(dT)17 ODN
The combination of loxoribine and ODN activates TLR8-expressing HEK293 cells
but
suppresses TLR7-mediated signaling. It was hypothesized that human TLR8-
positive
immune cells are the primary source for IL-12 and TNF-a and TLR7-positive pDCs
are the
primary source for IFN-a. Intracellular FACS staining revealed that the
majority of CD14+
cells produced TNF-a upon co-incubation of human PBMC with loxoribine and ODN
6056
(FIG. 5A). Loxoribine alone or in combination with ODN 1982 yielded only few
TNF-
positive monocytes. The percentage of IL-12-positive monocytes was less than
that for TNF-
a. Nevertheless, the synergistic effect of the combination of loxoribine with
ODN 6056 was
also clearly detectable for intracellular IL-12 (FIG. 5B). In none of these
experiments could
intracellular IL-12 or TNF-a be detected in CD 19+ B cells upon stimulation
with ODN and
loxoribine (unpublished data). The cellular source of IFN-y was also
investigated. The
combination of loxoribine with ODN stimulated high IL-12 levels and previous
studies
showed that IL-12 induces IFN-y secretion in NK cells (21-23). Therefore, IFN-
'y production
was evaluated in human NK cells (FIG. 6). Indeed, IFN-y production in NK cells
could be
observed when human PBMC were co-incubated with loxoribine and ODN 6056,
although
neither of the stimuli alone induced considerable IFN-y production as already
demonstrated
by IFN-y protein ELISA from supernatants of human PBMC.
To demonstrate direct TLR-mediated stimulation of monocytes in response to
loxoribine and ODN 6056, monocytes from human PBMC were purified and incubated
with
loxoribine in the presence or absence of ODN 6056 or 1982. Isolated monocytes
produced
IL-12p40 (FIG. 7A) or TNF-a (unpublished data) only in response to the
combination of both
stimuli, loxoribine and ODN 6056.
Incubation of human PBMC with small immunomodulating compounds like R-848 or
loxoribine induced IFN-a production by pDCs (FIG. 7B and (24,25)). Therefore,
the
suppressive effect of ODN 6056 on IFN-a production stimulated by loxoribine
could have
CA 02623764 2008-03-26
WO 2007/038720-- PCT/US2006/037987
-74-
been a direct result of inhibition of TLR7-mediated signaling in pDCs. To
investigate this
possibility, human pDCs were isolated and stimulated with loxoribine in the
presence of
either oligo(dT)17 homopolymer or heteropolymer ODN (FIG. 7B). Enriched human
pDC
produced high amounts of IFN-a when incubated with loxoribine alone. In
contrast, the IFN-
a production was abolished when co-incubated with either ODN 6056 or ODN 1982.
Again,
the suppressive effect of ODN 6056 seemed to be stronger than that of ODN 1982
since lower
ODN concentrations were needed to abrogate IFN-a production.
FIG. 8 shows data generated using TLR8-LUC-293 cells. A constant concentration
of
R-848 (50 micromolar) and increasing amounts of the specified ODN were used.
Luciferase
readout of R-848 alone was set to 100% for standardization. The results are
shown in the left
panel. FIG. 8 right panel shows the effect of increasing T content in a I
micromolar 17mer
poly C oligonucleotide in the presence of increasing amounts of R-848. The
activity is
enhanced even with just two thymidines and increases dramatically with 6 or
more
thymidines.
DISCUSSION
The present data demonstrate that the activity of hTLR8 can be modulated by
the
presence of certain phosphorothioate ODN. hTLR8 is most closely related to
hTLR7 (26), as
reflected by the ability of the small immune stimulatory compound R-848 to
activate both
hTLR7 and hTLR8 (16). However, hTLR7 is considerably more sensitive to
stimulation with
R-848 than hTLR8 as lower concentrations of R-848 are needed for TLR7
activation. Co-
incubation of R848 with a phosphorothioate oligo(dT)17 rendered TLR8 more
sensitive to
stimulation with R848 and yet inhibited TLR7 activation, despite the fact that
this T
homopolymer alone had no considerable effect on either of these TLRs.
