Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOSTIMULATORY PROPERTIES OF OLIGONUCLEOTIDE-BASED
COMPOUNDS COMPRISING MODIFIED IMMUNOSTIMULATORY
DINUCLEOTIDES
(Attorney Docket No. IDR-039PC)
BACKGROUND OF THE INVENTION
Field of the Invention
The invention relates to immunology and immunotherapy applications using
oligonucleotides as immunostimulatory agents.
Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology,
being used in a wide variety of techniques, ranging from diagnostic probing
methods to PCR
to antisense inhibition of gene expression and immunotherapy applications.
This widespread
use of oligonucleotides has led to an increasing demand for rapid, inexpensive
and efficient
methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications
can now
be routinely accomplished. See, e.g., Methods in Molecular Biology, Vol. 20:
Protocols for
Oligonucleotides andAnalogs pp. 165-189 (S. Agrawal, ed., Humana Press, 1993);
Oligonucleotides andAnalogues, A Practical Approach, pp. 87-108 (F. Eckstein,
ed., 1991);
and Uhlmann and Peyman, supra; Agrawal and Iyer, Curr. Op. in Biotech. 6:12
(1995); and
Antisense Research and Applications (Crooke and Lebleu, eds., CRC Press, Boca
Raton,
1993). Early synthetic approaches included phosphodiester and phosphotriester
chemistries.
For example, Khorana et al., J. Molec. Biol. 72:209 (1972) discloses
phosphodiester
chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34:3143-3179
(1978),
discloses phosphotriester chemistry for synthesis of oligonucleotides and
polynucleotides.
These early approaches have largely given way to the more efficient
phosphoramidite and H-
phosphonate approaches to synthesis. For example, Beaucage and Caruthers,
Tetrahedron
Lett. 22:1859-1862 (1981), discloses the use of deoxyribonucleoside
phosphoramidites in
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polynucleotide synthesis. Agrawal and Zamecnik, U.S. Patent No. 5,149,798
(1992),
discloses optimized synthesis of oligonucleotides by the H-phosphonate
approach. Both of
these modem approaches have been used to synthesize oligonucleotides having a
variety of
modified intemucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett.
28:3539-3542
(1987), teaches synthesis of oligonucleotide methylphosphonates using
phosphoramidite
chemistry. Connolly et al., Biochem. 23:3443 (1984), discloses synthesis of
oligonucleotide
phosphorothioates using phosphoramidite chemistry. Jager et al., Biochem.
27:7237 (1988),
discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite
chemistry.
Agrawal et al., Proc. Natl. Acad. Sci. (USA) 85:7079-7083 (1988), discloses
synthesis of
oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate
cheniistry.
More recently, several researchers have demonstrated the validity of the use
of
oligonucleotides as immunostimulatory agents in immunotherapy applications.
The
observation that phosphodiester and phosphorothioate oligonucleotides can
induce immune
stimulation has created interest in developing this side effect as a
therapeutic tool. These
efforts have focused on phosphorothioate oligonucleotides containing the
dinucleotide
natural CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches
that
phosphodiester oligonucleotides containing a palindrome that includes a CpG
dinucleotide
can induce interferon-alpha and gamma synthesis and enhance natural killer
activity. Krieg
et al., Nature 371:546-549 (1995) discloses that phosphorothioate CpG-
containing
oligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest. 98:1119-
1129 (1996)
discloses that such oligonucleotides activate human B cells. Moldoveanu et
al., Vaccine
16:1216-124 (1998) teaches that CpG-containing phosphorothioate
oligonucleotides enhance
immune response against influenza virus. McCluskie and Davis, J. Immunol.
161:4463-4466
(1998) teaches that CpG-containing oligonucleotides act as potent adjuvants,
enhancing
immune response against hepatitis B surface antigen. Hartman et al., J.
Immuno1164: 1617-
1624 (2000) teaches that the immunostimulatory sequence is species
specific,,and different
between mice and primates.
Other modifications of CpG-containing phosphorothioate oligonucleotides can
also
affect their ability to act as modulators of immune response. See, e.g., Zhao
et al., Biochem.
Pharmacol. (1996) 51:173-182= Z-han Pt al_, - Rlo-al,_z- v1 nv ! r, Qn/_~ Z~
iS27-1 ~r - A A.
'
-2-
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Zhao et al., Antisense Nucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al.,
Bioorg. Med.
Chem. Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000)
10:1051-1054;
Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588; Yu et al., Bioorg.
Med. Chem.
Lett. (2001) 11:2263-2267; and Kandimalla et al., Bioorg. Med. Chem. (2001)
9:807-813.
These reports make clear that there remains a need to be able to modulate the
immune
response caused by immunostimulatory oligonucleotides and to overcome species
specificity
of the immunostimulatory sequences.
