U.S. patent application number 11/183253 was filed with the patent office on 2006-01-05 for methods and products related to treatment and prevention of hepatitis c virus infection.
This patent application is currently assigned to Coley Pharmaceutical Group, Ltd.. Invention is credited to Navneet K. Ahluwalia, Heather L. Davis, Susan M. Efler, Jorg Vollmer.
Application Number | 20060003962 11/183253 |
Document ID | / |
Family ID | 32230300 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003962 |
Kind Code |
A1 |
Ahluwalia; Navneet K. ; et
al. |
January 5, 2006 |
Methods and products related to treatment and prevention of
hepatitis C virus infection
Abstract
The invention provides methods for identifying and treating
subjects having hepatitis C infections. In some instances, the
subjects are those that are non-responsive to non-CpG therapy.
Preferably, the subjects are treated with C class CpG
immunostimulatory nucleic acids having a semi-soft backbone.
Inventors: |
Ahluwalia; Navneet K.;
(Ottawa, CA) ; Efler; Susan M.; (Constance Bay,
CA) ; Davis; Heather L.; (Dunrobin, CA) ;
Vollmer; Jorg; (Dusseldorf, DE) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC;FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Assignee: |
Coley Pharmaceutical Group,
Ltd.
Kanata (Ottawa)
CA
Coley Pharmaceutical GmbH
Langenfeld
DE
|
Family ID: |
32230300 |
Appl. No.: |
11/183253 |
Filed: |
July 15, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10532746 |
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PCT/IB03/05520 |
Oct 29, 2003 |
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11183253 |
Jul 15, 2005 |
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60421987 |
Oct 29, 2002 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 31/14 20180101;
A61P 43/00 20180101; A61K 31/7088 20130101; A61K 31/7056 20130101;
G01N 33/5767 20130101; A61K 39/39 20130101; A61P 31/00 20180101;
C07H 21/00 20130101; A61K 2039/55561 20130101; A61K 38/212
20130101; A61K 38/212 20130101; A61K 2039/55522 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/044 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1-71. (canceled)
72. A branched immunostimulatory CpG oligonucleotide comprising at
least one unmethylated CpG dinucleotide (5'CG 3') and one or more
unusual linkages involving a sugar moiety (Su), a heterocyclic base
(Ba) or a phosphate backbone (Ph), wherein the one or more unusual
linkages are selected from the group consisting of Su-Su, Su-Ph,
Su-Ba, Ba-Ba, Ba-Su, Ba-Ph, Ph-Ph, Ph-Su, Ph-Ba, and any
combination thereof.
73. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein the unusual linkages are selected from the group consisting
of 2'5'-, 5'5'-, 3'3'-, 2'2'-, and 2'3'-linkages.
74. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein one part of the oligonucleotide is connected at a 3'-end
via a 3'3'-linkage to a second oligonucleotide part and at a 2'-end
via a 2'3'-linkage to a third part of the molecule.
75. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein the branched immunostimulatory CpG oligonucleotide has an
activity selected from the group consisting of (i) an ability to
stimulate IFN-.gamma. production from human peripheral blood
mononuclear cells, (ii) an ability to stimulate IFN-.alpha.
production from human peripheral blood mononuclear cells, and (iii)
an ability to stimulate B cell proliferation.
76. The branched immunostimulatory CpG oligonucleotide of claim 72,
further comprising one or more non-nucleotidic linker selected from
the group consisting of abasic linkers, triethylene glycol units,
hexaethylene glycol units, C3, C6, C12 alkylamino linkers, linkers
derived from ethanediol, linkers derived from propanediol,
triethylene phosphate, and tetraethylene phosphate.
77. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein the branched immunostimulatory CpG oligonucleotide
comprises a sequence selected from the group consisting of TCG,
TTCG, TTTCG, TTTTCGT, TTTTTCG, TCGT, TTCGT, TTCGT, TTTCGT, and
TCGTCGT.
78. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein the branched immunostimulatory CpG oligonucleotide has a
length between 4 and 100 nucleotides.
79. The branched immunostimulatory CpG oligonucleotide of claim 72,
wherein the branched immunostimulatory CpG oligonucleotide has a
length between 6 and 40 nucleotides.
80. A pharmaceutical composition comprising the branched
immunostimulatory CpG oligonucleotide of claim 72 and a
pharmaceutically acceptable excipient.
81. A nucleic acid delivery complex comprising the branched
immunostimulatory CpG oligonucleotide of claim 72 associated with a
cationic lipid.
82. A pharmaceutical composition comprising the branched
immunostimulatory CpG oligonucleotide of claim 72 and a
microsphere.
83. A method of stimulating an immune response in an individual,
the method comprising administering to an individual the branched
immunostimulatory CpG oligonucleotide of claim 72 in an amount
sufficient to stimulate an immune response in the individual.
84. The method of claim 83, wherein the individual suffers from a
disorder associated with a Th2-type immune response.
85. The method of claim 84, wherein the disorder associated with a
Th2-type immune response is chronic HCV infection.
86. A method of increasing the secretion of interferon-gamma
(IFN-.gamma.) by blood cells, comprising administering the branched
immunostimulatory CpG oligonucleotide of claim 72 in an amount
sufficient to increase the secretion of IFN-.gamma. by blood
cells.
87. A method of increasing the secretion of interferon-alpha
(IFN-.alpha.) by blood cells, comprising administering the branched
immunostimulatory CpG oligonucleotide of claim 72 in an amount
sufficient to increase the secretion of IFN-.alpha. by blood cells.
Description
FIELD OF THE INVENTION
[0001] The invention provides methods and products for the
treatment of subjects chronically infected with hepatitis C
virus.
BACKGROUND OF THE INVENTION
[0002] The hepatitis C virus (HCV) is a positive strand RNA virus
of the Flavivirus family that infects hepatocytes of humans and
some other primates. First characterized in 1989 (1), HCV has a 9.5
kb genome that encodes for three structural proteins: core and two
envelope glycoproteins (E1 and E2), as well as several
non-structural (NS) proteins that are involved in the viral
replication and interaction with the host cell (2).
[0003] HCV is a serious public health concern, causing >90% of
parenteral non-A, non-B hepatitis (1). From 0.4 to 1.5% of the
world's population is infected (3, 4), including about 300,000
Canadians (Health Canada). Epidemiological statistics are difficult
to compile since the vast majority of acute infections are
subclinical; however it is estimated that 50-80% of HCV infected
individuals fail to clear the virus, and most of these become
life-long carriers. About 50% of carriers develop chronic hepatitis
and 20% of these will develop liver cirrhosis, many of whom will
subsequently develop hepatocellular carcinoma (5-9). Hepatitis C
causes an estimated 8,000 to 10,000 deaths annually in the United
States (CDC).
[0004] In the United States and Canada, there are two different
regimens, which have been approved as therapy for hepatitis C:
monotherapy with alpha interferon and combination therapy with
alpha interferon and Ribavirin. Although more expensive and
associated with more side effects, combination therapy consistently
yields higher rates of sustained response than monotherapy.
[0005] Several forms of alpha interferon are available (alpha-2a,
alpha-2b, and consensus interferon (Alfacon)). These interferons
are typically given subcutaneously three times weekly. Pegylated
interferon, i.e., alpha interferon modified by addition of
polyethylene glycol (PEG) in order to increase the duration in the
circulation, is another of interferon, and it is given only once
weekly. Ribavirin, in contrast, is an oral antiviral agent that is
given twice a day in 200 mg capsules.
[0006] Side effects of alpha interferon include: fatigue, muscle
aches, headaches, nausea and vomiting, skin irritation at the
injection site, low-grade fever, weight loss, irritability,
depression, suicide, mild bone marrow suppression and hair loss
(reversible). For Ribavirin the side effects include; anemia
fatigue and irritability, itching, skin rash, nasal stuffiness,
sinusitis and cough.
[0007] Treatment with interferon alone or in combination with
interferon and Ribavirin leads to rapid improvements in serum ALT
levels in 50-75% of patients and the disappearance of detectable
HCV RNA from the serum in 30-50% of patients. Long-term improvement
in liver disease usually occurs only if HCV RNA disappears during
therapy and stays undetectable for at least 6 months after therapy
is completed. Combination treatment results in both a higher rate
of loss of HCV RNA on treatment and a lower rate of relapse when
treatment is complete. However, results depend strongly on the
genotype of virus, with better results being obtained for genotypes
2 and 3 (about 90% with 1 year of treatment with pegylated
IFN-.alpha. and Ribavirin), but much poorer results (about 40%
sustained response) for genotype 1 HCV. The majority of HCV chronic
carriers in North America now are of genotype 1.
[0008] The optimal duration of treatment varies depending on
whether interferon monotherapy or combination therapy is used, as
well as by HCV genotype. Typically, the duration ranges from 6 to
12 months.
[0009] There is currently no vaccine against HCV, or highly
effective therapy for chronic infection. Thus there is an urgent
need for an effective treatment that could be used to treat chronic
carriers.
SUMMARY OF THE INVENTION
[0010] The invention is premised in part on several surprising
findings including the observation that CpG immunostimulatory
nucleic acids can be used to treat subjects that are chronically
infected with hepatitis C virus (HCV) and that are non-responsive
to previously administered non-CpG therapies. The invention is
further premised in part on the observation that a synergistic
response can be had in such subjects from the combined use of CpG
immunostimulatory nucleic acids and an anti-viral agent such as
IFN-alpha.
[0011] In one aspect, the invention provides a method of treating a
subject having an HCV infection that was not successfully treated
using a previous non-CpG therapy comprising administering to a
subject in need of such treatment a CpG immunostimulatory nucleic
acid in an amount effective to treat the infection.
[0012] In one embodiment, the non-CpG therapy includes
interferon-alpha. In a related embodiment, the interferon-alpha is
interferon-alpha-2b, interferon-alpha-2a or consensus
interferon-alpha. In another embodiment, the non-CpG therapy
includes interferon-alpha and Ribavirin, or interferon-alpha and
Ribavirin and emantidine. In some important embodiments, the
non-CpG therapy includes pegylated interferon-alpha and an
anti-viral such as Ribavirin.
[0013] In one embodiment, the CpG immunostimulatory nucleic acid is
an A class CpG immunostimulatory nucleic acid. In another
embodiment, the CpG immunostimulatory nucleic acid is a B class CpG
immunostimulatory nucleic acid.
[0014] In yet a further embodiment, the CpG immunostimulatory
nucleic acid is a C class CpG immunostimulatory nucleic acid.
[0015] The method may optionally comprise administration of an
anti-viral such as interferon-alpha to the subject along with the
CpG immunostimulatory nucleic acid. The interferon-alpha may be
interferon-alpha-2b, interferon-alpha-2a or consensus interferon
alpha, but is not so limited. In one embodiment, the anti-viral is
administered substantially simultaneously with the CpG
immunostimulatory nucleic acid.
[0016] In one embodiment, the CpG immunostimulatory nucleic acid
comprises a backbone modification. In a related embodiment, the
backbone modification is a phosphorothioate backbone modification.
In some important embodiment, the CpG immunostimulatory nucleic
acid comprises a semi-soft backbone. In other important
embodiments, the CpG immunostimulatory nucleic acid is a C class
immunostimulatory nucleic acid having a semi-soft backbone.
[0017] Thus, in another aspect, a method is provided for treating a
subject having an HCV infection that was not successfully treated
using a previous non-CpG therapy comprising administering to a
subject in need of such treatment a C class CpG immunostimulatory
nucleic acid having a semi-soft backbone in an amount effective to
treat the infection.
[0018] In yet another aspect, a method is provided for treating a
subject having an HCV infection that was not successfully treated
using a previous non-CpG therapy comprising contacting peripheral
blood mononuclear cells from a subject in need of such treatment,
with a CpG immunostimulatory nucleic acid in an amount effective to
stimulate an immune response, and re-infusing the cells into the
subject.
[0019] In one embodiment, the peripheral blood mononuclear cells
comprise dendritic cells. In another embodiment, the dendritic
cells comprise plasmacytoid dendritic cells. In one embodiment, the
CpG immunostimulatory nucleic acid is a C class immunostimulatory
nucleic acid. In a related embodiment, the C class
immunostimulatory nucleic acid has a semi-soft backbone.
[0020] In another aspect, the invention provides a method of
treating a subject having an HCV infection and likely to be
non-responsive to a non-CpG therapy comprising administering to a
subject in need of such treatment a CpG immunostimulatory nucleic
acid in an amount effective to treat the infection.
[0021] In one embodiment, the method further comprises identifying
a subject likely to be non-responsive to a non-CpG therapy. In one
embodiment, the subject is identified as likely to be
non-responsive based on an assay of interferon-alpha produced per
dendritic cell. In another embodiment, the subject is identified as
likely to be non-responsive based on HCV genotype.
[0022] In one embodiment, the non-CpG therapy includes IFN-alpha.
In a related embodiment, the non-CpG therapy includes
interferon-alpha and Ribavirin.
[0023] In one embodiment, the method further comprises
administering to the subject an anti-viral agent. In important
embodiments, the anti-viral agent is interferon-alpha. The
interferon-alpha may be interferon-alpha-2b, interferon-alpha-2a or
consensus interferon alpha, but it is not so limited. In one
embodiment, the interferon-alpha is administered in a
sub-therapeutic amount, and optionally the combination of the CpG
immunostimulatory nucleic acid and the interferon-alpha is
synergistic.
[0024] In one embodiment, the CpG immunostimulatory nucleic acid
used to treat the subject is an A class CpG immunostimulatory
nucleic acid, a B class CpG immunostimulatory nucleic acid, or a C
class CpG immunostimulatory nucleic acid.
[0025] In one embodiment, the CpG immunostimulatory nucleic acid
used to identify whether a subject is likely to be non-responsive
to a non-CpG therapy is an A class CpG immunostimulatory nucleic
acid, or a C class CpG immunostimulatory nucleic acid.
[0026] In one embodiment, the anti-viral agent is administered to
the subject substantially simultaneously with the CpG
immunostimulatory nucleic acid. In other embodiments, the
interferon-alpha is administered for a period prior to treatment
with the CpG immunostimulatory nucleic acid.
[0027] In certain embodiments, the CpG immunostimulatory nucleic
acid comprises a backbone modification. In related embodiments, the
backbone modification is a phosphorothioate backbone modification.
In some preferred embodiments, the CpG immunostimulatory nucleic
acid comprises a semi-soft backbone, and some even more preferred
embodiments, the CpG immunostimulatory nucleic acid is a C class
CpG immunostimulatory nucleic acid having a semi-soft backbone.
[0028] In another aspect, a method is provided for treating a
subject having an HCV infection and likely to be non-responsive to
a non-CpG therapy comprising administering to a subject in need of
such treatment a C class CpG immunostimulatory nucleic acid having
a semi-soft backbone in an amount effective to treat the
infection.
[0029] In yet another aspect, the invention provides a method for
screening CpG immunostimulatory nucleic acids useful in the
treatment of chronic hepatitis C viral infection. The method
involves contacting peripheral blood mononuclear cells from a
subject having a chronic hepatitis C viral infection, to a CpG
immunostimulatory nucleic acid, and measuring a test response of
the blood mononuclear cells after exposure. The subject from which
the peripheral blood mononuclear cells wherein the subject was not
successfully treated using a previous therapy.
[0030] In one embodiment, the test response is selected from the
group consisting of B cell stimulation, secretion of IL-6,
secretion of IL-10, secretion of IL-12, secretion of
interferon-gamma, secretion of type 1 interferons (alpha+beta),
secretion of IP-10, NK activity, expression of CD80, expression of
CD 86, expression of CD83, and upregulation of class II MHC
expression.
[0031] In another embodiment, the peripheral blood mononuclear
cells comprise dendritic cells. In a related embodiment, the
dendritic cells comprise plasmacytoid dendritic cells. In still
another embodiment, the cells are dendritic cells and the test
response is selected from the group consisting of secretion of
IL-12, secretion of type 1 interferons, expression of CD80,
expression of CD 86, expression of CD83, and upregulation of class
II MHC expression.
[0032] In one embodiment, the contacting occurs in vitro. In
another embodiment, the peripheral blood mononuclear cells are
cultured. In yet another embodiment, the CpG immunostimulatory
nucleic acid is added to the cultured peripheral blood mononuclear
cells.
[0033] In one embodiment, the previous therapy is a non-CpG
therapy. In another embodiment, the non-CpG therapy comprises
interferon-alpha. In another embodiment, the non-CpG therapy
further comprises Ribavirin. In other embodiments, the
interferon-alpha is pegylated interferon-alpha. In one embodiment,
the previous therapy is therapy with a CpG nucleic acid of a
different sequence or class.
[0034] In other embodiments, the method further comprises screening
the CpG immunostimulatory nucleic acid for the ability to stimulate
a control response from peripheral blood mononuclear cells from a
normal subject.