To determine whether the T homopolymer would influence TLR activation by other
stimuli, its effects on two TLR7 ligands, loxoribine and 7-deaza-guanosine,
which do not
stimulate TLRB (18), were studied. Surprisingly, for both ligands the presence
of an
oligo(dT)17 led to a complete switch from a TLR7- to a TLR8-dependent NF-xB
activation,
with suppression of TLR7-mediated signaling by the oligo(dT)17. It is possible
that human
TLR8 may have a weak binding affinity to loxoribine which probably cannot be
detected in
the biological assays available. The presence of certain oligonucleotides may
enhance affinity
of loxoribine to TLR8 by a yet unknown mechanism, so that loxoribine-mediated
stimulation
of TLRB in stably transfected cells can be detected.
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The possibility of an influence of oligonucleotides on a general signaling
pathway or
uptake mechanism cannot be ruled out. However, the fact that the oligo(dT)17
had a different
mode of action on HEK293 cells transfected with hTLR7 (inhibition of NF-xB
signaling),
hTLR9 (no effect) or hTLR8 (strong enhancement of signaling) strongly argues
against this
possibility. The observed synergistic effect was not limited to cells
expressing TLR8
ectopically. Human TLR7 is mainly expressed in pDCs and B cells. These cells
do not
express TLR8 whereas TLR8 is expressed in myeloid cells (27-30). Indeed,
synergistic
effects of loxoribine and ODN on cytokine production (IL-12 and TNF-a) were
measured in
human immune cells with endogenous TLR8 expression such as monocytes. In
contrast, the
majority of loxoribine and R-848 induced IFN-a is produced by activation of
TLR7 in human
pDC (our data and (4,24,25)). Activation of IFN-a production in pDC by these
TLR7 stimuli
was completely suppressed by incubation with phosphorothioate ODN, consistent
with the
inhibitory effects seen in TLR7-transfected cells. There was no apparent
sequence-
dependence in the inhibition of TLR7-mediated signaling by phosphorothioate
ODN in
transfected cells or in pDC, in contrast to the sequence-selective stimulatory
effect seen on
TLR8. The combination of ODN and loxoribine not only led to a switch from pDC-
derived
IFN-a to monocyte-derived IL-12 and TNF-a but also to secretion of IFN-y from
NK cells.
Human NK cells lack TLR7 and TLR8 expression (31), so that stimulation of NK
cell-derived
IFN-y appears to be an indirect effect, probably mediated by IL-12 (32). Taken
together, a
complete alteration of the profile of loxoribine-mediated immune effects was
observed upon
simple addition of certain oligonucleotides. Furthermore, these data point to
a somewhat
sequence-dependent effect with phosphorothioate T-rich ODN being the most
efficient ODN
that can be combined with TLR7-specific ligands to activate TLR8.
Some reports indicate that myeloid dendritic cells and monocytes express both
TLR7
and TLR8 at the mRNA level (27-29). No data are available showing actual TLR
protein
expression in these cells. No direct stimulation of monocytes by loxoribine
was detected in
these experiments. In contrast, only the combination of loxoribine and
oligonucleotide
induced monocyte-derived cytokine production. These results argue against a
functional
expression of TLR7 in monocytes and point to TLR8 as the receptor targeted by
oligonucleotide and loxoribine. Furthermore, loxoribine alone did not induce
significant
amounts of IL-12, although production of IL-12 in human PBMC induced by R-848
has been
reported (29), which is consistent with the ability of R-848 to stimulate both
TLRs (16). A
clear distinction as to which TLR is activated in cells that may express both
TLR7 and TLR8
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with compounds stimulating both receptors is very difficult. Ito et al (33)
reported that mDC
produce IL-12 but not IFN-a when stimulated with R-848. In contrast, pDC do
not produce
IL-12 but do produce IFN-a (25) upon stimulation with R-848 or other TLR
ligands
indicating a TLR8-mediated induction of IL-12 in mDCs and a TLR7-mediated
induction of
IFN-a in pDCs. Even co-incubation of R-848 or loxoribine with oligo(dT)17 ODN
did not
result in production of IL-12 by human pDC suggesting that this signal is not
sufficient to
activate the adequate pathways in pDC.