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BRIEF SUMMARY OF THE INVENTION
The invention provides methods for modulating the immune response caused by
oligonucleotide compounds. The methods according to the invention enable
modifying the
cytokine profile produced by immunostimulatory oligonucleotides for
immunotherapy
applications. The present inventors have surprisingly discovered that
modification of
immunostimulatory dinucleotides allows flexibility in the nature of the immune
response
produced and that certain modifications overcome the species specificities
observed to date
of the immunostimulatory sequences.
In a first aspect the invention provides an immunostimulatory oligonucleotide
having
a structure from the group of 5'-TCTGTR'GTTCT-X-TCTTGR'TGTCT-5'; 5'-
TCTGTR'GTTC1U1-X-UIC1TTGR'TGTCT-5'; 5'-CTGTR'GTTCTC-X-CTCTTGR'TGTC-
5'; 5'-CTGTR'GTTCU1C1-X-C1U1CTTGR'TGTC-5'; 5'-CTGTR'GTTC1U1C1-X-
C1U1C1TTGR'TGTC-5'; 5'-TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 5'-
TCTGTR'GACAG-X-GACAGR'TGTCT-5'; 5'-TCTGTR'GACA1G1-X-
G1A1CAGR'TGTCT-5'; 5'-TCAGTR'GTTAG-X-GATTGR'TGACT-5'; 5'-
TCAGTR'GACTG-X-GTCAGR'TGACT-5'; 5'-TR'GTR'GAR'GAT-X-
TAGR'AGR'TGR'T-5'; 5'-TR'GTR'GTAGTA-X-ATGATGR'TGR'T-5'; 5'-
TR' GAAR' GTTCT-X-TCTTGR' AAGR' T-5' ; 5' -TR' GTAR' GTACT-X-
TCATGR'ATGR'T-5' and 5'-TCRAACRTTCR-X-RCTTRCAARCT-5', wherein R' = 1-
(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; Al/C1/Gl/U1= 2'-O-
methyl-
ribonucleotides; R= 2'-deoxy-7-deazaguanosine and X= glycerol linker.
In a second aspect the invention provides pharmaceutical compositions. These
compositions comprise any one of the compositions disclosed in the first
aspect of the
invention and a pharmaceutically acceptable carrier.
In a third aspect the invention provides a method for generating an immune
response
in a vertebrate, the method comprising administering to the vertebrate an
immunostimulatory
oligonucleotide having a structure from the group of 5'-TCTGTR'GTTCT-X-
TCTTGR'TGTCT-5'; 5'-TCTGTR'GTTC1U1-X-U1C1TTGR'TGTCT-5'; 5'-
CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'; 5'-CTGTR'GTTCU1C1-X-
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CiUiCTTGR'TGTC-5'; 5'-CTGTR'GTTC1UiC1-X-C1U1C1TTGR'TGTC-5'; 5'-
TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 5'-TCTGTR'GACAG-X-GACAGR'TGTCT-
5'; 5'-TCTGTR'GACA1G1-X-G1A1CAGR'TGTCT-5'; 5'-TCAGTR'GTTAG-X-
GATTGR'TGACT-5'; 5'-TCAGTR'GACTG-X-GTCAGR'TGACT-5'; 5'-
TR' GTR' GAR' GAT-X-TAGR'AGR' TGR' T-5'; 5 ' -TR' GTR' GTAGTA-X-
ATGATGR'TGR'T-5'; 5'-TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-5'; 5'-
TR'GTAR'GTACT-X-TCATGR'ATGR'T-5' and 5'-TCRAACRTTCR-X-
RCTTRCAARCT-5', wherein R' = 1-(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-
methyl-purine; Al/Cl/Gl/U1= 2'-O-methyl-ribonucleotides; R = 2'-deoxy-7-
deazaguanosine
and X= glycerol linker.
In a fourth aspect the invention provides a method for therapeutically
treating a
vertebrate having cancer, an autoimmune disorder, airway inflammation,
inflammatory
'disorders, skin disorders, allergy, asthma or a disease caused by a pathogen,
such method
comprising administering to the patient an immunostimulatory oligonucleotide
having a
structure from the group of 5'-TCTGTR'GTTCT-X-TCTTGR'TGTCT-5'; 5'-
TCTGTR'GTTC1U1-X-UiC1TTGR'TGTCT-5'; 5'-CTGTR'GTTCTC-X-CTCTTGR'TGTC-
5'; 5'-CTGTR'GTTCU1C1-X-C1U1CTTGR'TGTC-5'; 5'-CTGTR'GTTCiU1C1-X-
CiU1C1TTGR'TGTC-5'; 5'-TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 5'-
TCTGTR'GACAG-X-GACAGR'TGTCT-5'; 5'-TCTGTR'GACA1G1-X-
G1A1CAGR'TGTCT-5'; 5'-TCAGTR'GTTAG-X-GATTGR'TGACT-5'; 5'-
TCAGTR'GACTG-X-GTCAGR'TGACT-5'; 5'-TR'GTR'GAR'GAT-X-
TAGR' AGR' TGR' T-5' ; 5 ' -TR' GTR' GTAGTA-X-ATGATGR' TGR' T-5' ; 5' -
TR' GAAR' GTTCT-X-TCTTGR' AAGR' T-5' ; 5' -TR' GTAR' GTACT-X-
TCATGR'ATGR'T-5' and 5'-TCRAACRTTCR-X-RCTTRCAARCT-5', wherein R' = 1-
(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; Al/C1/Gi/Ui = 2'-
O-methyl-
ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X = glycerol linker.