[0035] The method may further comprise contacting peripheral blood
mononuclear cells to interferon-alpha substantially simultaneously
with the CpG immunostimulatory nucleic acid.
[0036] In one embodiment, the CpG immunostimulatory nucleic acid
comprises a backbone modification. In a related embodiment, the
backbone modification is a phosphorothioate backbone modification.
In important embodiments, the CpG immunostimulatory nucleic acid
comprises a semi-soft backbone. The CpG immunostimulatory nucleic
acid may be an A class CpG immunostimulatory nucleic acid, a B
class CpG immunostimulatory nucleic acid, or a C class CpG
immunostimulatory nucleic acid. In some embodiments, the CpG
immunostimulatory nucleic acid is a C class immunostimulatory
nucleic acid, and in other embodiments, the CpG immunostimulatory
nucleic acid is a C class immunostimulatory nucleic acid with a
semi-soft backbone.
[0037] In another aspect, the invention provides a method for
identifying a subject having an HCV infection and likely to be
non-responsive to a non-CpG therapy. The method involves exposing
peripheral blood mononuclear cells harvested from a subject having
a hepatitis C viral infection to a CpG immunostimulatory nucleic
acid, measuring interferon-alpha produced from the cells, and
determining an amount of interferon-alpha produced per dendritic
cell, wherein an amount that is below 1.0 pg/ml is indicative of a
subject that is likely to be non-responsive to a non-CpG therapy.
In one embodiment, an amount that is below 0.5 pg/ml is indicative
of a subject that is likely to be non-responsive to a non-CpG
therapy.
[0038] In one embodiment, the non-CpG therapy comprises
interferon-alpha. In another embodiment, the non-CpG therapy
comprises Ribavirin. In another embodiment, the IFN-alpha is
pegylated IFN-alpha.
[0039] In some important embodiments, the CpG immunostimulatory
nucleic acid is an A class or a C class CpG immunostimulatory
nucleic acid.
[0040] In still other embodiments, the peripheral blood mononuclear
cells are further exposed to an anti-viral agent together with a
CpG immunostimulatory nucleic acid. The anti-viral agent may be
interferon-alpha, but it is not so limited. In one embodiment, the
interferon-alpha is interferon-alpha-2b, interferon-alpha-2a or
consensus interferon alpha.
[0041] In one embodiment, the peripheral blood mononuclear cells
comprise dendritic cells. In another embodiment, the dendritic
cells comprise plasmacytoid dendritic cells.
[0042] In another embodiment, the hepatitis C viral infection is an
acute hepatitis C viral infection.
[0043] In another embodiment, the method further comprises
determining a genotype of the HCV.
[0044] In still a further aspect, a method is provided for
identifying a subject having an HCV infection and likely to be
non-responsive to a non-CpG therapy comprising exposing peripheral
blood mononuclear cells harvested from a subject having a hepatitis
C viral infection to an A class or a C class CpG immunostimulatory
nucleic acid, measuring interferon-alpha produced from the cells,
and determining an amount of interferon-alpha produced per
dendritic cell, wherein an amount that is below 1.0 pg/ml is
indicative of a subject that is likely to be non-responsive to a
non-CpG therapy.
[0045] In yet another aspect, the invention provides a method of
treating a subject having a hepatitis C viral infection comprising
administering to a subject, identified according to the method
described above, a CpG immunostimulatory nucleic acid molecule in
an amount effective to treat the infection.
[0046] In one embodiment, the method further comprises
administering to the subject interferon-alpha. In one embodiment,
the interferon-alpha is interferon-alpha-2b, interferon-alpha-2a or
consensus interferon-alpha.
[0047] In one embodiment, the CpG immunostimulatory nucleic acid
used to treat the subject is an A class CpG immunostimulatory
nucleic acid, a B class CpG immunostimulatory nucleic acid, or a C
class CpG immunostimulatory nucleic acid.
[0048] In another embodiment, the CpG immunostimulatory nucleic
acid comprises a backbone modification. In a related embodiment,
the backbone modification is a phosphorothioate backbone
modification. In yet another embodiment, the CpG immunostimulatory
nucleic acid comprises a semi-soft backbone.
[0049] In one embodiment, the hepatitis C viral infection is a
chronic hepatitis C viral infection. In another embodiment, the
hepatitis C viral infection is an acute hepatitis C viral
infection.
[0050] Each of the limitations of the invention can encompass
various embodiments, of the invention. It is, therefore,
anticipated that each of the limitations of the invention involving
any one element or combinations of elements can be included in each
aspect of the invention.
[0051] These and other aspects of the invention are described in
greater detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0052] FIG. 1 shows the induction of IFN-.alpha. secretion from
HCV-infected and normal PBMCs following stimulation with 3 classes
of CpG. PBMCs from normal or HCV-infected subjects were incubated
with different classes of CpG for 48 h. Cell supernatants were
collected and assayed for IFN-.alpha. secretion by commercial ELISA
kits. The average IFN-.alpha. secretion for 10 normal subjects and
10 HCV-infected subjects are shown by the black bars.
[0053] FIG. 2 shows the flow cytometric analysis of freshly
isolated PBMCs from chronic HCV carriers and normal subjects. PBMCs
were isolated from the blood of HCV infected subjects and from
normal healthy donors and immunostained with fluorescent-tagged
anti-plasmacytoid dendritic cell (pDC) antibodies. Cells were
analyzed on a flow cytometer and results were compared to
IFN-.alpha. secretion data on these same subjects when stimulated
with CpG.
[0054] FIG. 3 shows the IFN-.alpha. induction by stimulation of
PBMCs with C-class and soft-C oligonucleotides. PBMCs from normal
or HCV-infected subjects were incubated with different classes of
CpG for 48 h. Cell supernatants were collected and assayed for
IFN-.alpha. secretion by commercial ELISA kits. The average
IFN-.alpha. secretion for 10 normal subjects and 10 HCV-infected
subjects are shown by the black bars.
[0055] FIG. 4 shows the IFN-.alpha. induction following stimulation
with a panel of semi-soft C-class CpG. PBMCs isolated from 5
HCV-infected subjects were incubated with a panel of semi-soft
C-class oligonucleotides for 48 h. Cell supernatants were collected
and assayed for IFN-.alpha. secretion by commercial ELISA kits. The
average IFN-.alpha. secretion for 5 HCV-infected subjects are shown
by the black bars.
[0056] FIG. 5 shows the IFN-.gamma. secretion following stimulation
with three classes of CpG. PBMCs from normal or HCV-infected
subjects were incubated with different classes of CpG for 48 h.
Cell supernatants were collected and assayed for IFN-.gamma.
secretion by commercial ELISA kits. The average IFN-.gamma.
secretion for 10 normal subjects and 10 HCV-infected subjects are
shown by the black bars.
[0057] FIG. 6 shows the IFN-.gamma. induction following stimulation
with a panel of semi-soft C-class CpG. PBMCs isolated from 5
HCV-infected subjects were incubated with a panel of semi-soft
C-class oligonucleotides for 48 h. Cell supernatants were collected
and assayed for IFN-.gamma. secretion by commercial ELISA kits. The
average IFN-.gamma. secretion for 5 HCV subjects are shown by the
black bars.
[0058] FIG. 7 shows the IP-10 secretion following stimulation with
three classes of CpG. PBMCs from normal or HCV-infected subjects
were incubated with different classes of CpG for 48 h. Cell
supernatants were collected and assayed for IP-10 secretion by
commercial ELISA kits. The average IP-10 secretion for 10 normal
subjects and 10 HCV-infected subjects are shown by the black
bars.
[0059] FIG. 8 shows the effect of CpG on B cell proliferation.
PBMCs from HCV-infected or normal donors were incubated with class
A, B or C CpG for 5 days. Cells were then pulsed with
.sup.3H-thymidine for 16 to 18 hours before measuring
radioactivity. Values are represented as stimulation indices in
comparison with media control (SI=cpm incubated with CpG/cpm of
cells incubated with media alone).
[0060] FIG. 9 shows the effect of semi-soft C-class CpG on B cell
proliferation. PBMCs from 5 HCV-infected subjects were incubated
with A, B, C and semi-soft-C class CpG for 5 days. Cells were then
pulsed with .sup.3H-thymidine for 16 to 18 hours before measuring
radioactivity. Values are represented as stimulation indices in
comparison with media control (SI=cpm incubated with CpG/cpm of
cells incubated with media alone).
[0061] FIG. 10 shows the IL-10 secretion following stimulation with
three classes of CpG. PBMCs from normal or HCV-infected subjects
were incubated with different classes of CpG for 48 h. Cell
supernatants were collected and assayed for IL-10 secretion by
commercial ELISA kits. The average IL-10 secretion for 10 normal
subjects and 10 HCV-infected subjects are shown by the black
bars.
[0062] FIG. 11 shows IFN-.alpha. secretion following stimulation of
HCV-infected cells with Ribavirin and CpG alone or in combination
with Intron A. PBMCs from 10 HCV-infected subjects and 10 normal
healthy donors were incubated with Intron A, Ribavirin or C-class
CpG alone and also with and without Intron A (a purified exogenous
source of IFN-.alpha.) for 48 hours. Cell supernatants were
collected and assayed for IFN-.alpha. secretion by commercial ELISA
kits. The amount of IFN-.alpha. measured for Intron A alone for
each subject, was considered background and was subtracted from
Intron A, Ribavirin+Intron A and C-Class+Intron A for these same
subjects before the data was included in the graph. Mean values for
normal and HCV subjects are indicated by black and white bars
respectively.
[0063] FIG. 12. Synergistic effect of CpG combined with Intron A on
IFN-.alpha.secretion by HCV-infected cells. PBMCs from 15
HCV-infected subjects were incubated with C-class CpG alone or
together with Intron A (a purified exogenous source of IFN-.alpha.)
for 48 hours. Cell supernatants were collected and assayed for
IFN-.alpha. secretion by commercial ELISA kits. The amount of
IFN-.alpha. measured for Intron A alone for each subject, was
subtracted from CpG+Intron A for these same subjects before the
data was included in the graph.
[0064] It is to be understood that the figures are not required for
enablement of the claimed invention.
DETAILED DESCRIPTION OF THE INVENTION
[0065] It has been discovered according to the invention, that CpG
oligonucleotides can activate PBMCs from patients chronically
infected with HCV, including those who have failed previous
interferon-alpha (IFN-.alpha.) therapy, in a manner similar to
PBMCs from healthy subjects.
[0066] It was discovered that endogenous IFN-.alpha. secretion was
strongly induced from plasmacytoid dendritic cells (PDC), which are
thought to be infected by HCV resulting in their dysfunction and
reduced ability to respond to other stimuli. In some instances, the
A and C CpG classes, which induce high levels of IFN-.alpha. in
PBMCs from healthy volunteers, were found to induce the highest
levels of IFN-.alpha. from pDCs. It was further discovered that the
semi-soft C class CpG ODN are also particularly useful for this
effect. These ODN may be preferred in some embodiments since they
will not accumulate in the kidney with repeat dosing.
[0067] It has been further discovered according to the invention
that neither exogenous IFN-.alpha. (Intron A) nor Ribavirin have
any detectable direct immune stimulatory effects on PBMCs from
normal subjects or HCV chronic carriers, when used alone or
together. However, when Intron A and CpG ODN (e.g., B or C classes)
are used together, then a strong synergy for production of
endogenous IFN-.alpha. is observed.
[0068] These results indicate that CpG ODN are an effective
treatment alone, or together with IFN-.alpha., to treat chronic HCV
infection. The invention provides methods and products for
preventing and treating HCV infection, based on these findings.
[0069] Chronic infection appears to be due, at least in part, to
the rapid mutation rate of HCV, resulting in the production of
quasi-species that can escape immune surveillance (10, 11). Both
humoral and cell-mediated immune (CMI) responses can be detected in
chronically infected individuals. While neutralizing antibodies are
critical to protection from infection, cell-mediated immunity (CMI)
appears to play the major role in viral clearance once infection is
established.
[0070] In one aspect, the invention provides a method of treating a
subject infected with hepatitis C virus (HCV) who is not
successfully treated with a previous non-CpG therapy. The method
comprises administering to a non-responsive subject in need of such
treatment a CpG immunostimulatory nucleic acid in an amount
effective to inhibit the infection.
[0071] A non-CpG therapy, as used herein, is a therapy that uses
active or inactive compounds that are not CpG immunostimulatory
nucleic acids. In various embodiments, the non-CpG therapy includes
interferon-alpha. Pegylated IFN-alpha is commonly administered to
HCV subjects (e.g., human HCV patients), preferably in combination
with Ribavirin and optionally amantadine. The interferon-alpha can
be interferon-alpha-2b, interferon-alpha-2a or consensus
interferon-alpha. All of the foregoing interferon-alpha treatments
are included in the definition of non-CpG therapy.
[0072] A subject who is not successfully treated with a previous
non-CpG therapy is a subject who notwithstanding prior treatment
still has detectable viral load in their bloodstream 6 months after
the cessation of therapy. These subjects include those that may
respond to a previous non-CpG therapy, but who fail to control the
infection and subsequently relapse as indicated by detectable viral
load. As used herein and for the sake of simplicity, these subjects
are referred to as "non-responders", however this term is to be
understood as defined herein, and not as defined in a clinical
setting. In other words, although in a clinical setting a
"non-responder" defines only that narrow subset of subjects that
fail to show any response to a treatment, the invention is directed
to a broader category of subjects that while perhaps responding at
some level to a previous treatment, are still not successfully
treated. A subject that is successfully treated is one that has no
detectable viral load in its bloodstream 6 months after the
cessation of treatment. Successful treatment means treatment that
leads to an undetectable level of viral load in the bloodstream
that is sustained for at least 6 months after cessation of
treatment. It is to be understood that a non-responder, as used
herein, implicitly is also chronically infected with HCV.
[0073] As used herein, when referring to treatment using CpG
nucleic acids, the methods are used to achieve a successful
treatment of subjects. Successful treatment of subjects using CpG
treatment is defined as a reduction of viral load to undetectable
levels in the bloodstream 6 months after the cessation of therapy.
Interestingly, viral loads may not be observed to decrease during
or immediately after CpG treatment, but rather may only decrease
with time after treatment, with the ultimate result that there is
no detectable virus in the bloodstream of these subjects 6 months
following the cessation of treatment. To treat an infection
therefore means to reduce viral load to an undetectable level in
the bloodstream of a subject and to sustain that level for 6 months
following the cessation of treatment. Effective amounts of agents
are therefore administered to achieve this end result.
[0074] In some embodiments, the potential non-responders may be
identified prospectively (i.e., prior to actual in vivo treatment
with a non-CpG therapy), and the invention provides methods not
only for the identification of such subjects but also for their
treatment. Potential non-responders may be identified by assessing
their ability to respond to CpG immunostimulatory nucleic acids,
particularly A class and C class. The ability to respond to CpG
immunostimulatory nucleic acids will be assessed by the amount of
interferon-alpha that is produced per pDC in HCV infected subjects.
It was discovered, according to the invention, that HCV infected
subjects that would be unlikely to respond to non-CpG therapy such
as IFN-alpha therapy could be identified prior to receiving such
treatment. The ability to identify such subjects prior to in vivo
therapy eliminates unnecessary treatment and places the subjects in
a therapeutically advantageous position for treatment with the CpG
immunostimulatory nucleic acids of the invention either alone or in
combination with other anti-HCV therapies including but not limited
to IFN-alpha. These subjects would suffer from less cytotoxicity
and the time period for viral growth would be reduced by not
undergoing a treatment that will be unsuccessful. Subjects having
below a reasonable level of IFN-alpha induction per pDC are likely
not to be successfully treated with IFN-alpha and thus should be
treated using the methods provided herein. Measurement of IFN-alpha
induction and pDC numbers are described in more detail in the
Examples.
[0075] It is to be understood that an HCV infected subject that is
successfully treated with any of the therapeutic agents and methods
discussed herein will probably still have virus in their body.
However, while the subject is not able to completely eradicate the
virus, it is able to control viral load (to undetectable levels).
Although not intending to be bound by any particular theory, it is
expected that the maintenance of undetectable viral loads in such
subjects involve an immune system that is able to control viral
replication and spread.
[0076] One of ordinary skill given the teachings provided herein
will be able to determine whether a subject is likely to be a
"non-responder" to IFN-alpha therapy. As an example, if the
IFN-alpha induction were performed with an A class nucleic acid
such as nucleic acid designated SEQ ID NO 1, under the culture
conditions described in the Examples, then a normal response
indicative of the ability to respond to IFN-alpha therapy would be
at least 1 pg/ml per pDC. An amount less than this is indicative of
some pDC dysfunction. Amounts that are less than 0.5 pg/ml per pDC
correlate with a higher probability of non-response to IFN-alpha
treatment. One of ordinary skill will be able to determine such
cutoffs for the particular type of nucleic acid used in the assay,
and will therefore be capable of identifying subjects expected not
to be successfully treated with IFN-alpha therapy (at least) prior
to actually treating such subjects in that manner.