It is tempting to postulate a model where certain TLRs may possess a (possibly
allosteric) regulatory site to which oligonucleotides could bind and function
as effector
molecules. In the case of TLR7, phosphorothioate oligonucleotides appear to
act as
antagonists. Therefore, oligonucleotide binding to TLR7 may inhibit either
proper binding of
a TLR7 ligand to its binding pocket or correct downstream signaling, e.g., by
aggravating
correct conformational changes within the receptor. Binding of T-rich
oligonucleotides to
TLRB, on the other hand, may function as an (allosteric) activator enabling
increased binding
of R-848 or other small molecule ligands like loxoribine to the active TLRB
binding pocket or
increased downstream signaling. Whether there may be two binding domains
involved or
binding of small molecule ligand and effector ODN may occur at the same site
cannot
currently be determined. Allosteric enhancers are known for receptors from
several different
families. For example, Knoflach et al. (34) reported the identification of two
classes of small
molecules that behave as allosteric enhancers for the metabotropic glutamate
receptor, a
member of the G protein-coupled receptor family. Detailed analysis of receptor
structure
localized the enhancer binding site within the transmembrane domain. Other
studies show
that small molecules can act as allosteric enhancers for the muscarinic
acetylcholine receptor
(35). Certain 2-Aminothiophenes act as allosteric enhancers of Adenosine
receptor Al
(36,37) by stabilizing the ligand-receptor-G-protein ternary complex and
increasing
association of unoccupied receptor and G-protein (38). Interestingly, Gao et
al. (39) found a
series of imidazoquinoline derivates to act as allosteric enhancers of agonist
binding at human
A3 adenosine receptors. Recently, Rutz et al. (40) demonstrated direct
blockade of CpG
ODN binding to purified TLR9 protein by the small molecules chloroquine and
quinacrine
using SPR biosensor analysis. Their findings indicate direct binding of these
molecules to the
extracellular domain of TLR9 acting as antagonists of TLR9-mediated signaling.
Therefore,
the described oligonucleotides and small molecules may directly bind to TLR7
and S.
Binding studies using isolated proteins and the respective receptor ligands
could give further
CA 02623764 2008-03-26
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insight into the exact mechanism of action of the combination of small
molecule TLR ligands
and oligonucleotides on TLR7 or TLR8. These findings may have important
implications for
understanding the molecular signaling mechanisms of the TLRs, as well as for
pharmaceutical
research and drug development. It has been demonstrated, according to the
invention, that the
signaling activity of two members of the TLR family, TLR8 and TLR7, can be
manipulated
by the presence or absence of certain oligonucleotides. Taken together, these
results suggest
new ways to modify an immune response using loxoribine (or other small
molecules) in
combination with certain oligonucleotides. By combination of these molecules
it is possible
to alter the cytokine profile of the TLR small molecule ligand by redirecting
its signaling
activity to a different TLR. An altered or enforced immune response could be
beneficial for
the treatment of a variety of diseases.
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TABLE 1
SEQ ID NO: SEQUENCE % R-848
(ODN) ACTIViTY
3 T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T
271f 12
6056
11 U*U*U*U*U*U*U*U*U*U*U*U*U*U*U*U*U
236 14
12 A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A
228f37
13 C*C*C*C*C*C*C*C*C*C*C*C*C*C*C*C*C
183f5
15 A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A*A
160 16
N*N*N*N*N*N*N*N*N*N*N*N*N*N*N
156 119
4 T*C*C*A*G*G*A*C*T*T*C*T*C*T*C*A*G*G*T*T
121 21
1982
16 TTTTTTTTTTTTTTTTT 98f7
14 G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G*G
76 12
Table 1: Comparison of synergistic effects of different ODN on R-848 activity.
HEK293 cells expressing hTLR8 and a NF-xB reporter construct were incubated
with 50 M
ofR-848 intheabseiiceorpresenceof5 Moftheind.icat,edODN. NF-xB stimulation
byR-848
in the absence of ODN was set to 100% and effect on R-848 activity was
calculated
accordingly. Values represent the mean ( SD) of2-4 experiinents. "N"
represenis A, C, G or T
in random order, "*" depicts a phosphorothioate backbone, "_" depicts a
phosphodiester
backbone.