In a fifth aspect the invention provides a method for preventing cancer, an
autoimmune disorder, airway inflammation, inflammatory disorders, skin
disorders, allergy,
asthma or a disease caused by a pathogen in a vertebrate, such method
comprising
administering to the vertebrate an immunostimulatory oligonucleotide having a
structure
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from the group of 5'-TCTGTR'GTTCT-X-TCTTGR'TGTCT-5'; 5'-TCTGTR'GTTC1U1-X-
UiC1TTGR'TGTCT-5'; 5'-CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'; 5'-
CTGTR'GTTCU1C1-X-CiU1CTTGR'TGTC-5'; 5'-CTGTR'GTTCiU1C1-X-
C1U1C1TTGR'TGTC-5'; 5'-TCTGTR'GTTCT-X-CGTTCGAACGT-5'; 5'-
TCTGTR'GACAG-X-GACAGR'TGTCT-5'; 5'-TCTGTR'GACA1G1-X-
G1A1CAGR'TGTCT-5'; 5'-TCAGTR'GTTAG-X-GATTGR'TGACT-5'; 5'-
TCAGTR' GACTG-X-GTCAGR' TGACT-5'; 5' -TR' GTR' GAR' GAT-X-
TAGR'AGR'TGR'T-5'; 5'-TR'GTR'GTAGTA-X-ATGATGR'TGR'T-5'; 5'-
TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-5'; 5'-TR'GTAR'GTACT-X-
TCATGR'ATGR'T-5' and 5'-TCRAACRTTCR-X-RCTTRCAARCT-5', wherein R' =1-
(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; Al/Ci/Gl/U1= 2'-O-
methyl-
ribonucleotides; R = 2'-deoxy-7-deazaguanosine and X = glycerol linker.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a group of representative small molecule linkers suitable for
linear
synthesis of immunostimulatory oligonucleotides of the invention.
Figure 2 depicts a group of representative small molecule linkers suitable for
parallel
synthesis of immunostimulatory oligonucleotides of the invention.
Figure 3 is a synthetic scheme for the linear synthesis of immunostimulatory
oligonucleotides of the invention. DMTr = 4,4'-dimethoxytrityl; CE =
cyanoethyl.
Figure 4 is a synthetic scheme for the parallel synthesis of immunostimulatory
oligonucleotides of the invention. DMTr = 4,4'-dimethoxytrityl; CE =
cyanoethyl.
Figure 5 is a schematic representation of the 3'-terminal nucleoside of an
oligonucleotide, showing that a non-nucleotidic linkage can be attached to the
nucleoside at
the nucleobase, at the 3' position, or at the 2' position.
Figure 6 shows IL-12 induction in C57BL/6 mouse spleen cell cultures by
immunostimulatory oligonucleotides of the invention.
Figure 7 shows IL-6induction in C57BL/6 mouse spleen cell cultures by
immunostimulatory oligonucleotides of the invention.
Figure 8 shows IFN-a induction in human pDC cultures by immunostimulatory
oligonucleotides of the invention.
Figure 9 shows IFN-a induction in human PBMC cultures by immunostimulatory
oligonucleotides of the invention.
Figure 10 shows Human B cell proliferation by immunostimulatory
oligonucleotides
of the invention.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention relates to the therapeutic use of oligonucleotides as
immunostimulatory
agents for immunotherapy applications. The issued patents, patent
applications, and
references that are cited herein are hereby incorporated by reference to the
same extent as if
each was specifically and individually indicated to be incorporated by
reference. In the event
of inconsistencies between any teaching of any reference cited herein and the
present
specification, the latter shall prevail for purposes of the invention.
The invention provides methods for enhancing the immune response caused by
immunostimulatory compounds used for immunotherapy applications such as, but
not limited
to, treatment of cancer, autoimmune disorders, asthma, respiratory allergies,
food allergies,
and bacteria, parasitic, and viral infections in adult and pediatric human and
veterinary
applications. Thus, the invention further provides compounds having optimal
levels of
immunostimulatory effect for immunotherapy and methods for making and using
such
compounds. In addition, compounds of the invention are useful as adjuvants in
combination
with DNA vaccines, antibodies, and allergens; and in combination with
chemotherapeutic
agents and/or antisense oligonucleotides.