[0077] In still other embodiments, the method of identifying a
subject who is likely to be a non-responder to non-CpG therapy
(e.g., IFN-alpha therapy) may further include identification of the
genotype of HCV he/she is infected with. It is more likely that a
subject infected with a genotype 1 HCV will not be successfully
treated with IFN-alpha therapy, for example. Therefore, in addition
to assessing the production of IFN-alpha per DC in such subjects,
their HCV genotype can also be determined (using methods known in
the art), and this combination of information can be used to
identify a subject that is likely to be non-responsive to IFN-alpha
therapy.
[0078] It is to be further understood that in some aspects, the
invention provides a method for identifying a subject that is
unlikely to be successfully treated using a non-CpG therapy
(without actually treating the subject with a non-CpG therapy) and
then treating the subject using either CpG immunostimulatory
nucleic acids alone or in combination with an anti-viral agent such
as but not limited to IFN-alpha.
[0079] The above methods can also be used to screen subjects for
their response to particular CpG immunostimulatory nucleic
acids.
[0080] In still other embodiments, the methods may involve the
additional step of identifying subjects having received previous
non-CpG therapy but not successfully treated. Those of ordinary
skill, given the teachings provided herein, will be able to
identify such subjects. As an example, such subjects would have
detectable viral loads in their bloodstream 6 months after the
cessation of treatment. In some embodiments, these subjects may
also demonstrate a reduction in viral load immediately following
treatment, but this reduction is not sustained.
[0081] The invention intends to treat subjects not successfully
treated with a previous non-CpG therapy using, inter alia, CpG
immunostimulatory nucleic acids alone or in combination with other
active agents such as those previously described for HCV infection.
As broadly defined, CpG immunostimulatory nucleic acids are nucleic
acids having at least one CpG dinucleotide motif in which at least
the C of the dinucleotide is unmethylated. CpG immunostimulatory
nucleic acids include but are not limited to A class, B class and C
class CpG immunostimulatory nucleic acids, as described more fully
herein and in the patent and patent applications cited herein and
incorporated by reference. These classes of CpG immunostimulatory
nucleic acid have differing properties and activation profiles.
[0082] In important embodiments, the CpG immunostimulatory nucleic
acid is a C class immunostimulatory nucleic acid. It was
surprisingly found, according to the invention, that C class
immunostimulatory nucleic acids were preferred in some embodiments,
even though these nucleic acids possessed properties intermediate
to those of A class and B class. The Examples provided herein
demonstrate that, even though pDC of chronically infected subjects
not successfully treated with a previous non-CpG therapy are
themselves infected with HCV and thereby dysfunctional in some
aspects, exposure of such cells to CpG immunostimulatory nucleic
acids, and in particular C class immunostimulatory nucleic acids,
restores their function. In some embodiments, it is also preferred
that the C class immunostimulatory nucleic acids be either of a
"soft" or "semi-soft" variety, as described in greater detail
herein. In some preferred embodiments, the CpG immunostimulatory is
a semi-soft C class nucleic acid.
[0083] In other aspects, the CpG immunostimulatory nucleic acids
are used in combination with active agents which preferably include
those previously described for HCV treatment. Of particular
importance is the use of CpG immunostimulatory nucleic acids with
interferon-.alpha. (e.g., Intron A). The interferons that can be
used in combination with the CpG immunostimulatory nucleic acids of
the invention include but are not limited to interferon-alpha-2b,
interferon-alpha-2a or consensus interferon alpha. Other
anti-virals are described herein. Any of the CpG classes can be
used in these combinations. As an example, it was unexpectedly
found, according to the invention, that although exogenously
administered interferon-.alpha. fails to treat these subjects
successfully, when combined with CpG immunostimulatory nucleic
acids it is therapeutically efficacious. In some embodiments, the
CpG immunostimulatory nucleic acid is a C class immunostimulatory
nucleic acid. In come preferred embodiments, it is a semi-soft C
class nucleic acid.
[0084] The timing of administration of the CpG nucleic acid and
anti-viral agent (e.g., interferon-alpha) may vary depending upon
the subject and the severity of infection. The CpG nucleic acid may
be administered substantially simultaneously with the CpG
immunostimulatory nucleic acid. This means that the two agents may
be combined prior to administration, or may be combined in the
process of administration (e.g., with both feeding into an
intravenous line in a subject), or they may be administered
separately but within a period of time that it would take someone
to perform two administrations (e.g., the time to inject a subject
twice). Regardless of whether the agents are administered
substantially simultaneously or in staggered fashion, the order may
vary. Accordingly, in some embodiments, the CpG immunostimulatory
nucleic acid may be administered prior to an anti-viral agent such
as IFN-alpha while in others it may be administered following the
anti-viral agent.
[0085] When CpG nucleic acids are used together with other
anti-virals (e.g., IFN-alpha), these compounds may be administered
in a combined amount that is therapeutically efficacious. The
amount of either compound may therefore be sub-therapeutic or
supra-therapeutic (i.e., below or above the amount that would be
therapeutically efficacious when administered alone).
Alternatively, the compounds each may be administered in a
therapeutic amount, but the combination of those agents creates a
therapeutic benefit such as a reduction of side effects. In
preferred embodiments, if the anti-viral is IFN-alpha, it is
administered in a therapeutic amount. Regardless of the actual
amounts administered, the combination of agents may be synergistic.
A synergistic response is one that is greater than the additive
response expected by the combination of the agents.
[0086] In still other aspects and in keeping with the description
provided above, the invention provides methods for screening CpG
nucleic acids for the ability to stimulate immune cells isolated
from a subject chronically infected with HCV and not successfully
treated with a non-CpG therapy or likely to be non-responders to
non-CpG therapy. These screening methods are generally performed in
vitro by contacting peripheral blood mononuclear cells (PBMCs) with
a CpG immunostimulatory nucleic acid in an effective amount
sufficient to stimulate an immune response. The immune response can
be measured by any number of markers, including IFN-alpha
production, B cell stimulation, secretion of cytokines such as
IL-6, IL-10, IL-12, interferon-gamma, type 1 interferons
(alpha+beta), chemokine secretion such as IP-10, NK activity,
expression of costimulatory molecules (e.g., CD80, CD 86) and
maturation molecules (e.g., CD83) and upregulation of class II MHC
expression.
[0087] In some important embodiments, the immune cells are
dendritic cells, and preferably plasmacytoid dendritic cells (pDCs)
and the immune response markers are specific to this cell type.
These include but are not limited to expression of costimulatory
molecules (e.g., CD80 and CD86) expression of maturation molecules
(e.g., CD83), expression and/or secretion of IL-12 and type 1
interferons (alpha+beta), and upregulation of class II MHC
expression. It is to be understood that these in vitro assays are
not dependent upon isolation of dendritic cells such as pDCs from
the remainder of PBMCs. Rather the assays can be carried out in
homogeneous populations of PBMCs.
[0088] In still another aspect, the invention provides a method for
identifying a subject having a chronic hepatitis C viral infection
to be treated with a CpG immunostimulatory nucleic acid. The method
involves exposing peripheral blood mononuclear cells harvested from
a subject having a chronic hepatitis C viral infection to i) a CpG
immunostimulatory nucleic acid, and ii) a CpG immunostimulatory
nucleic acid and an anti-viral (e.g., interferon-alpha), and
measuring response of the peripheral blood mononuclear cells after
exposure. A response to a CpG immunostimulatory nucleic acid is
indicative of a subject to be treated with a CpG immunostimulatory
nucleic acid either following or in place of a non-CpG therapy (as
described above, but only after identifying a subject that is
unlikely to respond to a non-CpG therapy). A response to a CpG
immunostimulatory nucleic acid together with an anti-viral agent
(e.g., interferon-alpha) that is greater than the response to CpG
immunostimulatory nucleic acid alone is indicative of a subject to
be treated with the combination. As described herein, the
anti-viral agent can be an interferon-alpha including but not
limited to interferon-alpha-2b, interferon-alpha-2a or consensus
interferon-alpha. Preferably, the peripheral blood mononuclear
cells comprise dendritic cells such as plasmacytoid dendritic
cells. The invention further includes treatment of subjects
identified as just described using either CpG immunostimulatory
nucleic acids alone or in combination with an anti-viral agent
(e.g., IFN-alpha), depending upon the outcome of the screening
assay. Clinical strategies comprise local and systemic in vivo
administration of such nucleic acids, as well as ex vivo strategies
in which pDCs isolated from non-responsive HCV infected subjects
are activated in vitro with immunostimulatory nucleic acids and
then reinfused into the patient locally or systemically. These
therapeutic strategies may include the combination with other
growth factors (IL-3, GM-CSF, flt3-ligand, etc.) as well as with
other stimuli (superantigens, viral products). Since natural
IFN-.alpha. is a family of more than a dozen separate gene
products, the individual products of which have unique activity
profiles, the clinical use of natural interferon may be preferable
compared to recombinant IFN-.alpha. derived from a single
recombinant IFN-.alpha. gene.
[0089] The invention further provides a method activating pDCs from
an Hepatitis C infected subject. The method involves isolating pDCs
from the subject in need of such treatment, culturing the isolated
pDCs in vitro, contacting the pDCs in vitro with an effective
amount of an isolated immunostimulatory nucleic acid, and returning
the contacted cells to the subject. The cells can also be contacted
in vitro with a growth factor or with a cytokine. The
immunostimulatory nucleic acids and conditions calling for
treatment with IFN-.alpha. according to this aspect of the
invention are as described above.
[0090] IFN-alpha itself represents a family of more than a dozen
related, homologous proteins (isoforms, see Table 1 below), each
encoded by a unique gene and each exhibiting a unique activity
profile. The activities of the different alpha-interferon species
on viruses can vary as much as twenty-fold or more. IFN-alpha
products in clinical use are recombinant proteins or highly
purified natural proteins of a single isoform. In the United States
IFN-.alpha. is available as recombinant human IFN-.alpha.2a
(ROFERON-A), recombinant human IFN-.alpha.2b (INTRON A), and as
purified natural IFN-.alpha.n3 (ALFERON N). Outside the United
States, IFN-.alpha. is also available as purified natural
IFN-.alpha.n1 (WELLFERON). TABLE-US-00001 TABLE 1 Family of Human
IFN-.alpha. IFN-.alpha.A (IFN-.alpha.2a) IFN-.alpha.2
(IFN-.alpha.2b) IFN-.alpha.4b (IFN-.alpha.4) IFN-.alpha.B2
(IFN-.alpha.8) IFN-.alpha.C (IFN-.alpha.10) IFN-.alpha.D
(IFN-.alpha.1) IFN-.alpha.F (IFN-.alpha.21) IFN-.alpha.G
(IFN-.alpha.5) IFN-.alpha.H2 (IFN-.alpha.14) IFN-.alpha.I
(IFN-.alpha.17) IFN-.alpha.J1 (IFN-.alpha.7) IFN-.alpha.K
(IFN-.alpha.6) IFN-.alpha.M1 IFN-.alpha.N IFN-.alpha.WA
(IFN-.alpha.16)
[0091] Some of the methods of the invention require measurement of
immune responses including detecting the presence of IFN-.alpha..
Assays for IFN-.alpha. are well known in the art. These include
direct tests, e.g., enzyme-linked immunosorbent assay (ELISA)
specific for at least one IFN-.alpha., and indirect tests, e.g.,
functional tests including NK cell activation/cytotoxicity
(Trinchieri G Adv Immunol 47:187-376 (1989) and phenotyping by
fluorescence-activated cell sorting (FACS) analysis for class I
MHC. Additional specific assay methods well known in the art can be
particularly useful in settings where local concentration or local
presence of IFN-.alpha. is of interest. These methods include, for
example, immunohistochemistry, nucleic acid hybridization (e.g.,
Northern blotting), Western blotting, reverse
transcriptase/polymerase chain reaction (RT/PCR), and in situ
RT/PCR. Intracellular IFN-.alpha. can also be detected using flow
cytometry.
[0092] The invention in some aspects involves measuring pDC
activation. pDC activation can be assayed in a number of ways.
These include IFN-.alpha. production, expression of costimulatory
molecules (e.g., CD80 and CD86), expression of maturation molecules
(e.g., CD83), expression of IL-12, and upregulation of class II MHC
expression. Unlike administration of exogenous IFN-.alpha.,
activation of pDC leads to the production of various if not all the
forms of IFN-.alpha., as well as other type I IFN such as
IFN-.beta.. In some embodiments, therefore, the pDC are activated
as measured by their ability to produce type I interferons
including IFN-.alpha..
[0093] The invention provides various methods that involve
immunostimulatory nucleic acids. An immunostimulatory nucleic acid
is a nucleic acid molecule which, upon contacting cells of the
immune system, is itself capable of inducing contacted cells of the
immune system to proliferate and/or to become activated. The
contacting can be direct or indirect, e.g., the immunostimulatory
nucleic acid may directly stimulate a first type of immune cell to
express a product which may in turn stimulate a second type of
immune cell which has not been exposed to, or is not responsive to,
the immunostimulatory nucleic acid. The immunostimulatory effect of
the immunostimulatory nucleic acid is separate from any product
that might happen to be encoded by the sequence of the
immunostimulatory nucleic acid. Similarly, the immunostimulatory
effect of an immunostimulatory nucleic acid is distinct from and
does not rely upon any antisense mechanism.
[0094] Only certain nucleic acids are immunostimulatory nucleic
acids. Originally it was believed that certain palindromic
sequences were immunostimulatory. Tokunaga T et al. Microbiol
Immunol 36:55-66 (1992); Yamamoto T et al. Antisense Res Dev
4:119-22 (1994). Further work demonstrated that non-palindromic
sequences are also immunostimulatory provided they contained CpG
dinucleotides within particular sequence contexts (CpG motifs).
Krieg AM et al. Nature 374:546-9 (1995).
[0095] The immunostimulatory nucleic acids can be single-stranded
or double-stranded. Generally, double-stranded nucleic acid
molecules are more stable in vivo, while single-stranded nucleic
acid molecules have increased immune activity. Thus in some aspects
of the invention it is preferred that the immunostimulatory nucleic
acid be single-stranded and in other aspects it is preferred that
the immunostimulatory nucleic acid be double-stranded.
[0096] The methods and products provided in accordance with the
invention relate to the use of CpG oligonucleotides. CpG ODN
trigger most (>95%) B-cells to proliferate, secrete
immunoglobulin (Ig), IL-6 and IL-12, and to be protected from
apoptosis. In addition, CpG ODN cause DC maturation and also
directly activate DCs, monocytes, and macrophages to secrete
IFN-.alpha./.beta., IL-6, IL-12, GM-CSF, chemokines and
TNF-.alpha.. These cytokines stimulate natural killer (NK) cells to
secrete IFN-.gamma. and have increased lytic activity. Overall, CpG
induces a strong Th1-like pattern of cytokine production dominated
by IL-12 and IFN-.gamma. with little secretion of Th2
cytokines.
[0097] In addition to induction of innate immune responses, CpG DNA
also augments antigen-specific responses due to (i) a strong
synergy between the B-cell signalling pathways triggered through
the B-cell antigen receptor and by CpG, (ii) Th1-like cytokines
that replace or augment antigen-specific T-help augmenting both B-
and T-cell antigen-specific responses and (iii) up-regulation of
co-stimulatory molecules that are required for cellular
responses.
[0098] CpG ODN has been shown to be a potent adjuvant to HBsAg in
BALB/c mice with clear Th1-like responses (predominantly IgG2a
antibodies and strong CTL) (49). CpG ODN was found to be superior
to other Th1 adjuvants such as monophosphoryl lipid A (MPL, Corixa)
or even complete Freund's adjuvant (CFA) which is too toxic for
human use. Similar results have been reported using CpG ODN with a
variety of other antigens (47, 50-53). CpG ODN have also been
reported to redirect a Th2 response previously established by
immunization with a Th2 antigen (i.e., Schistosomiasis surface
antigen) (54) or a Th2 adjuvant (i.e., alum).
[0099] There are at least three basic classes of CpG ODNs found to
be effective at stimulating healthy human PBMCs (Table 1). These
have differential effects that are likely associated with the
different modes of by which CpG ODNs can stimulate immune
cells.
[0100] The B class of CpG ODN are synthesized with nuclease
resistant phosphorothioate backbones and are generally
characterized by good B-cell and DC activation, leading to the
production of IL-12 and antibody, but only limited NK cell
activation. This class of ODN functions well as a vaccine adjuvant,
as has already been demonstrated in a phase I/II clinical trial
testing CpG (a member of this class) (SEQ ID NO.: 2) as an adjuvant
to a commercial hepatitis B vaccine (60).