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TABLE 2
T2 T*T -'
T4 T*T*T*T
T6 T*T*T*T*T*T
T8 T*T*T*T*T*T*T*T
T10 T*T*T*T*T*T*T*T*T*T )C~
T12 T*T*T*T*T*T*T*T*T*T*T*T
T14 T*T*T*T*T*T*T*T*T*T*T*T*T*T 2-0
T17 T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T ~
C17 C*C*C*C*C*C*C*C*C*C*C*C*C*C*C*C*C I~
C7T2C8 C*C*C*C*C*C*C*T*T*C*C*C*C*C*C*C*C
C6T4C7 C*C*C*C*C*C*T*T*T*T*C*C*C*C*C*C*C ~:f
C5T6C6 C*C*C*C*C*T*T*T*T*T*T*C*C*C*C*C*C
C4T8C5 C*C*C*C*T*T*T*T*T*T*T*T*C*C*C*C*C -2-
C3T10C4 C*C*C*T*T*T*T*T*T*T*T*T*T*C*C*C*C ~
T17 T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T ~
* indicates a phosphorothioate bond
References
1. Schetter, C. and J. Volhner. 2004. Toll-like receptors involved in the
response to microbial
pathogens: Development of agonists for toll-like receptor 9. Current pinion
in Drug
Discovery & Development 7:204-210.
2. Krieg, A. M. 2003. CpG motifs: the active ingredient in bacterial extracts?
Nat.Med. 9:831-
835.
3. Akira, S. 2003. Malnmalian Toll-like receptors. Curr. pin.Immunol. 15:5-
11.
4. Heil, F., P. Ahmaci Nej ad, H. Hemmi, H. Hochrein, F. Ampenberger, T.
Gellert, H. Dietrich, G.
Lipford, K Takeda, S. Aldra, H. Wagner, and S. Bauer. 2003. The Toll-like
receptor 7('TLR7)-
specific stimulus loxon'bine uncovers a strong relationship within the TLR7, 8
and 9 subfamily.
Eur.JImmunol. 33: 2987-2997.
5. Diebold, S. S., T. K-asha, H. Henuni, S. Akira, and Reis e Sousa 2004.
Itmate antiviral responses by
means of TLR7mediated recognition of single-stranded RNA. Science 303:1529-
1531.
6. Lund, J. M., L. Alexopoulou, A. Sato, M. Karow, N. C. Adams, N. W. Gale, A.
Iwasald, and R
A. Flavell. 2004. Recognition of single-stranded RNA vi~ses by Toll-like
receptor 7.
Proc.Natl.Acad.}S'ci. U.,SA.101:5598-5603.
7. Matsumoto, M., K Funami, M. Tanabe, H. Osbiumi, M. Shingai, Y. Seto, A.
Yainamoto, and T.
Seya 2003. Subcellular localization of toll-like receptor 3 in human dendritic
cells. J.Irnmun.ol.
171:3154-3162.
CA 02623764 2008-03-26
WO 2007/038720._ PCT/US2006/037987
-80-
8. Latz, E., A. Schoenemeyer, A. Visintin, K A. Fitzgerald, B. G. Monks, C. F.
Knetter, E. Lien, N.
J. Nilsen, T. Espevik, and D. T. Golenboclc. 2004. TLR9 signals after
translocating from the ER to
CpG DNA in the lysosome. Nat.Immunol.
9. Abmad-Nejad, P., H. Hacker, M. Rutz, S. Bauer, R. M. Vabulas, and H.
Wagner. 2002. Bacterial
CpG DNA and lipopolysaccharides activate Toll -like receptors at distinct
cellular comparhmen.ts.
Eur.J.Immunol. 32:1958-1968.
10. Akua, S. ax1K. Takeda. 2004. Toll-like rec,eptor signalling.
Nat.Rev.Inzmunol. 4:499511.
11. Kobe, B. and A.V. Kajava. 2001. The leucine-rich repeat as a pivtein
recognition motif.
Curr. Op i n. Stru c t. B i o l. 11: 725 - 732.
12. Bell, J. K, G. E. Mullen, C. A. Leifer, A. Mazzoni, D. R Davies, and D. M.
Segal. 2003.
Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends
Immunol.
24:528-533.
13. Ulilmann, E. and J. Vollmer. 2003. Recent advances in the development of
immunostimulatory oligonucleotides. Curr. Opin.Drug Discov.Devel. 6: 204-217.
14. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kiischning, S. Akira,
G. Lipford, H.
Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded
RNA via toll-like
receptor 7 and S. Science 303:1526-1529.
15. Ulevitch, R. J. 2004. Therapeutics targeting the innate immune systein.
Nat.Rev.Immunol. 4:512-520.
16. Jurk, M., F. Heil, J. Vollmer, C. Schetter, A. M. Krieg, H. Wagner, G.
Lipford, and S. Bauer.