[0101] The A class of CpG ODNs are synthesized with a chimeric
backbone where the 5' and 3' ends are phosphorothioate and the
central CpG motif region is phosphodiester. These ODNs are
characterized by good NK cell and DC activation leading to greater
production of IFN-.alpha. but limited B-cell activation.
[0102] The C class of CpG ODN are synthesized with a
phosphorothioate backbone and have stimulatory properties
intermediate to the other two classes of CpG ODNs (e.g., good
activation of B-cells as well as activation of NK cells and DCs).
TABLE-US-00002 TABLE 1 Pattern of in vitro immune activation
induced by the three different classes of CpG ODNs Natural Class
Backbone B-cells Killer cells Dendritic cells IFN-.alpha. A SOS
.sup.2 + ++++ ++++ ++++ B S .sup.1 ++++ ++ ++++ + C S .sup.1 +++
+++ +++ +++ .sup.1 S-ODN are made with a phosphorothioate backbone
.sup.2 SOS-ODN are made with a chimeric backbone where the central
CpG-containing region has phosphodiester linkages and the 3' and 5'
ends of the ODN are made with phosphorothioate linkages
[0103] The methods of the invention may embrace the use of A class,
B class and C class CpG immunostimulatory nucleic acids. As to CpG
nucleic acids, it has recently been described that there are
different classes of CpG nucleic acids. One class is potent for
activating B cells but is relatively weak in inducing IFN-.alpha.
and NK cell activation; this class has been termed the B class. The
B class CpG nucleic acids typically are fully stabilized and
include an unmethylated CpG dinucleotide within certain preferred
base contexts. See, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646;
6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class is
potent for inducing IFN-.alpha. and NK cell activation but is
relatively weak at stimulating B cells; this class has been termed
the A class. The A class CpG nucleic acids typically have
stabilized poly-G sequences at 5' and 3' ends and a palindromic
phosphodiester CpG dinucleotide-containing sequence of at least 6
nucleotides. See, for example, published patent application
PCT/US00/26527 (WO 01/22990). Yet another class of CpG nucleic
acids activates B cells and NK cells and induces IFN-.alpha.; this
class has been termed the C-class. The C-class CpG nucleic acids,
as first characterized, typically are fully stabilized, include a B
class-type sequence and a GC-rich palindrome or near-palindrome.
This class has been described in U.S. provisional patent
application 60/313,273, filed Aug. 17, 2001, U.S. Ser. No.
10/224,523 filed on Aug. 19, 2002, and US the entire contents of
which are incorporated herein by reference.
[0104] "A class" CpG immunostimulatory nucleic acids have been
described in U.S. Non-Provisional patent application Ser. No.
09/672,126 and published PCT application PCT/US00/26527 (WO
01/22990), both filed on Sep. 27, 2000. These nucleic acids are
characterized by the ability to induce high levels of
interferon-alpha while having minimal effects on B cell activation.
The A class CpG immunostimulatory nucleic acid do not necessarily
contain a hexamer palindrome GACGTC, AGCGCT, or AACGTT described by
Yamamoto and colleagues. Yamamoto S et al. J Immunol 148:4072-6
(1992).
[0105] Exemplary sequences of A class immunostimulatory nucleic
acids are described in U.S. Non-Provisional patent application Ser.
No. 09/672,126 and published PCT application PCT/US00/26527 (WO
01/22990), both filed on Sep. 27, 2000.
[0106] B class CpG immunostimulatory nucleic acids strongly
activate human B cells but have minimal effects inducing
interferon-.alpha.. B class CpG immunostimulatory nucleic acids
have been described in U.S. Pat. Nos. 6,194,388 B1 and 6,239,116
B1, issued on Feb. 27, 2001 and May 29, 2001 respectively.
[0107] The CpG oligonucleotides of the invention are
oligonucleotides which include at least one unmethylated CpG
dinucleotide. An oligonucleotide containing at least one
unmethylated CpG dinucleotide is a nucleic acid molecule which
contains an unmethylated cytosine-guanine dinucleotide sequence
(i.e., "CpG DNA" or DNA containing a 5' cytosine followed by 3'
guanine and linked by a phosphate bond) and activates the immune
system. The entire CpG oligonucleotide can be unmethylated or
portions may be unmethylated but at least the C of the 5'CG 3' must
be unmethylated. The terms CpG oligonucleotide or CpG nucleic acid
as used herein refer to an immunostimulatory CpG oligonucleotide or
a nucleic acid unless otherwise indicated.
[0108] In one embodiment the invention provides a B class CpG
oligonucleotide represented by at least the formula: 5'
X.sub.1X.sub.2CGX.sub.3X.sub.4 3' wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides. In one embodiment X.sub.2 is
adenine, guanine, or thymine. In another embodiment X.sub.3 is
cytosine, adenine, or thymine.
[0109] In another embodiment the invention provides an isolated B
class CpG oligonucleotide represented by at least the formula: 5'
N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.2 3' wherein X.sub.1,
X.sub.2, X.sub.3, and X.sub.4 are nucleotides and N is any
nucleotide and N.sub.1 and N.sub.2 are nucleic acid sequences
composed of from about 0-25 N's each. In one embodiment
X.sub.1X.sub.2 is a dinucleotide selected from the group consisting
of: GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG;
and X.sub.3X.sub.4 is a dinucleotide selected from the group
consisting of: TpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA,
and CpA. Preferably X.sub.1X.sub.2 is GpA or GpT and X.sub.3X.sub.4
is TpT. In other embodiments X.sub.1 or X.sub.2 or both are purines
and X.sub.3 or X.sub.4 or both are pyrimidines or X.sub.1X.sub.2 is
GpA and X.sub.3 or X.sub.4 or both are pyrimidines. In another
preferred embodiment X.sub.1X.sub.2 is a dinucleotide selected from
the group consisting of: TpA, ApA, ApC, ApG, and GpG. In yet
another embodiment X.sub.3X.sub.4 is a dinucleotide selected from
the group consisting of: TpT, TpA, TpG, ApA, ApG, GpA, and CpA.
X.sub.1X.sub.2 in another embodiment is a dinucleotide selected
from the group consisting of: TpT, TpG, ApT, GpC, CpC, CpT, TpC,
GpT and CpG; X.sub.3 is a nucleotide selected from the group
consisting of A and T and X.sub.4 is a nucleotide, but wherein when
X.sub.1X.sub.2 is TpC, GpT, or CpG, X.sub.3X.sub.4 is not TpC, ApT
or ApC.
[0110] In another preferred embodiment the CpG oligonucleotide has
the sequence 5' TCN.sub.1TX.sub.1X.sub.2CGX.sub.3X.sub.4 3' (SEQ ID
NO.:26). The CpG oligonucleotides of the invention in some
embodiments include X.sub.1X.sub.2 selected from the group
consisting of GpT, GpG, GpA and ApA and X.sub.3X.sub.4 is selected
from the group consisting of TpT, CpT and TpC.
[0111] The B class CpG nucleic acid sequences of the invention are
those broadly described above as well as disclosed in PCT Published
Patent Applications PCT/US95/01570 and PCT/US97/19791, and U.S.
Pat. No. 6,194,388 B1 and U.S. Pat. No. 6,239,116 B1, issued Feb.
27, 2001 and May 29, 2001 respectively. Exemplary sequences include
but are not limited to those disclosed in these latter applications
and patents.
[0112] The C class immunostimulatory nucleic acids contain at least
two distinct motifs have unique and desirable stimulatory effects
on cells of the immune system. Some of these ODN have both a
traditional "stimulatory" CpG sequence and a "GC-rich" or "B-cell
neutralizing" motif. These combination motif nucleic acids have
immune stimulating effects that fall somewhere between those
effects associated with traditional "class B" CpG ODN, which are
strong inducers of B cell activation and dendritic cell (DC)
activation, and those effects associated with a more recently
described class of immune stimulatory nucleic acids ("class A" CpG
ODN) which are strong inducers of IFN-.alpha. and natural killer
(NK) cell activation but relatively poor inducers of B-cell and DC
activation. Krieg A M et al. (1995) Nature 374:546-9; Ballas Z K et
al. (1996) J Immunol 157:1840-5; Yamamoto S et al. (1992) J Immunol
148:4072-6. While preferred class B CpG ODN often have
phosphorothioate backbones and preferred class A CpG ODN have mixed
or chimeric backbones, the C class of combination motif immune
stimulatory nucleic acids may have either stabilized, e.g.,
phosphorothioate, chimeric, or phosphodiester backbones, and in
some preferred embodiments, they have semi-soft backbones.
[0113] In one aspect the invention provides immune stimulatory
nucleic acids belonging to this new class of combination motif
immune-stimulatory nucleic acids. The B cell stimulatory domain is
defined by a formula: 5' X.sub.1DCGHX.sub.2 3'. D is a nucleotide
other than C. C is cytosine. G is guanine. H is a nucleotide other
than G.
[0114] X.sub.1 and X.sub.2 are any nucleic acid sequence 0 to 10
nucleotides long. X.sub.1 may include a CG, in which case there is
preferably a T immediately preceding this CG. In some embodiments
DCG is TCG. X.sub.1 is preferably from 0 to 6 nucleotides in
length. In some embodiments X.sub.2 does not contain any poly G or
poly A motifs. In other embodiments the immunostimulatory nucleic
acid has a poly-T sequence at the 5' end or at the 3' end. As used
herein, "poly-A" or "poly-T" shall refer to a stretch of four or
more consecutive A's or T's respectively, e.g., 5' AAAA 3' or 5'
TTTT 3'.
[0115] As used herein, "poly-G end" shall refer to a stretch of
four or more consecutive G's, e.g., 5' GGGG 3', occurring at the 5'
end or the 3' end of a nucleic acid. As used herein, "poly-G
nucleic acid" shall refer to a nucleic acid having the formula 5'
X.sub.1X.sub.2GGGX.sub.3X.sub.4 3' wherein X.sub.1, X.sub.2,
X.sub.3, and X.sub.4 are nucleotides and preferably at least one of
X.sub.3 and X.sub.4 is a G.
[0116] Some preferred designs for the B cell stimulatory domain
under this formula comprise TTTTTCG, TCG, TTCG, TTTCG, TTTTCG,
TCGT, TTCGT, TTTCGT, TCGTCGT.
[0117] The second motif of the nucleic acid is referred to as
either P or N and is positioned immediately 5' to X.sub.1 or
immediately 3' to X.sub.2.
[0118] N is a B-cell neutralizing sequence that begins with a CGG
trinucleotide and is at least 10 nucleotides long. A B-cell
neutralizing motif includes at least one CpG sequence in which the
CG is preceded by a C or followed by a G (Krieg A M et al. (1998)
Proc Natl Acad Sci USA 95:12631-12636) or is a CG containing DNA
sequence in which the C of the CG is methylated. As used herein,
"CpG" shall refer to a 5' cytosine (C) followed by a 3' guanine (G)
and linked by a phosphate bond. At least the C of the 5'CG 3' must
be unmethylated. Neutralizing motifs are motifs which has some
degree of immunostimulatory capability when present in an otherwise
non-stimulatory motif, but, which when present in the context of
other immunostimulatory motifs serve to reduce the
immunostimulatory potential of the other motifs.
[0119] P is a GC-rich palindrome containing sequence at least 10
nucleotides long. As used herein, "palindrome" and, equivalently,
"palindromic sequence" shall refer to an inverted repeat, i.e., a
sequence such as ABCDEE'D.degree. C.'B'A' in which A and A', B and
B', etc., are bases capable of forming the usual Watson-Crick base
pairs.
[0120] As used herein, "GC-rich palindrome" shall refer to a
palindrome having a base composition of at least two-thirds G's and
C's. In some embodiments the GC-rich domain is preferably 3' to the
"B cell stimulatory domain". In the case of a 10-base long GC-rich
palindrome, the palindrome thus contains at least 8 G's and C's. In
the case of a 12-base long GC-rich palindrome, the palindrome also
contains at least 8 G's and C's. In the case of a 14-mer GC-rich
palindrome, at least ten bases of the palindrome are G's and C's.
In some embodiments the GC-rich palindrome is made up exclusively
of G's and C's.
[0121] In some embodiments the GC-rich palindrome has a base
composition of at least 81 percent G's and C's. In the case of such
a 10-base long GC-rich palindrome, the palindrome thus is made
exclusively of G's and C's. In the case of such a 12-base long
GC-rich palindrome, it is preferred that at least ten bases (83
percent) of the palindrome are G's and C's. In some preferred
embodiments, a 12-base long GC-rich palindrome is made exclusively
of G's and C's. In the case of a 14-mer GC-rich palindrome, at
least twelve bases (86 percent) of the palindrome are G's and C's.
In some preferred embodiments, a 14-base long GC-rich palindrome is
made exclusively of G's and C's. The C's of a GC-rich palindrome
can be unmethylated or they can be methylated.
[0122] In general this domain has at least 3 Cs and Gs, more
preferably 4 of each, and most preferably 5 or more of each. The
number of Cs and Gs in this domain need not be identical. It is
preferred that the Cs and Gs are arranged so that they are able to
form a self-complementary duplex, or palindrome, such as CCGCGCGG.
This may be interrupted by As or Ts, but it is preferred that the
self-complementarity is at least partially preserved as for example
in the motifs CGACGTTCGTCG (SEQ ID NO: 27) or CGGCGCCGTGCCG (SEQ ID
NO: 28). When complementarity is not preserved, it is preferred
that the non-complementary base pairs be TG. In a preferred
embodiment there are no more than 3 consecutive bases that are not
part of the palindrome, preferably no more than 2, and most
preferably only 1. In some embodiments the GC-rich palindrome
includes at least one CGG trimer, at least one CCG trimer, or at
least one CGCG tetramer. In other embodiments the GC-rich
palindrome is not CCCCCCGGGGGG (SEQ ID NO: 29) or GGGGGGCCCCCC (SEQ
ID NO: 30), CCCCCGGGGG (SEQ ID NO: 31) or GGGGGCCCCC (SEQ ID NO:
32).
[0123] At least one of the G's of the GC rich region may be
substituted with an inosine (I). In some embodiments P includes
more than one I.
[0124] In certain embodiments the immunostimulatory nucleic acid
has one of the following formulas 5' NX.sub.1DCGHX.sub.2 3', 5'
X.sub.1DCGHX.sub.2N 3', 5' PX.sub.1DCGHX.sub.2 3', 5'
X.sub.1DCGHX.sub.2P 3', 5' X.sub.1DCGHX.sub.2PX.sub.3 3', 5'
X.sub.1DCGHPX.sub.3 3', 5' DCGHX.sub.2PX.sub.3 3', 5'
TCGHX.sub.2PX.sub.3 3', 5' DCGHPX.sub.3 3', or 5' DCGHP 3'.
[0125] In other aspects the invention provides immune stimulatory
nucleic acids which are defined by a formula: 5' N.sub.1PyGN.sub.2P
3'. N.sub.1 is any sequence 1 to 6 nucleotides long. Py is a
pyrimidine. G is guanine. N.sub.2 is any sequence 0 to 30
nucleotides long. P is a GC-rich palindrome containing sequence at
least 10 nucleotides long.
[0126] N.sub.1 and N.sub.2 may contain more than 50% pyrimidines,
and more preferably more than 50% T. N.sub.1 may include a CG, in
which case there is preferably a T immediately preceding this CG.
In some embodiments N.sub.1PyG is TCG (such as ODN 5376, which has
a 5' TCGG), and most preferably a TCGN.sub.2, where N.sub.2 is not
G.
[0127] N.sub.1PyGN.sub.2P may include one or more inosine (I)
nucleotides. Either the C or the G in N.sub.1 may be replaced by
inosine, but the CpI is preferred to the IpG. For inosine
substitutions such as IpG, the optimal activity may be achieved
with the use of a "semi-soft" or chimeric backbone, where the
linkage between the IG or the CI is phosphodiester. N.sub.1 may
include at least one CI, TCI, IG or TIG motif.
[0128] In certain embodiments N.sub.1PyGN.sub.2 is a sequence
selected from the group consisting of TTTTTCG, TCG, TTCG, TTTCG,
TTTTCG, TCGT, TTCGT, TTTCGT, and TCGTCGT.
[0129] Some non limiting examples of C-Class nucleic acids include:
TABLE-US-00003 SEQ ID NO: Sequence 17
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 18
T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 19
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 20
T*C_G*G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 21
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 22
T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 23
T*C_G*A*C_G*T*T*C_G*G*C*G*C*G*C*C*G 24
T*C_G*C_G*T*C_G*T*T*C_G*G*C*G*C*C*G 25
T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G
[0130] For facilitating uptake into cells, immunostimulatory
nucleic acids, including CpG-containing oligonucleotides, are
preferably in the range of 8 to 100 bases in length. However,
nucleic acids of any size greater than 8 nucleotides (even many kb
long) are capable of inducing an immune response according to the
invention if sufficient immunostimulatory motifs are present, since
larger nucleic acids are degraded into oligonucleotides inside of
cells. Preferably the immunostimulatory nucleic acid is in the
range of between 8 and 100 nucleotides in length. In some preferred
embodiments the immunostimulatory nucleic acids is between 12 and
40 nucleotides in length. In more preferred embodiments the
immunostimulatory nucleic acids is between 8 and 30 nucleotides in
length. In most preferred embodiments the immunostimulatory nucleic
acids is between 8 and 24 nucleotides in length.
[0131] "Palindromic sequence" shall mean an inverted repeat, i.e.,
a sequence such as ABCDEE'D.degree. C'B'A' in which A and A', B and
B', C and C', D and D', and E and E' are bases capable of forming
the usual Watson-Crick base pairs. In vivo, such palindromic
sequences may form double-stranded structures. In one embodiment
the CpG oligonucleotide contains a palindromic sequence. A
palindromic sequence used in this context refers to a palindrome in
which the CpG is part of the palindrome, and preferably is the
center of the palindrome. In another embodiment the CpG
oligonucleotide is free of a palindrome. A CpG oligonucleotide that
is free of a palindrome is one in which the CpG dinucleotide is not
part of a palindrome. Such an oligonucleotide may include a
palindrome in which the CpG is not the center of the
palindrome.
[0132] In some embodiments of the invention the immunostimulatory
oligonucleotides include immunostimulatory motifs which are "CpG
dinucleotides". A CpG dinucleotide can be methylated or
unmethylated. An immunostimulatory nucleic acid containing at least
one unmethylated CpG dinucleotide is a nucleic acid molecule which
contains an unmethylated cytosine-guanine dinucleotide sequence
(i.e., an unmethylated 5' cytidine followed by 3' guanosine and
linked by a phosphate bond) and which activates the immune system;
such an immunostimulatory nucleic acid is a CpG nucleic acid. CpG
nucleic acids have been described in a number of issued patents,
published patent applications, and other publications, including
U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371;
6,239,116; and 6,339,068. An immunostimulatory nucleic acid
containing at least one methylated CpG dinucleotide is a nucleic
acid which contains a methylated cytosine-guanine dinucleotide
sequence (i.e., a methylated 5' cytidine followed by a 3' guanosine
and linked by a phosphate bond) and which activates the immune
system. In other embodiments the immunostimulatory oligonucleotides
are free of CpG dinucleotides. These oligonucleotides which are
free of CpG dinucleotides are referred to as non-CpG
oligonucleotides, and they have non-CpG immunostimulatory motifs.
The invention, therefore, also encompasses nucleic acids with other
types of immunostimulatory motifs, which can be methylated or
unmethylated. The immunostimulatory oligonucleotides of the
invention, further, can include any combination of methylated and
unmethylated CpG and non-CpG immunostimulatory motifs.
[0133] The immunostimulatory nucleic acid molecules may have a
chimeric backbone. For purposes of the instant invention, a
chimeric backbone refers to a partially stabilized backbone,
wherein at least one internucleotide linkage is phosphodiester or
phosphodiester-like, and wherein at least one other internucleotide
linkage is a stabilized internucleotide linkage, wherein the at
least one phosphodiester or phosphodiester-like linkage and the at
least one stabilized linkage are different. Since boranophosphonate
linkages have been reported to be stabilized relative to
phosphodiester linkages, for purposes of the chimeric nature of the
backbone, boranophosphonate linkages can be classified either as
phosphodiester-like or as stabilized, depending on the context. For
example, a chimeric backbone according to the instant invention
could in one embodiment include at least one phosphodiester
(phosphodiester or phosphodiester-like) linkage and at least one
boranophosphonate (stabilized) linkage. In another embodiment a
chimeric backbone according to the instant invention could include
boranophosphonate (phosphodiester or phosphodiester-like) and
phosphorothioate (stabilized) linkages. A "stabilized
internucleotide linkage" shall mean an internucleotide linkage that
is relatively resistant to in vivo degradation (e.g., via an exo-
or endo-nuclease), compared to a phosphodiester internucleotide
linkage. Preferred stabilized internucleotide linkages include,
without limitation, phosphorothioate, phosphorodithioate,
methylphosphonate, and methylphosphorothioate. Other stabilized
internucleotide linkages include, without limitation: peptide,
alkyl, dephospho, and others as described above.
[0134] 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, e.g., 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 backbone modifications and substitutions have been
described. Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J
(1990) Bioconjugate Chem 1:165. Methods for preparing chimeric
oligonucleotides are also known. For instance patents issued to
Uhlmann et al have described such techniques.
[0135] Mixed backbone modified ODN may be synthesized using a
commercially available DNA synthesizer and standard phosphoramidite
chemistry. (F. E. Eckstein, "Oligonucleotides and Analogues--A
Practical Approach" IRL Press, Oxford, UK, 1991, and M. D.
Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719 (1980))
After coupling, PS linkages are introduced by sulfurization using
the Beaucage reagent (R. P. Iyer, W. Egan, J. B. Regan and S. L.
Beaucage, J. Am. Chem. Soc. 112, 1253 (1990)) (0.075 M in
acetonitrile) or phenyl acetyl disulfide (PADS) followed by capping
with acetic anhydride, 2,6-lutidine in tetrahydrofurane (1:1:8;
v:v:v) and N-methylimidazole (16% in tetrahydrofurane). This
capping step is performed after the sulfurization reaction to
minimize formation of undesired phosphodiester (PO) linkages at
positions where a phosphorothioate linkage should be located. In
the case of the introduction of a phosphodiester linkage, e.g. at a
CpG dinucleotide, the intermediate phosphorous-III is oxidized by
treatment with a solution of iodine in water/pyridine. After
cleavage from the solid support and final deprotection by treatment
with concentrated ammonia (15 hrs at 50.degree. C.), the ODN are
analyzed by HPLC on a Gen-Pak Fax column (Millipore-Waters) using a
NaCl-gradient (e.g. buffer A: 10 mM NaH.sub.2PO.sub.4 in
acetonitrile/water=1:4/v:v pH 6.8; buffer B: 10 mM
NaH.sub.2PO.sub.4, 1.5 M NaCl in acetonitrile/water=1:4/v:v; 5 to
60% B in 30 minutes at 1 ml/min) or by capillary gel
electrophoresis. The ODN can be purified by HPLC or by FPLC on a
Source High Performance column (Amersham Pharmacia).
HPLC-homogeneous fractions are combined and desalted via a C18
column or by ultrafiltration. The ODN was analyzed by MALDI-TOF
mass spectrometry to confirm the calculated mass.
[0136] The nucleic acids of the invention can also include other
modifications. These include nonionic DNA analogs, such as alkyl-
and aryl-phosphates (in which the charged phosphonate oxygen is
replaced by an alkyl or aryl group), phosphodiester and
alkylphosphotriesters, in which the charged oxygen moiety is
alkylated. Nucleic acids which contain diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
[0137] In some embodiments the oligonucleotides may be soft or
semi-soft oligonucleotides. A soft oligonucleotide is an
immunostimulatory oligonucleotide having a partially stabilized
backbone, in which phosphodiester or phosphodiester-like
internucleotide linkages occur only within and immediately adjacent
to at least one internal pyrimidine-purine dinucleotide (YZ).
Preferably YZ is YG, a pyrimidine-guanosine (YG) dinucleotide. The
at least one internal YZ dinucleotide itself has a phosphodiester
or phosphodiester-like internucleotide linkage. A phosphodiester or
phosphodiester-like internucleotide linkage occurring immediately
adjacent to the at least one internal YZ dinucleotide can be 5',
3', or both 5' and 3' to the at least one internal YZ
dinucleotide.
[0138] In particular, phosphodiester or phosphodiester-like
internucleotide linkages involve "internal dinucleotides". An
internal dinucleotide in general shall mean any pair of adjacent
nucleotides connected by an internucleotide linkage, in which
neither nucleotide in the pair of nucleotides is a terminal
nucleotide, i.e., neither nucleotide in the pair of nucleotides is
a nucleotide defining the 5' or 3' end of the oligonucleotide. Thus
a linear oligonucleotide that is n nucleotides long has a total of
n-1 dinucleotides and only n-3 internal dinucleotides. Each
internucleotide linkage in an internal dinucleotide is an internal
internucleotide linkage. Thus a linear oligonucleotide that is n
nucleotides long has a total of n-1 internucleotide linkages and
only n-3 internal internucleotide linkages. The strategically
placed phosphodiester or phosphodiester-like internucleotide
linkages, therefore, refer to phosphodiester or phosphodiester-like
internucleotide linkages positioned between any pair of nucleotides
in the nucleic acid sequence. In some embodiments the
phosphodiester or phosphodiester-like internucleotide linkages are
not positioned between either pair of nucleotides closest to the 5'
or 3' end.
[0139] Preferably a phosphodiester or phosphodiester-like
internucleotide linkage occurring immediately adjacent to the at
least one internal YZ dinucleotide is itself an internal
internucleotide linkage. Thus for a sequence N.sub.1 YZ N.sub.2,
wherein N.sub.1 and N.sub.2 are each, independent of the other, any
single nucleotide, the YZ dinucleotide has a phosphodiester or
phosphodiester-like internucleotide linkage, and in addition (a)
N.sub.1 and Y are linked by a phosphodiester or phosphodiester-like
internucleotide linkage when N.sub.1 is an internal nucleotide, (b)
Z and N.sub.2 are linked by a phosphodiester or phosphodiester-like
internucleotide linkage when N.sub.2 is an internal nucleotide, or
(c) N.sub.1 and Y are linked by a phosphodiester or
phosphodiester-like internucleotide linkage when N.sub.1 is an
internal nucleotide and Z and N.sub.2 are linked by a
phosphodiester or phosphodiester-like internucleotide linkage when
N.sub.2 is an internal nucleotide.
[0140] Soft oligonucleotides according to the instant invention are
believed to be relatively susceptible to nuclease cleavage compared
to completely stabilized oligonucleotides. Without meaning to be
bound to a particular theory or mechanism, it is believed that soft
oligonucleotides of the invention are cleavable to fragments with
reduced or no immunostimulatory activity relative to full-length
soft oligonucleotides. Incorporation of at least one
nuclease-sensitive internucleotide linkage, particularly near the
middle of the oligonucleotide, is believed to provide an "off
switch" which alters the pharmacokinetics of the oligonucleotide so
as to reduce the duration of maximal immunostimulatory activity of
the oligonucleotide. This can be of particular value in tissues and
in clinical applications in which it is desirable to avoid injury
related to chronic local inflammation or immunostimulation, e.g.,
the kidney.
[0141] A semi-soft oligonucleotide is an immunostimulatory
oligonucleotide having a partially stabilized backbone, in which
phosphodiester or phosphodiester-like internucleotide linkages
occur only within at least one internal pyrimidine-purine (YZ)
dinucleotide. Semi-soft oligonucleotides generally possess
increased immunostimulatory potency relative to corresponding fully
stabilized immunostimulatory oligonucleotides. Due to the greater
potency of semi-soft oligonucleotides, semi-soft oligonucleotides
may be used, in some instances, at lower effective concentations
and have lower effective doses than conventional fully stabilized
immunostimulatory oligonucleotides in order to achieve a desired
biological effect.
[0142] It is believed that the foregoing properties of semi-soft
oligonucleotides generally increase with increasing "dose" of
phosphodiester or phosphodiester-like internucleotide linkages
involving internal YZ dinucleotides. Thus it is believed, for
example, that generally for a given oligonucleotide sequence with
five internal YZ dinucleotides, an oligonucleotide with five
internal phosphodiester or phosphodiester-like YZ internucleotide
linkages is more immunostimulatory than an oligonucleotide with
four internal phosphodiester or phosphodiester-like YG
internucleotide linkages, which in turn is more immunostimulatory
than an oligonucleotide with three internal phosphodiester or
phosphodiester-like YZ internucleotide linkages, which in turn is
more immunostimulatory than an oligonucleotide with two internal
phosphodiester or phosphodiester-like YZ internucleotide linkages,
which in turn is more immunostimulatory than an oligonucleotide
with one internal phosphodiester or phosphodiester-like YZ
internucleotide linkage. Importantly, inclusion of even one
internal phosphodiester or phosphodiester-like YZ internucleotide
linkage is believed to be advantageous over no internal
phosphodiester or phosphodiester-like YZ internucleotide linkage.
In addition to the number of phosphodiester or phosphodiester-like
internucleotide linkages, the position along the length of the
nucleic acid can also affect potency.
[0143] The soft and semi-soft oligonucleotides will generally
include, in addition to the phosphodiester or phosphodiester-like
internucleotide linkages at preferred internal positions, 5' and 3'
ends that are resistant to degradation. Such degradation-resistant
ends can involve any suitable modification that results in an
increased resistance against exonuclease digestion over
corresponding unmodified ends. For instance, the 5' and 3' ends can
be stabilized by the inclusion there of at least one phosphate
modification of the backbone. In a preferred embodiment, the at
least one phosphate modification of the backbone at each end is
independently a phosphorothioate, phosphorodithioate,
methylphosphonate, or methylphosphorothioate internucleotide
linkage. In another embodiment, the degradation-resistant end
includes one or more nucleotide units connected by peptide or amide
linkages at the 3' end.
[0144] A phosphodiester internucleotide linkage is the type of
linkage characteristic of nucleic acids found in nature. As shown
in FIG. 20, the phosphodiester internucleotide linkage includes a
phosphorus atom flanked by two bridging oxygen atoms and bound also
by two additional oxygen atoms, one charged and the other
uncharged. Phosphodiester internucleotide linkage is particularly
preferred when it is important to reduce the tissue half-life of
the oligonucleotide.
[0145] A phosphodiester-like internucleotide linkage is a
phosphorus-containing bridging group that is chemically and/or
diastereomerically similar to phosphodiester. Measures of
similarity to phosphodiester include susceptibility to nuclease
digestion and ability to activate RNAse H. Thus for example
phosphodiester, but not phosphorothioate, oligonucleotides are
susceptible to nuclease digestion, while both phosphodiester and
phosphorothioate oligonucleotides activate RNAse H. In a preferred
embodiment the phosphodiester-like internucleotide linkage is
boranophosphate (or equivalently, boranophosphonate) linkage. U.S.
Pat. No. 5,177,198; U.S. Pat. No. 5,859,231; U.S. Pat. No.
6,160,109; U.S. Pat. No. 6,207,819; Sergueev et al., (1998) J Am
Chem Soc 120:9417-27. In another preferred embodiment the
phosphodiester-like internucleotide linkage is diasteromerically
pure Rp phosphorothioate. It is believed that diasteromerically
pure Rp phosphorothioate is more susceptible to nuclease digestion
and is better at activating RNAse H than mixed or
diastereomerically pure Sp phosphorothioate. Stereoisomers of CpG
oligonucleotides are the subject of co-pending U.S. patent
application Ser. No. 09/361,575 filed Jul. 27, 1999, and published
PCT application PCT/US99/17100 (WO 00/06588). It is to be noted
that for purposes of the instant invention, the term
"phosphodiester-like internucleotide linkage" specifically excludes
phosphorodithioate and methylphosphonate internucleotide
linkages.
[0146] As described above the soft and semi-soft oligonucleotides
of the invention may have phosphodiester like linkages between C
and G. One example of a phosphodiester-like linkage is a
phosphorothioate linkage in an Rp conformation. Oligonucleotide
p-chirality can have apparently opposite effects on the immune
activity of a CpG oligonucleotide, depending upon the time point at
which activity is measured. At an early time point of 40 minutes,
the R.sub.p but not the S.sub.P stereoisomer of phosphorothioate
CpG oligonucleotide induces JNK phosphorylation in mouse spleen
cells. In contrast, when assayed at a late time point of 44 hr, the
S.sub.P but not the R.sub.p stereoisomer is active in stimulating
spleen cell proliferation. This difference in the kinetics and
bioactivity of the R.sub.p and S.sub.P stereoisomers does not
result from any difference in cell uptake, but rather most likely
is due to two opposing biologic roles of the p-chirality. First,
the enhanced activity of the Rp stereoisomer compared to the Sp for
stimulating immune cells at early time points indicates that the Rp
may be more effective at interacting with the CpG receptor, TLR9,
or inducing the downstream signalling pathways. On the other hand,
the faster degradation of the Rp PS-oligonucleotides compared to
the Sp results in a much shorter duration of signalling, so that
the Sp PS-oligonucleotides appear to be more biologically active
when tested at later time points.
[0147] A surprisingly strong effect is achieved by the p-chirality
at the CpG dinucleotide itself. In comparison to a stereo-random
CpG oligonucleotide the congener in which the single CpG
dinucleotide was linked in Rp was slightly more active, while the
congener containing an Sp linkage was nearly inactive for inducing
spleen cell proliferation.