2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral
compound R
848. Nat.Immunol. 3:499.
17. Hemmi, H., T. Y~aisho, 0. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T.
Horiuchi, H. Tomizawa, K
Takeda, and S. Akira 2002. Small anti-viral compounds activate immune cells
via the TLR7
MyD88-dependent signaling pathway. Nat.Irnmunol. 3:196-200.
18. Lee, J., T. H. Chuang, V. Redecke, L. She, P. M. Pitha, D. A. Carson, E.
Raz, and H. B. Cottam.
2003. Molecular basis for the immunostimulatory activity of guanine nucleoside
analogs: activation
of Toll-like receptor 7. Proc.Natl.Acad.Sci. U. S A.100: 6646-6651.
19. Goodman, IVL G., A. B. Reitz, R. Chen, M. D. Bobardt, J. H. Goodman, and
B. L. Pope. 1995.
Selective modulation of elements of the immune system by low molecular weight
nucleosides.
JPharmacol.Exp. Ther. 274:1552-1557.
20. Bauer, S., C. J. Kirschning, H. Hacker, V. Redecke, S. Hausniarn, S.
Akira, H. Wagner, and G. B.
Lipford. 2001. Human TLR9 confers responsiveness to bacterial DNA via species-
specific CpG
motifrecognition. Proc.Natl.Acad.Sci. U.S.A. 98: 9237-9242.
CA 02623764 2008-03-26
WO 2007/038720 PCT/US2006/037987
-81-
21. Magram, J., S. E. Connaughton, R. R VtTarner, D. M. Carvajal, C. Y. Wu, J.
Fen-ante, C. Stewart,
U. Sanniento, D. A. Faherty, and M. K. Gately. 1996. IL-12-deficient mice are
defective in IFN
garrnnaproductionandtype I cytokineresponses. Iinnzunity. 4:471481.
22. Thierfelder, W. E., J. M. van Deursen, K. Yam:arnoto, R A. Tzipp, S. R
Sarawar, R. T. Carson,
M. Y. Sangster, D. A. Vignali, P. C. Doherty, G. C. Grosveld, and S. N. Ihle.
1996.
Requiiemen.t for Stat4 ininterleulcin 12mediatedresponses ofnaturalkiller and
T cells. Nature
382:171-174.
23. Trinchieri, G. 2003. Interleuldn-12 and the regulation of innate
resistance and adaptive ianmmunity.
Nat. Rev.Inamunol. 3:133-146.
24. Lore, K., M. R Bett.s, J. M. Brenchley, J. Ktuuppu, S. Khojasteh, S.
Perfetto, M. Roederer, R
A. Seder, and R A. Koup. 2003. Toll-Like Receptor Ligands Modulate Dendritic
Ce1Ls to
Augment Cytomegalovirus- and HIV-1-Specific T Ce1I Responses. J.rmmunol.
171:4320-
4328.
25. Gibson, S. J., J. M. Lindh, T. R Riter, R. M. Gleason, L. M. Rogers, A. E.
Fuller, J. L.
Oesterich, K. B. Gorden, X. Qiu, S. W. McKane, R J. Noelle, R L. Miller, R. M.
Ked1, P.
Fitzgerald-Bocarsly, M. A. Tomai, and J. P. Vasilakos. 2002. Plasmacytoid
dendritic cells produce
cytokines andinatiue in iesponse to the TLR7 agonists, imiqunmod and
resiquimod Cell Iinfnunol.
218: 74-86.
26. Da, X., A. Poltoralc, Y. Wei, and B. Beutler. 2000. Three novel mammalian
toll-like receptors:
gene structure, expression, and evolution. Eur. Cytokine Netw. 11: 362-371
27. Jarrossay, D., G. Napolitani, M. Colonna, F. Sallusto, and A.
Lanzavecchia. 2001.
Specialization and complementarityin microbialmolecule recognition by human
myeloid
and plasmacytoid dendritic cells. Eur.J.Imfnunol. 31:3388-3393.
28. Krug, A., A. Towarowski, S. Britsch, S. Rothenfusser, V. Hornung, R. Bals,
T. Giese, H.
Engelmann, S. Endres, A. M. Krieg, and G. Hartmann. 2001. Toll-like receptor
expression
reveals CpG DNA as a urzique micmbial stimulus for plasmacytoid dendritic
cells which synergizes
withCD40ligandtoinchzcebighamounts of IL-12. EurJ.Immunol. 31:3026-3037.