[0148] The size (i.e., the number of nucleotide residues along the
length of the nucleic acid) of the immunostimulatory
oligonucleotide may also contribute to the stimulatory activity of
the oligonucleotide. For facilitating uptake into cells
immunostimulatory oligonucleotides preferably have a minimum length
of 6 nucleotide residues. Nucleic acids of any size greater than 6
nucleotides (even many kb long) are capable of inducing an immune
response according to the invention if sufficient immunostimulatory
motifs are present, since larger nucleic acids are degraded inside
of cells. It is believed by the instant inventors that semi-soft
oligonucleotides as short as 4 nucleotides can also be
immunostimulatory if they can be delivered to the interior of the
cell. In certain preferred embodiments according to the instant
invention, the immunostimulatory oligonucleotides are between 4 and
100 nucleotides long. In typical embodiments the immunostimulatory
oligonucleotides are between 6 and 40 nucleotides long. In certain
preferred embodiments according to the instant invention, the
immunostimulatory oligonucleotides are between 6 and 19 nucleotides
long.
[0149] The immunostimulatory oligonucleotides generally have a
length in the range of between 4 and 100 and in some embodiments 10
and 40. The length may be in the range of between 16 and 24
nucleotides.
[0150] The terms "nucleic acid" and "oligonucleotide" also
encompass nucleic acids or oligonucleotides with substitutions or
modifications, such as in the bases and/or sugars. For example,
they include nucleic acids having backbone sugars that are
covalently attached to low molecular weight organic groups other
than a hydroxyl group at the 2' position and other than a phosphate
group or hydroxy group at the 5' position. Thus modified nucleic
acids may include a 2'-O-alkylated ribose group. In addition,
modified nucleic acids may include sugars such as arabinose or
2'-fluoroarabinose instead of ribose. Thus the nucleic acids may be
heterogeneous in backbone composition thereby containing any
possible combination of polymer units linked together such as
peptide-nucleic acids (which have an amino acid backbone with
nucleic acid bases).
[0151] Nucleic acids also include substituted purines and
pyrimidines such as C-5 propyne pyrimidine and
7-deaza-7-substituted purine modified bases. Wagner R W et al.
(1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but
are not limited to adenine, cytosine, guanine, thymine,
5-methylcytosine, 5-hydroxycytosine, 5-fluorocytosine,
2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine,
hypoxanthine, and other naturally and non-naturally occurring
nucleobases, substituted and unsubstituted aromatic moieties. Other
such modifications are well known to those of skill in the art.
[0152] The immunostimulatory oligonucleotides of the instant
invention can encompass various chemical modifications and
substitutions, in comparison to natural RNA and DNA, involving a
phosphodiester internucleotide bridge, a .beta.-D-ribose unit
and/or a natural nucleotide 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
S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and
Hunziker J et al. (1995) Mod Synth Methods 7:331-417. An
oligonucleotide according to the invention may have one or more
modifications, wherein each modification is located at a particular
phosphodiester internucleotide bridge and/or at a particular
.beta.-D-ribose unit and/or at a particular natural nucleotide base
position in comparison to an oligonucleotide of the same sequence
which is composed of natural DNA or RNA.
[0153] For example, the invention relates to an oligonucleotide
which may comprise one or more modifications and wherein each
modification is independently selected from: [0154] a) the
replacement of a phosphodiester internucleotide bridge located at
the 3' and/or the 5' end of a nucleotide by a modified
internucleotide bridge, [0155] b) the replacement of phosphodiester
bridge located at the 3' and/or the 5' end of a nucleotide by a
dephospho bridge, [0156] c) the replacement of a sugar phosphate
unit from the sugar phosphate backbone by another unit, [0157] d)
the replacement of a .beta.-D-ribose unit by a modified sugar unit,
and [0158] e) the replacement of a natural nucleotide base by a
modified nucleotide base.
[0159] More detailed examples for the chemical modification of an
oligonucleotide are as follows.
[0160] A phosphodiester internucleotide bridge located at the 3'
and/or the 5' end of a nucleotide can be replaced by a modified
internucleotide bridge, wherein the modified internucleotide bridge
is for example selected from phosphorothioate, phosphorodithioate,
NR.sup.1R.sup.2-phosphoramidate, boranophosphate,
.alpha.-hydroxybenzyl phosphonate,
phosphate-(C.sub.1-C.sub.21)-O-alkyl ester,
phosphate-[(C.sub.6-C.sub.12)aryl-(C.sub.1-C.sub.21)-O-alkyl]ester,
(C.sub.1-C.sub.8)alkylphosphonate and/or
(C.sub.6-C.sub.12)arylphosphonate bridges,
(C.sub.7-C.sub.12)-.alpha.-hydroxymethyl-aryl (e.g., disclosed in
WO 95/01363), wherein (C.sub.6-C.sub.12)aryl,
(C.sub.6-C.sub.20)aryl and (C.sub.6-C.sub.14)aryl are optionally
substituted by halogen, alkyl, alkoxy, nitro, cyano, and where
R.sup.1 and R.sup.2 are, independently of each other, hydrogen,
(C.sub.1-C.sub.18)-alkyl, (C.sub.6-C.sub.20)-aryl,
(C.sub.6-C.sub.14)-aryl-(C.sub.1-C.sub.8)-alkyl, preferably
hydrogen, (C.sub.1-C.sub.8)-alkyl, preferably
(C.sub.1-C.sub.4)-alkyl and/or methoxyethyl, or R.sup.1 and R.sup.2
form, together with the nitrogen atom carrying them, a 5-6-membered
heterocyclic ring which can additionally contain a further
heteroatom from the group O, S and N.
[0161] The replacement of a phosphodiester bridge located at the 3'
and/or the 5' end of a nucleotide by a dephospho bridge (dephospho
bridges are described, for example, in Uhlmann E 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.
[0162] A sugar phosphate unit (i.e., a .beta.-D-ribose and
phosphodiester internucleotide 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 E P et al. (1989) Nucleic Acids
Res 17:6129-41), that is, e.g., the replacement by a
morpholino-derivative unit; or to build up a polyamide nucleic acid
("PNA"; as described for example, in Nielsen P E et al. (1994)
Bioconjug Chem 5:3-7), that is, e.g., the replacement by a PNA
backbone unit, e.g., by 2-aminoethylglycine.
[0163] A .beta.-ribose unit or a .beta.-D-2'-deoxyribose unit can
be replaced by a modified sugar unit, wherein the modified sugar
unit is for example selected from .beta.-D-ribose,
.alpha.-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose,
2'-F-arabinose, 2'-O-(C.sub.1-C.sub.6)alkyl-ribose, preferably
2'-O-(C.sub.1-C.sub.6)alkyl-ribose is 2'-O-methylribose, 2'-O-
(C.sub.2-C.sub.6)alkenyl-ribose,
2'-[O-(C.sub.1-C.sub.6)alkyl-O-(C.sub.1-C.sub.6)alkyl]-ribose,
2'-NH.sub.2-2'-deoxyribose, .beta.-D-xylo-furanose,
.alpha.-arabinofuranose,
2,4-dideoxy-.beta.-D-erythro-hexo-pyranose, and carbocyclic
(described, for example, in Froehler J (1992) Am Chem 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) Helv Chim Acta 76:481).
[0164] In some preferred embodiments the sugar is
2'-O-methylribose, particularly for one or both nucleotides linked
by a phosphodiester or phosphodiester-like internucleotide
linkage.
[0165] Nucleic acids also include substituted purines and
pyrimidines such as C-5 propyne pyrimidine and
7-deaza-7-substituted purine modified bases. Wagner R W et al.
(1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but
are not limited to adenine, cytosine, guanine, and thymine, and
other naturally and non-naturally occurring nucleobases,
substituted and unsubstituted aromatic moieties.
[0166] 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
nucleotide base may be, for example, selected from hypoxanthine,
uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil,
5-aminouracil, 5-(C.sub.1-C.sub.6)-alkyluracil,
5-(C.sub.2-C.sub.6)-alkenyluracil,
5-(C.sub.2-C.sub.6)-alkynyluracil, 5-(hydroxymethyl)uracil,
5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine,
5-(C.sub.1-C.sub.6)-alkylcytosine,
5-(C.sub.2-C.sub.6)-alkenylcytosine,
5-(C.sub.2-C.sub.6)-alkynylcytosine, 5-chlorocytosine,
5-fluorocytosine, 5-bromocytosine, N.sup.2-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-ethylcytosine, 5-hydroxydeoxycytidine,
5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,
N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and
deoxyribonucleotides 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 nucleotide bases. This list is meant to
be exemplary and is not to be interpreted to be limiting.
[0167] In particular formulas described herein a set of modified
bases is defined. For instance the letter Y is used to refer to a
nucleotide containing a cytosine or 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
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).
[0168] The letter Z is used to refer to guanine or 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-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen
atom (dSpacer).
[0169] The oligonucleotides may have one or more accessible 5'
ends. It is possible to create modified oligonucleotides having two
such 5' ends. This may be achieved, for instance by attaching two
oligonucleotides through a 3'-3' linkage to generate an
oligonucleotide having one or two accessible 5' ends. The
3'3'-linkage may be a phosphodiester, phosphorothioate or any other
modified internucleotide bridge. Methods for accomplishing such
linkages are known in the art. For instance, such linkages have
been described in Seliger, H.; et al., Oligonucleotide analogs with
terminal 3'-3'- and 5'-5'-internucleotidic linkages as antisense
inhibitors of viral gene expression, Nucleotides & Nucleotides
(1991), 10(1-3), 469-77 and Jiang, et al., Pseudo-cyclic
oligonucleotides: in vitro and in vivo properties, Bioorganic &
Medicinal Chemistry (1999), 7(12), 2727-2735.
[0170] Additionally, 3'-3'-linked nucleic acids where the linkage
between the 3'-terminal nucleotides is not a phosphodiester,
phosphorothioate or other modified bridge, can be prepared using an
additional spacer, such as tri- or tetra-ethylenglycol phosphate
moiety (Durand, M. et al, Triple-helix formation by an
oligonucleotide containing one (dA)12 and two (dT)12 sequences
bridged by two hexaethylene glycol chains, Biochemistry (1992),
31(38), 9197-204, U.S. Pat. No. 5,658,738, and U.S. Pat. No.
5,668,265). Alternatively, the non-nucleotidic linker may be
derived from ethanediol, propanediol, or from an abasic deoxyribose
(dSpacer) unit (Fontanel, Marie Laurence et al., Sterical
recognition by T4 polynucleotide kinase of non-nucleosidic moieties
5'-attached to oligonucleotides; Nucleic Acids Research (1994),
22(11), 2022-7) using standard phosphoramidite chemistry. The
non-nucleotidic linkers can be incorporated once or multiple times,
or combined with each other allowing for any desirable distance
between the 3'-ends of the two ODNs to be linked.
[0171] For use in the instant invention, the oligonucleotides of
the invention can be synthesized de novo using any of a number of
procedures well known in the art. For example, the b-cyanoethyl
phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet.
Let. 22:1859, 1981); nucleotide H-phosphonate method (Garegg et
al., Tet. Let. 27:4051-4054, 1986; Froehler et al., Nucl. Acid.
Res. 14:5399-5407, 1986,; Garegg et al., Tet. Let. 27:4055-4058,
1986, Gaffney et al., Tet. Let. 29:2619-2622, 1988). These
chemistries can be performed by a variety of automated nucleic acid
synthesizers available in the market. 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.
[0172] The oligonucleotides are partially resistant to degradation
(e.g., are stabilized). A "stabilized oligonucleotide molecule"
shall mean an oligonucleotide that is relatively resistant to in
vivo degradation (e.g. via an exo- or endo-nuclease). Nucleic acid
stabilization can be accomplished via backbone modifications.
Oligonucleotides having phosphorothioate linkages provide maximal
activity and protect the oligonucleotide from degradation by
intracellular exo- and endo-nucleases. Other modified
oligonucleotides include phosphodiester modified nucleic acids,
combinations of phosphodiester and phosphorothioate nucleic acid,
methylphosphonate, methylphosphorothioate, phosphorodithioate,
p-ethoxy, and combinations thereof.
[0173] 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, e.g., 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 backbone modifications and substitutions have been
described (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. 90:544,
1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).
[0174] Other stabilized oligonucleotides include: nonionic DNA
analogs, such as alkyl- and aryl-phosphates (in which the charged
phosphonate oxygen is replaced by an alkyl or aryl group),
phosphodiester and alkylphosphotriesters, in which the charged
oxygen moiety is alkylated. Nucleic acids which contain diol, such
as tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
[0175] The immunostimulatory oligonucleotides may also contain one
or more unusual linkages between the nucleotide or
nucleotide-analogous moieties. The usual internucleoside linkage is
the 3'5'-linkage. All other linkages are considered as unusual
internucleoside linkages, such as 2'5'-, 5'5'-, 3'3'-, 2'2'-,
2'3'-linkages. Thereby, the nomenclature 2' to 5' is chosen
according to the carbon atom of ribose. However, if unnatural sugar
moieties are employed, such as ring-expanded sugar analogs (e.g.
hexanose, cylohexene or pyranose) or bi- or tricyclic sugar
analogs, then this nomenclature changes according to the
nomenclature of the monomer. In 3'-deoxy-.beta.-D-ribopyranose
analogs (also called p-DNA), the mononucleotides are e.g. connected
via a 4'2'-linkage.
[0176] If the nucleotide contains one 3'3'-linkage, then this
oligonucleotide analog will have two unlinked 5'-ends. Similarly,
if the nucleotide contains one 5'5'-linkage, then this
oligonucleotide analog will have two unlinked 3'-ends. The
accessibility of unlinked ends of nucleotides may be better
accessible by their receptors. Both types of unusual linkages
(3'3'- and 5'5') were described by Ramalho Ortigao et al.
(Antisense Research and Development (1992) 2, 129-46), whereby
oligonucleotides having a 3'3'-linkage were reported to show
enhanced stability towards cleavage by nucleases.
[0177] Different types of linkages can also be combined in one
molecule which may lead to branching of the oligomer. If one part
of the oligonucleotide is connected at the 3'-end via a
3'3'-linkage to a second oligonucleotide part and at the 2'-end via
a 2'3'-linkage to a third part of the molecule, this results e.g.
in a branched oligonucleotide with three 5'-ends (3'3'-,
2'3'-branched).
[0178] In principle, linkages between different parts of an
oligonucleotide or between different oligonucleotides,
respectively, can occur via all parts of the molecule, as long as
this does not negatively interfere with the recognition by its
receptor. According to the nature of the nucleic acid, the linkage
can involve the sugar moiety (Su), the heterocyclic nucleobase (Ba)
or the phosphate backbone (Ph). Thus, linkages of the type Su-Su,
Su-Ph, Su-Ba, Ba-Ba, Ba-Su, Ba-Ph, Ph-Ph, Ph-Su, and Ph-Ba are
possible. If the oligonucleotides are further modified by certain
non-nucleotidic substituents, the linkage can also occur via the
modified parts of the oligonucleotides. These modifications include
also modified nucleic acids, e.g. PNA, LNA, or Morpholino
Oligonucleotide analogs.
[0179] The linkages are preferably composed of C, H, N, O, S, B, P,
and Halogen, containing 3 to 300 atoms. An example with 3 atoms is
an acetal linkage (ODN1-3'-O--CH.sub.2--O-3'-ODN2; Froehler and
Matteucci) connecting e.g. the 3'-hydroxy group of one nucleotide
to the 3'-hydroxy group of a second oligonucleotide. An example
with about 300 atoms is PEG-40 (tetraconta polyethyleneglycol).
Preferred linkages are phosphodiester, phosphorothioate,
methylphosphonate, phosphoramidate, boranophosphonate, amide,
ether, thioether, acetal, thioacetal, urea, thiourea, sulfonamide,
Schiff Base and disulfide linkages. Another possibility is the use
of the Solulink BioConjugation System (www.trilinkbiotech.com).
[0180] If the oligonucleotide is composed of two or more sequence
parts, these parts can be identical or different. Thus, in an
oligonucleotide with a 3'3'-linkage, the sequences can be identical
5'-ODN1-3'3'-ODN1-5' or different 5'-ODN1-3'3'-ODN2-5'.
Furthermore, the chemical modification of the various
oligonucleotide parts as well as the linker connecting them may be
different. Since the uptake of short oligonucleotides appears to be
less efficient than that of long oligonucleotides, linking of two
or more short sequences results in improved immune stimulation. The
length of the short oligonucleotides is preferably 2-20
nucleotides, more preferably 3-16 nucleotides, but most preferably
5-10 nucleotides. Preferred are linked oligonucleotides which have
two or more unlinked 5'-ends.
[0181] The oligonucleotide partial sequences may also be linked by
non-nucleotidic linkers, in particular abasic linkers (dSpacers),
trietyhlene glycol units or hexaethylene glycol units. Further
preferred linkers are alkylamino linkers, such as C3, C6, C12
aminolinkers, and also alkylthiol linkers, such as C3 or C6 thiol
linkers. The oligonucleotides can also 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 (www.glenres.com), in particular those
oligonucleotides with a 3'3'-linkage. Branching of the
oligonucleotides by multiple doubler, trebler, or other multiplier
units leads to dendrimers which are a further embodiment of this
invention. The oligonucleotides may also contain linker units
resulting from peptide modifying reagents or oligonucleotide
modifying reagents (www.glenres.com). Furthermore, it may contain
one or more natural or unnatural amino acid residues which are
connected by peptide (amide) linkages.
[0182] Another possibility for linking oligonucleotides is via
crosslinking of the heterocyclic bases (Verma and Eckstein; Annu.
Rev. Biochem. (1998) 67: 99-134; page 124). Yet another possibility
is a linkage between the sugar moiety of one sequence part with the
heterocyclic base of another sequence part (Iyer et al. Curr. Opin.
Mol. Therapeutics (1999) 1: 344-358; page 352).
[0183] 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
post-synthesis of the individual partial sequences.
[0184] A "subject" shall mean a human or vertebrate animal
including but not limited to a dog, cat, horse, cow, pig, sheep,
goat, chicken, non-human primate (e.g., monkey), fish (aquaculture
species, e.g., salmon), rabbit, rat, and mouse.
[0185] A "subject having a viral infection" is a subject that has
been exposed to a virus and has acute or chronic manifestations or
detectable levels of the virus in the body. In preferred
embodiments of the invention, the subject is one having a chronic
viral infection, more preferably a chronic hepatitis C infection.
In important aspects of the invention, the subject is one that is
non-responsive to prior therapy for hepatitis C infection. For
example, a non-responsive subject includes one that was previously
treated for hepatitis C infection with, for example, IFN-.alpha.
(e.g., Intron A), and but such treatment was not successful, as
described herein. The invention intends to treat subjects that are
non-responsive, and in some instances to identify subjects that
would be non-responsive in order to triage effective treatment.
[0186] Immunostimulatory nucleic acids can be effective in any
vertebrate. Different immunostimulatory nucleic acids can cause
optimal immune stimulation depending on the mammalian species. Thus
an immunostimulatory nucleic acid causing optimal stimulation or
inhibition in humans may not cause optimal stimulation or
inhibition in a mouse, and vice versa. One of skill in the art can
identify the most appropriate immunostimulatory nucleic acids
useful for a particular mammalian species of interest using routine
assays described herein and/or known in the art, using the guidance
supplied herein.
[0187] The immunostimulatory nucleic acid 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 (e.g., pDCs or B cells) and/or increased
cellular uptake by target cells. 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 nucleic acid is released in a functional
form.
[0188] The immunostimulatory nucleic acid or other therapeutics may
be administered alone (e.g., in saline or buffer) or using any
delivery vehicles known in the art. For instance the following
delivery vehicles have been described: cochleates; emulsomes;
ISCOMs; liposomes; live bacterial vectors (e.g., Salmonella,
Escherichia coli, Bacillus Calmette-Guerin, Shigella,
Lactobacillus); live viral vectors (e.g., Vaccinia, adenovirus,
Herpes Simplex); microspheres; nucleic acid vaccines; polymers
(e.g., carboxymethylcellulose, chitosan); polymer rings;
Proteosomes; sodium fluoride; transgenic plants; virosomes;
virus-like particles. Those skilled in the art will recognize that
other delivery vehicles that are known in the art may also be
used.
[0189] 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 preferred mode of administration, an
effective therapeutic treatment regimen can be planned which does
not cause substantial toxicity and yet is entirely effective to
treat the particular subject as described above. The effective
amount for any particular application can vary depending on such
factors as the disease or condition being treated, the particular
immunostimulatory nucleic acid being administered (e.g., the class
of CpG immunostimulatory nucleic acid, the number of unmethylated
CpG motifs or their location in the nucleic acid, the degree of
chirality to the oligonucleotide, etc.), whether an antigen is also
administered and the nature of such antigen, 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 immunostimulatory nucleic acids and/or other
therapeutic agent without necessitating undue experimentation.
[0190] For adult human subjects, doses of the immunostimulatory
nucleic acids compounds described herein typically range from about
50 .mu.g/dose to 20 mg/dose, more typically from about 80
.mu.g/dose to 8 mg/dose, and most typically from about 800
.mu.g/dose to 4 mg/dose. Stated in terms of subject body weight,
typical dosages range from about 0.5 to 500 .mu.g/kg/dose, more
typically from about 1 to 100 .mu.g/kg/dose, and most typically
from about 10 to 50 .mu.g/kg/dose. Doses will depend on factors
including the route of administration, e.g., oral administration
may require a substantially larger dose than subcutaneous
administration.
[0191] The formulations of the invention are administered in
pharmaceutically acceptable solutions, which may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, adjuvants, and
optionally other therapeutic ingredients.
[0192] The immunostimulatory nucleic acids can be given in
conjunction with other agents known in the art to be useful in
treating viral infections. Of particular importance is the
combination of immunostimulatory nucleic acids with anti-viral
agents such as IFN-.alpha., as demonstrated in the Examples
section, to provide a synergistic response. Immunostimulatory
nucleic acids can be used as a substitute for Ribavirin, which
currently is administered together with IFN-.alpha.. Examples of
such other agents currently used or under investigation for use in
combination with IFN-.alpha. include amantadine, and cytokines,
including IL-2, IL-10, IL-12, and IFN-.gamma..
[0193] Antiviral 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, including translation initiation (e.g., interferon,
antisense, and ribozymes), virus enzymes (e.g., nonstructural
serine proteases, RNA polymerases, reverse transcriptases and
helicases), replication of viral RNA or DNA (e.g., nucleoside
analogues), maturation of new virus proteins (e.g., protease
inhibitors such as serine protease inhibitor BILN2061ZW from
Boehringer Ingelheim), anti-oxidants such as Livfit (U.S. Pat. No.
6,136,316), and budding and release of the virus. Other anti-viral
agents are described in U.S. Pat. Nos. 6,130,326, and 6,440,985,
and published US patent application 20020095033. Ribavirin
analogues are also anti-viral agents embraced by the invention.
[0194] Nucleotide analogues are synthetic compounds which are
similar to nucleotides, but which have an incomplete or abnormal
deoxyribose or ribose group. Once the nucleotide analogues are in
the cell, they are phosphorylated, producing the triphosphate
formed which competes with normal nucleotides for incorporation
into the viral DNA or RNA. Once the triphosphate form of the
nucleotide analogue is incorporated into the growing nucleic acid
chain, it causes irreversible association with the viral polymerase
and thus chain termination.
[0195] Immunoglobulin therapy is typically used for the prevention
of viral infection, but can also be used to reduce levels of
circulating virus and preventing newly formed cells from becoming
infected. Immunoglobulin therapy for viral infections is different
than 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 reduction of viremia for the period of
time that the antibodies are present in the host. In general there
are two types of immunoglobulin therapies, normal immunoglobulin
therapy and hyper-immunoglobulin 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 human viruses,
such as hepatitis A, parvovirus, enterovirus (especially in
neonates). To use normal immune globulin therapy for HCV, the serum
would have to be obtained from people who were previously infected
with HCV and who have successfully cleared the infection, either
spontaneously or with some form of therapy. 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. For
HCV, hyper-immune globulins could be produced by vaccinating
volunteers with recombinant HCV proteins to produce hepatitis C
immune globulin.
[0196] Other anti-virals suitable in the methods of the invention
are manufactured by Triangle Pharmaceuticals, Inc., Gilead, ICN,
Procter and Gamble and ViroPharma Incorporated.
[0197] For use in therapy, an effective amount of the
immunostimulatory nucleic acid can be administered to a subject by
any mode that delivers the immunostimulatory nucleic acids to the
desired site, e.g., mucosal, systemic. "Administering" the
pharmaceutical composition of the present invention may be
accomplished by any means known to the skilled artisan. Preferred
routes of administration include but are not limited to oral,
parenteral, intralesional, topical, transdermal, intramuscular,
intranasal, intratracheal, inhalational, ocular, vaginal, and
rectal.
[0198] For oral administration, the compounds (i.e.,
immunostimulatory nucleic acids, or other therapeutic agents) can
be formulated readily by combining 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 administered without any
carriers.
[0199] 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.
[0200] 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 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.
[0201] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0202] For administration by inhalation, the compounds for use
according to the present invention may be conveniently 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. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0203] 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 may be 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.
[0204] Pharmaceutical 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.
[0205] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0206] 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.
[0207] 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.
[0208] The pharmaceutical compositions also may comprise 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.
[0209] 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
implantation 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 Science 249:1527 (1990), which is incorporated herein by
reference.
[0210] The immunostimulatory nucleic acids 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-pharmaceutically 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, sulfuric,
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.
[0211] Suitable buffering agents include: acetic acid and a salt
(1-2 percent w/v); citric acid and a salt (1-3 percent w/v); boric
acid and a salt (0.5-2.5 percent w/v); and phosphoric acid and a
salt (0.8-2 percent w/v). Suitable preservatives include
benzalkonium chloride (0.003-0.03 percent w/v); chlorobutanol
(0.3-0.9 percent w/v); parabens (0.01-0.25 percent w/v) and
thimerosal (0.004-0.02 percent w/v).
[0212] The pharmaceutical compositions of the invention contain a
pharmaceutically-acceptable carrier. The term
"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. 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 the present invention, and with each other,
in a manner such that there is no interaction which would
substantially impair the desired pharmaceutical efficiency.
[0213] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular adjuvants or antigen selected, the particular condition
being treated and the dosage required for therapeutic efficacy. The
methods of this invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of an immune
response without causing clinically unacceptable adverse effects.
Preferred modes of administration are discussed above.
[0214] The compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. All methods include the step of bringing the
compounds into association with a carrier which constitutes one or
more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing the compounds into
association with a liquid carrier, a finely divided solid carrier,
or both, and then, if necessary, shaping the product. Liquid dose
units are vials or ampoules. Solid dose units are tablets, capsules
and suppositories. For treatment of a patient, depending on
activity of the compound, manner of administration, purpose of the
immunization (i.e., prophylactic or therapeutic), nature and
severity of the disorder, age and body weight of the patient,
different doses may be necessary. The administration of a given
dose can be carried out both by single administration in the form
of an individual dose unit or else several smaller dose units.
[0215] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the compounds, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer-based systems such as
poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-, di- and tri-glycerides;
hydrogel release systems; silastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which an agent of the invention is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152, and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0216] In some embodiments, the immunostimulatory nucleic acid is
modified. In certain embodiments, the immunostimulatory nucleic
acid has a modified backbone with at least one nuclease-resistant
internucleotide linkage. A nuclease-resistant internucleotide
linkage can be selected from the group which includes a
phosphorothioate linkage, a phosphorodithioate linkage, a
methylphosphonate linkage, and a peptide linkage. In certain
embodiments a modified immunostimulatory nucleic acid includes at
least one nucleotide analog or at least one nucleotide analog. The
immunostimulatory nucleic acid is a palindrome in certain
embodiments, while in other embodiments, the immunostimulatory
nucleic acid is not a palindrome. In some preferred embodiments the
immunostimulatory nucleic acid is between 8 and 100 nucleotides in
length, while in other preferred embodiments the immunostimulatory
nucleic acid is between 12 and 40 nucleotides in length. Preferred
sizes, sequences and modifications are described in greater detail
below.
[0217] The following examples are included for purposes of
illustration and are not intended to limit the scope of the
invention.
EXAMPLES
[0218] The purpose of this study was to evaluate the ability of
different classes of CpG ODN to stimulate PBMC from HCV chronic
carriers. PBMC were isolated from whole blood collected from
normal, healthy volunteers and chronic carriers of HCV and the
ability of the different classes CpG ODNs as well as soft and
semi-soft molecules to stimulate B cell proliferation, cytokine
secretion (IFN-g, TNF-.alpha., IL-10 and IFN-.alpha.) and chemokine
secretion (IP-10) in vitro was evaluated.
[0219] Also evaluated were the immune stimulatory effects of
exogenous IFN-.alpha.-2b (Intron A) and Ribavirin, either alone, in
combination with each other, and in combination with CpG ODN (B and
C classes).
[0220] Materials and Methods
Oligonucleotides
[0221] All oligonucleotide stocks were resuspended in TE buffer at
pH 8.0 (OmniPer.RTM.; EM Science, Gibbstown, N.J.). Dilutions of
various ODNs were made in RPMI 1640 complete media (Gibco BRL,
Grand Island, N.Y.) containing 10% heat inactivated, normal human
AB serum (Wisent Inc, St. Bruno, QC) and 1% penicillin/streptomycin
(Gibco BRL, Grand Island, N.Y.) just prior to their use in cell
assays. For the exogenous IFN-.alpha. synergy experiments, Intron A
(Interferon Alfa-2b, DIN 02223406, Schering Canada Inc.,
Pointe-Claire, Quebec, Canada) was added to the ODN solutions to
give final concentrations of 125 or 1000 IU/ml. Ribavirin (CAS
36791-04-5, Calbiochem, CN Biosciences Inc., La Jolla, Calif., USA)
was reconstituted with sterile distilled water to produce a 500
.mu.m stock and diluted in media as described above, to give a
final concentration of 5 .mu.m in wells. Cells were incubated at
37.degree. C. with 5% CO.sub.2. After 48 h, cell supernatants were
collected from each well and frozen at -80.degree. C.
[0222] ODNs used in experiments are shown in the following table:
TABLE-US-00004 TABLE 2 Sequences of oligos used in experiments SEQ
ID NO: CLASS SEQUENCE 1 A G-G-GGACGACGTCGTGG-G-G-G-G-G 2 B TCG TCG
TTT TGT CGT TTT GTC GTT 3 Control for B TGC TGC TTT TTG CTG GCT TTT
T Class 4 C TCGTCGTTTTCGGCGGCCGCCG 5 B TCGTCGTTTCGTCGTTTTGTCGTT 6 B
TCG TCG TTT TTC GTG CGT TTT T 7 Soft C TCGTCGTTT-T-C-G-G-CGGCCGCCG
8 semi-soft B TC-GTC-GTTTT-GTC-GTTTTGTC-GTT 9 Semi-soft C
TCGTC-GTTTTCGGC-GGCCGCCG 10 Semi-soft C TCGTCGTTTTC-GGCGGCC-GCCG 11
Semi-soft C TCGTCG-TTTTC-GGCGCGC-GCCG 12 Semi-soft C
TCGTC-GTTTTC-GGC-GCGC-GCCG 13 Semi-soft C
TCGTCGTTTTAC-GGC-GCC-GTGCCG 14 Semi-soft C
TCGTCG-TTTTAC-GGCGCC-GTGCCG 15 Semi-soft C
TCGTC-GTTTTAC-GGCGCC-GTGCCG 16 Semi-soft C
TCGTC-GTTTTC-GGCGGCC-GCCG * A phosphodiester bond replacing a
phosphorothioate bond within the oligonucleotide backbone is
indicated by (-)
Isolation of PBMCs
[0223] Whole blood (200 ml) was collected by venous puncture into
heparinized green top vacutainers from ten (10) normal, healthy,
adult subjects and fifteen (15) adult subjects chronically infected
with HCV who had a previous 6 month course of an IFN-.alpha.-based
therapy and were either a treatment failure or a relapsed
responder. Peripheral blood mononuclear cells (PBMCs) were purified
by centrifugation over Ficoll-Hypaque (Amersham Pharmacia Biotech,
Uppsala, Sweden) at 400.times.g for 35 min. Cells were resuspended
at a concentration of 10.times.10.sup.6/ml in RPMI complete media
containing 10% normal human AB serum (heat inactivated) and 1%
penicillin/streptomycin.
B-Cell Proliferation
[0224] Cells were isolated as described above and resuspended at
1.times.10.sup.6/ml in complete RPMI media 100 .mu.l of cells were
added to each well of round-bottom 96 well plates. ODN solutions
(100 .mu.l) were added to wells to give the selected range of final
concentrations (1, 3, 6 .mu.g/ml). Cells were cultured for 5 days
and then pulsed with .sup.3H-Thymidine (1 .mu.Ci/well) for 18 h,
before harvesting onto filter paper for measuring radioactivity.
Results are reported as stimulation index (SI) with respect to
untreated media control.
Cytokine Assays
[0225] Freshly isolated PBMCs were resuspended at
10.times.10.sup.6/ml (2.times. final concentration) and 100 .mu.l
of cells were added to each well of a 96 well flat-bottom plate
containing an equal volume of ODN solution (2.times. final desired
concentration). A range of concentrations (1, 3, 6 .mu.g/ml) was
tested for each ODN. Cells were incubated at 37.degree. C. with 5%
CO.sub.2. After 48 h, cell supernatants were collected from each
well and frozen at -80.degree. C. until assayed.