29. Ito, T., R Amakawa, T. Kaisho, R Hemriv, K, Tajima, K. Uehira, Y. Ozaki,
H. Toxnizawa, S.
Akiza, and S. Fiikuhaa. 2002. Iiriterfenon alpha. and interleukin-12 are
induced differential.ly by
Toll-likereceptor7ligands in human blood dendritic cell subsets. J.Exp.Med.
195:1507-
1512.
30. Iwasaki, A. and R. Medaliitov. 2004. Toll-like receptor control of the
adaptive immune responses.
Nat.Imnzunol 5:987-995.
31. Homung, V., S. Rothenfiisser, S. Britsch, A. Krug, B. Jahrsdorfer, T.
Giese, S. Endres, and G.
Nmtmann. 2002. Quantitati.ve expres,sion of toll-like receptor 1-10 mRNA in
cellulat subsets of
humanperiphetalbloodmononuclearcells and sensitivityto CpG
oligodeoxynucleotides.
J.Immunol. 168:4531-4537.
CA 02623764 2008-03-26
WO 2007/038720 PCT/US2006/037987
-82-
32. Trinchieri, G. 2003. Interleuldrr12 andtheregulationof innate resistance
and adaptive immunity.
Nat.Rev.Inarnunol. 3:133-146.
33. Ito, T., R. Amakawa, M. Inaba, S. Ikehara, K. Inaba, and S. Fukuhara.
2001.
Differential regulation of human blood dendritic cell subsets by IFNs.
J.Irnrnunol. 166: 2961-
2969.
34. Knoflach, F., V. Mutel, S. Jolidon, J. N. Kew, P. Malherbe, E. Vieira, J.
Wichmann, and J. A.
Kemp. 2001. Positive allosteric modulators of metabotropic glutamate 1
receptor: charactetization,
mechanism of action, andbinding site. Proc.Natl.Acad. Sci. U. S.A. 98:13402-
13407.
35. Waelbroeck, M. 2003. Allosteric drugs acting at muscarinic acetylcholine
receptors.
Neurocliem.Res. 28:419-422.
36. Bnms,R F. andJ. H. Fergus. 1990. Allostericenhancement of adenosine Al
receptor binding
andfunctionby2-anino-3 benzoylthiophenes. Mol.Pharmacol. 38:939-949.
37. Bruns, R. F., J. H. Fergus, L. L. Coughenour, G. G. Courtland, T. A.
Pugsley, J.
H. Dodd, and F. J. Tinney. 1990. Stntchzre-activityrelationships for
enhancement of
adenosine Al receptor binding by 2-atnino-3-benzoylthiophenes. Mol.Pharmacol.
38:950-
958.
38. Bhattacharya, S. andJ. Lin.den. 1995. The allosteric enhancer, PD 81,723,
stabilizes human Al
adenosine receptor coupling to Gproteins. Biochim.Bioplzys.Acta 1265:1521.
39. Gao, Z. G., S. G. Kim, K A. Soltysi*N. Mehnan, A. P. IJzerman, and K. A.
Jacobson. 2002.
Selective allosteric enhancement of agonist binding and function at human A3
adenosine receptors by
aseriesofimidazoquinolinederivatives. Mol.Pharmacol. 62:8189.
40. Rutz, M., J. Metzger, T. Gellert, P. Luppa, G. B. Lipford, H. Wagner, and
S. Bauer. 2004.
Toll-like receptor 9 binds single-siranded CpG-DNA in a sequence- and
pHdependent manner.
Eur.J.Immunol. 34:2541-2550.
Equivalents
The foregoing written specification is considered to be sufficient to enable
one skilled
in the art to practice the invention. The present invention is not to be
limited in scope by
examples provided, since the examples are intended as a single illustration of
one aspect of
the invention and other functionally equivalent embodiments are within the
scope of the
invention. Various modifications of the invention in addition to those shown
and described
herein will become apparent to those skilled in the art from the foregoing
description and fall
within the scope of the appended claims. The advantages of the invention are
not necessarily
encompassed by each embodiment of the invention.
CA 02623764 2008-03-26
WO 2007/038720 PCT/US2006/037987
-83-
All references, patents and patent publications that are recited in this
application are
incorporated by reference in their entirety herein.
What is claimed is:
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