[0226] IFN-.alpha., IP-10, IL-10 and IFN-.lamda. levels in
supernatants were measured using commercial ELISA Kits (R&D
Systems, Minneapolis, Minn., USA; IP-10, Cat# DIP 100, IL-10, Cat#
D1000, IFN-g Cat# DIF50 or PBL Biomedical, IFN-.alpha. Cat# 4110S).
When measured ELISA values were below the detection limit of the
kit as specified by the manufacturer, a value equal to the lowest
detectable limit was entered into data tables.
[0227] Results
[0228] PBMCs isolated from blood collected from 15 chronically
infected HCV subjects and 10 normal healthy volunteers were
incubated at 37.degree. C. with different classes of CpG (e.g.,
class A, B, C, soft C, semi-soft B and semi-soft C), and cell
supernatants were assessed for cytokine presence, indicative of
cytokine secretion during the incubation period. Results of these
experiments are presented below.
Induction of IFN-.alpha. Secretion by PBMC
[0229] When the three classes of CpG ODN were tested on PBMC from
normal volunteers, very high levels of IFN-.alpha. were produced by
the A class (CpG SEQ ID NO. 1), moderately high levels by the C
class (CpG SEQ ID NO. 4) and only low levels were induced by the B
class (CpG SEQ ID NO. 2) (FIG. 1). The main cellular source of
IFN-.alpha. is pDC.
[0230] With the PBMC obtained from HCV chronic carriers, all three
classes of CpG could induce secretion of IFN-.alpha.. The levels
with the B and C classes were the same as those obtained with the
normal PBMCs. In contrast, the A class induced only about 50% of
the normal level (FIG. 1), suggesting that the dysfunction of the
HCV-infected pDC has some impact on the efficacy of A class CpG to
induce IFN-.alpha., but not the C class. Thus, either A or C class
CpG could be used to treat HCV chronic carriers, but in some
instances the C class may be preferred.
[0231] The number of pDC was determined by FACS analysis. A linear
regression was performed against this compared to the amount of
IFN-.alpha. secreted with either the A and C class CpG ODN, and a
reasonable correlation for the normal subjects (e.g., R=0.43 and
0.58, respectively) was found. It was further discovered that the
correlation was slightly better for the C class ODNs. In contrast,
no correlation was observed between number of pDC and amount of
IFN-.alpha. secreted for the HCV infected subjects (R=0.02 and
0.08, respectively) (FIG. 3). The HCV-infected DC are nevertheless
capable of secreting IFN-.alpha. in response to the CpG ODN.
[0232] The effect of soft and semi-soft alterations of these ODS
was also analyzed. Soft molecules were synthesized that had a row
of phosphodiester bonds in the central region of the molecule.
Semi-soft molecules were synthesized that had one or more
individual phosphodiester bonds that are between the cytosine and
guanine nucleotides of the CpG motifs. Both soft (FIG. 3) and
semi-soft (FIG. 4) C class CpG ODN were capable of stimulating
IFN-.alpha. secretion from normal or HCV PBMC in a manner similar
to the original C class CpG ODN. Several of the semi-soft C class
CpG ODN were even more potent that the regular C class CpG SEQ ID
NO. 4 (FIG. 4). This may be because the molecule is still
sufficient stable to have maximal immune stimulation and the
phosphodiester in the middle of the CpG motif?mass? increase its
activity.
Induction of IFN-.gamma. Secretion by PBMC
[0233] FIG. 5 compares the ability of different classes of CpG to
induce the secretion of Th1 cytokine, IFN-.gamma.. Class A induced
low levels of IFN-.gamma. while in comparison class B produced
moderate amounts and class C CpG stimulated high concentrations of
IFN-.gamma.. Both HCV-infected and normal PBMCs displayed a similar
Th1 response to all three classes of CpG. Similar results were
obtained with semi-soft class C CpG ODN (FIG. 6).
Induction of IP-10 Secretion by PBMC
[0234] IP-10, a chemokine associated with production of type 1 and
2 interferons, is also induced by CpG ODN. Highest levels are
induced with A class, next highest with C class and lowest with B
class CpG ODN. Regardless of the class of CpG ODN, similar levels
of IP-10 were induced with PBMCs from normal subjects and HCV
chronic carriers (FIG. 7).
B Cell Stimulation by CpG ODN
[0235] The effect of CpG on B cell stimulation was also
investigated. As shown in FIGS. 8 and 9, CpG Class A was a poor
stimulator of B cells for both HCV-infected and normal populations.
In contrast, classes B, C and semi-soft C CpG strongly activated B
cells. There were no differences between PBMCs from normal and
HCV-infected subjects.
IL-10 Secretion from PBMC after Stimulation with CpG ODN
[0236] The production of cytokine IL-10 following stimulation with
CpG was also assessed and these results are shown in FIG. 10. For
both HCV-infected and normal subjects, all classes of CpG induced
significant secretion of IL-10, and there were no differences
between PBMC from normal volunteers and those from HCV chronic
carriers. Several cell types can produce IL-10 after incubation
with CpG; however since B cells are the major producers of this
cytokine, IL-10 production can be used as an indicator of the level
of B cell activation.
Effects of Intron A and Ribavirin
[0237] The in vitro effect of Ribavirin and exogenous IFN-.alpha.
Intron A, alone or in combination with CpG was tested on
HCV-infected cells. Neither Ribavirin nor Intron A, on their own or
together, resulted in the induction of IFN-.alpha. secretion by
HCV-infected PBMCs (FIG. 11).
[0238] As has been discussed above, A and C class CpG ODN result in
strong induction of IFN-.alpha. secretion from pDC from normal and
HCV-infected subjects. Furthermore, when CpG and Intron-A were used
together, there was a synergistic response for the majority (60%)
of subjects (FIG. 12).
[0239] Discussion
[0240] CpG ODN are able to induce DC from patients chronically
infected with HCV, to secrete IFN-.alpha., with higher levels using
Class A and C CpG and lower levels with Class B CpG ODN. The levels
of secreted IFN-.alpha. are comparable to those observed with cells
from normal healthy volunteers. As well, IP-10 is induced from
stimulated HCV PBMC, further indicating a Th1-type immune
activation.
[0241] HCV antigen-specific immune responses are already present in
persons chronically infected with HCV. These are Th2-biased and
thus cannot bring about clearance of the HCV-infected cells. Th1
type responses would be required for viral clearance. Augmentation
of systemic levels of Th1 cytokines, without additional antigen,
allows persons chronically infected with HCV to develop Th1-type
HCV-specific immune responses that are instrumental in viral
clearance. All classes of CpG (A, B and C) are capable of
establishing Th1-type responses. These Th1-type responses are
essential for long-term clearance of HCV chronic infection, yet
they are difficult to induce with exogenous IFN-.alpha. therapy,
which has direct anti-viral effects but not direct effects on the
immune system. CpG ODN can therefore be used in combination with
exogenous IFN-.alpha. to treat HCV chronic carriers.
[0242] Alternatively, CpG ODN could also be used alone. Owing to
induction of cytokines such as IFN-.alpha. and IFN-.gamma., CpG ODN
on its own has direct anti-viral effects, in addition to the
induction of Th1-type HCV-specific immune responses. In some
instances, the A and C class molecules are preferred since they
induce higher levels of IFNs. Depending on their characteristic
sequence, CpG ODN can preferentially stimulate pDC functions,
maturation and type I IFN production (Krug, A et al., Eur J
Immunol, 2001; 31:2154-2163). Although according to the invention
two classes of CpG ODN were shown to be superior at stimulating
IFN-.alpha. production, any CpG ODN, regardless of backbone or CpG
sequence, could be used in the treatment of chronic HCV. The
controlled release of different type I IFN isoforms by specific CpG
ODN in vivo is superior to the systemic administration of
recombinant type I IFN that is of a single subtype (e.g., Intron A
is only IFN-.alpha.2b). Soft and semi-soft versions of CpG ODN are
capable of stimulating similar levels of IFN-.alpha. as their
parent molecule. Soft or semi-soft versions of the CpG ODN,
especially the C class, would preferentially be used for chronic
treatment of HCV, as they are more easily degraded and would
therefore not be expected to accumulate in the organs, specifically
the liver, spleen and kidney.
[0243] At least 50% of the HCV subjects failed to respond to
exogenous IFN-.alpha. therapy, however CpG ODN (especially A and C
class) were able to induce IFN-.alpha. secretion in vitro at levels
comparable to normal healthy volunteers in all subjects. CpG ODN
could therefore be used to treat patients who have failed to
respond to exogenous IFN-.alpha. therapy, whether the IFN is
pegylated or not, and whether the treatment also includes Ribavirin
or not. Classes of CpG ODN that induce high levels of IFN-.alpha.
would be preferred, and ever more preferred for long-term treatment
would be the semi-soft versions.
[0244] Neither commercial Intron-A (IFN-.alpha.-2b) nor Ribavirin,
alone or in combination, were capable of inducing IFN-.alpha.
secretion from PBMCs from normal or HCV infected subjects in vitro.
However when CpG ODN was used in combination with Intron-A, a
synergistic effect was observed for IFN-.alpha. secretion from
PBMCs from HCV infected subjects. The C class of ODN were shown to
have synergy with exogenous IFN-.alpha.; however treatment with any
class of CpG ODN with commercial alpha-interferons would be
therapeutically effective. As mentioned previously, due to their
relative ease of degradation into non-stimulatory metabolites,
semi-soft versions of the CpG ODN could be used for chronic
treatment without fear of accumulation in end-organs such as the
kidney.
[0245] Ribavirin is purported to have Th1 effects, but in these
studies it had no immunostimulatory activity on human PBMCs. Even
in combination with Intron A, Ribavirin did not enhance endogenous
IFN-.alpha. production. Thus, replacing Ribavirin in combination
therapy for HCV with a CpG ODN will increase the proportion of
sustained viral responses. When combined with CpG ODN, Ribavirin
reduced the efficacy of CpG. CpG ODN should therefore be given in
combination with alpha-interferons in the absence of Ribavirin.
[0246] CpG ODN have been administered IM, SC and IV to human
subjects and were determined to be well tolerated and safe
(clinical study, in progress). Any effective route of
administration would be acceptable such as SC, IM, IV, inhalation
etc. however subcutaneous administration would be the route of
choice. CpG ODN were diluted in TE buffer and added to PBMCs
however, CpG ODN could also be formulated in delivery systems such
as bioadhesive polymers (Sha et al., 1999), cochleates
(Gould-Fogerite et al., 1994, 1996), dendrimers (Kukowska-Latallo
et al., 1996, Qin et al, 1998), enteric-coated capsules (Czerkinsky
et al., 1987, Levine et al., 1987), emulsomes (Vancott et al.,
1998, Lowell et al., 1997), ISCOMs (Mowat et al., 1993, Morein et
al., 1999, Hu et al., 1998, Carlsson et al., 1991), liposomes
(Childers et al., 1999, Michalek et al., 1989, 1992), microspheres
(Gupta et al., 1998, Maloy et al., 1994, Eldridge et al., 1989),
nanospheres (Roy et al., 1999), polymer rings (Wyatt et al., 1998),
proteosomes (Lowell et al., 1988, 1996) and virosomes (Gluck et
al., 1992, Mengiardi et al., 1995, Cryz et al., 1998).
[0247] For treatment of HCV chronic carriers, CpG ODN could be
administered on a repeated basis from once daily to once monthly,
but preferably every 3-10 days, and most preferably weekly, for a
prolonged period. This period could be from one month to two years,
but preferably 3 to 12 months, and most preferably for 6 months.
Thus the most optimal therapy would be given twice weekly or weekly
for 6 months. It could also be given more frequently during an
inductive phase (daily or every other day or twice weekly or weekly
for the first 1-3 months), then less frequently for maintenance
(weekly, or every other week, or monthly for several more
months).
[0248] For combination therapy, CpG and alpha-interferons
(pegylated or not) could potentially be (i) mixed together and
given at the same time and by the same route (subcutaneous), (ii)
given at the same time and same route but not mixed, (iii) given at
the same time but by different routes (e.g., the alpha-interferon
could be given SC and the CpG could be IV, IM, ID, orally or
topically), (iv) given at different times and schedules with same
or different routes, or (v) given consecutively. In this latter
case, preferably the IFN-.alpha. would be given first in order to
reduce viral load, then the CpG ODN would be given afterwards to
induce and sustain Th1-type adaptive immunity for long term
control.
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Equivalents
[0309] It will be understood that various modifications may be made
to the embodiments disclosed herein. Therefore, the above
description should not be construed as limiting, but merely as
exemplifications of preferred embodiments. Those skilled in the art
will envision other modifications within the scope of the claims
appended hereto.
[0310] All references, patents and patent applications disclosed
herein are incorporated by reference in their entirety.
Sequence CWU 1
1
32 1 21 DNA Artificial sequence Synthetic oligonucleotide 1
ggggacgacg tcgtgggggg g 21 2 24 DNA Artificial sequence Synthetic
oligonucleotide 2 tcgtcgtttt gtcgttttgt cgtt 24 3 22 DNA Artificial
sequence Synthetic oligonucleotide 3 tgctgctttt tgctggcttt tt 22 4
22 DNA Artificial sequence Synthetic oligonucleotide 4 tcgtcgtttt
cggcggccgc cg 22 5 24 DNA Artificial sequence Synthetic
oligonucleotide 5 tcgtcgtttc gtcgttttgt cgtt 24 6 22 DNA Artificial
sequence Synthetic oligonucleotide 6 tcgtcgtttt tcgtgcgttt tt 22 7
22 DNA Artificial sequence Synthetic oligonucleotide 7 tcgtcgtttt
cggcggccgc cg 22 8 24 DNA Artificial sequence Synthetic
oligonucleotide 8 tcgtcgtttt gtcgttttgt cgtt 24 9 22 DNA Artificial
sequence Synthetic oligonucleotide 9 tcgtcgtttt cggcggccgc cg 22 10
22 DNA Artificial sequence Synthetic oligonucleotide 10 tcgtcgtttt
cggcggccgc cg 22 11 22 DNA Artificial sequence Synthetic
oligonucleotide 11 tcgtcgtttt cggcgcgcgc cg 22 12 22 DNA Artificial
sequence Synthetic oligonucleotide 12 tcgtcgtttt cggcgcgcgc cg 22
13 24 DNA Artificial sequence Synthetic oligonucleotide 13
tcgtcgtttt acggcgccgt gccg 24 14 24 DNA Artificial sequence
Synthetic oligonucleotide 14 tcgtcgtttt acggcgccgt gccg 24 15 24
DNA Artificial sequence Synthetic oligonucleotide 15 tcgtcgtttt
acggcgccgt gccg 24 16 22 DNA Artificial sequence Synthetic
oligonucleotide 16 tcgtcgtttt cggcggccgc cg 22 17 22 DNA Artificial
sequence Synthetic oligonucleotide 17 tcgcgtcgtt cggcgcgcgc cg 22
18 23 DNA Artificial sequence Synthetic oligonucleotide 18
tcgtcgacgt tcggcgcgcg ccg 23 19 21 DNA Artificial sequence
Synthetic oligonucleotide 19 tcggacgttc ggcgcgcgcc g 21 20 19 DNA
Artificial sequence Synthetic oligonucleotide 20 tcggacgttc
ggcgcgccg 19 21 20 DNA Artificial sequence Synthetic
oligonucleotide 21 tcgcgtcgtt cggcgcgccg 20 22 20 DNA Artificial
sequence Synthetic oligonucleotide 22 tcgacgttcg gcgcgcgccg 20 23
18 DNA Artificial sequence Synthetic oligonucleotide 23 tcgacgttcg
gcgcgccg 18 24 18 DNA Artificial sequence Synthetic oligonucleotide
24 tcgcgtcgtt cggcgccg 18 25 22 DNA Artificial sequence Synthetic
oligonucleotide 25 tcgcgacgtt cggcgcgcgc cg 22 26 10 DNA Artificial
sequence Synthetic oligonucleotide 26 tcntnncgnn 10 27 12 DNA
Artificial sequence Synthetic oligonucleotide 27 cgacgttcgt cg 12
28 13 DNA Artificial sequence Synthetic oligonucleotide 28
cggcgccgtg ccg 13 29 12 DNA Artificial sequence Synthetic
oligonucleotide 29 ccccccgggg gg 12 30 12 DNA Artificial sequence
Synthetic oligonucleotide 30 ggggggcccc cc 12 31 10 DNA Artificial
sequence Synthetic oligonucleotide 31 cccccggggg 10 32 10 DNA
Artificial sequence Synthetic oligonucleotide 32 gggggccccc 10
* * * * *