The present inventors have surprisingly discovered that modification of an
immunostimulatory oligonucleotide to optimally present its 5' ends
dramatically affects its
immunostimulatory capabilities. In addition, the present inventors have
discovered that the
cytokine profile and species specificity of an immune response can be
modulated by using
novel purine or pyrimidine structures as part of an immunostimulatory
oligonucleotide.
In a first aspect, the invention provides immunostimulatory oligonucleotides
alone or
comprising at least two oligonucleotides linked at their 3' ends, or an
intemucleoside linkage
or a functionalized nucleobase or sugar to a non-nucleotidic linker, at least
one of the
oligonucleotides being an immunostimulatory oligonucleotide and having an
accessible 5'
end. As used herein, the term."accessible 5' end" means that the 5' end of the
oligonucleotide is sufficiently available such that the factors that recognize
and bind to
oligonucleotide and stimulate the immune system have access to it. In
oligonucleotides
having an accessible 5' end, the 5' OH position of the terminal sugar is not
covalently linked
~? yry <.~ ~ u +L yy + ,= ;_;;,
to more than two nucleoside residues or anv othPr ~õoiP xr tha.t i f
- VYiLla
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the 5' end. Optionally, the 5' OH can be linked to a phosphate,
phosphorothioate, or
phosphorodithioate moiety, an aromatic or aliphatic linker, cholesterol, or
another entity
which does not interfere with accessibility. The immunostimulatory
oligonucleotides
according to the invention preferably further comprise an immunostimulatory
dinucleotide
comprising a novel purine or pyrimidine.
In certain embodiments, the immunostimulatory oligonucleotides include a
ribozyme
or a decoy oligonucleotide. As used herein, the term "ribozyme" refers to an
oligonucleotide
that possesses catalytic activity. Preferably, the ribozyme binds to a
specific nucleic acid
target and cleaves the target. As used herein, the term "decoy
oligonucleotide" refers to an
oligonucleotide that binds to a transcription factor in a sequence-specific
manner and arrests
transcription activity. Preferably, the ribozyme or decoy oligonucleotide
exhibits secondary
structure, including, without limitation, stem-loop or hairpin structures. In
certain
embodiments, at least one oligonucleotide comprises poly(I)-poly(C). In
certain
embodiments, at least one set of Nn includes a string of 3 to 10 dGs and/or Gs
or 2'-
substituted ribo or arabino Gs.
For purposes of the invention, the term "oligonucleotide" refers to a
polynucleoside
formed from a plurality of linked nucleoside units. Such oligonucleotides can
be obtained
from existing nucleic acid sources, including genomic or cDNA, but are
preferably produced
by synthetic methods. In preferred embodiments each nucleoside unit includes a
heterocyclic
base and a pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted
arabinose, 2'-O-
substituted arabinose or hexose sugar group. The nucleoside residues can be
coupled to each
other by any of the numerous known intemucleoside linkages. Such
internucleoside linkages
include, without limitation, phosphodiester, phosphorothioate,
phosphorodithioate,
alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate,
siloxane,
carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether,
bridged
phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and
sulfone
intemucleoside linkages. The term "oligonucleotide" also encompasses
polynucleosides
having one or more stereospecific intemucleoside linkage (e.g., (Rp)- or (Sp)-
phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used
herein, the terms
"oligonucleotide" and "dinucleotide" are expressly intended to include
polynucleosides and
dinucleosides having any such intemucleoside linkage, whether or not the
linkage comprises
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a phosphate group. In certain preferred embodiments, these intemucleoside
linkages may be
phosphodiester, phosphorothioate, or phosphorodithioate linkages, or
combinations thereof.
In some embodiments, the oligonucleotides each have from about 3 to about 35
nucleoside residues, preferably from about 4 to about 30 nucleoside residues,
more
preferably from about 4 to about 20 nucleoside residues. In some embodiments,
the
immunostimulatory oligonucleotides comprise oligonucleotides have from about 5
to about
18, or from about 5 to about 14, nucleoside residues. As used herein, the term
"about"
implies that the exact number is not critical. Thus, the number of nucleoside
residues in the
oligonucleotides is not critical, and oligonucleotides having one or two fewer
nucleoside
residues, or from one to several additional nucleoside residues are
contemplated as
equivalents of each of the embodiments described above. In some embodiments,
one or
more of the oligonucleotides have 11 nucleotides. In the context of
immunostimulatory
oligonucleotides, preferred embodiments have from about 13 to about 35
nucleotides, more
preferably from about 13 to about 26 nucleotides.
The term "oligonucleotide" also encompasses polynucleosides having additional
substituents including, without limitation, protein groups, lipophilic groups,
intercalating
agents, diamines, folic acid, cholesterol and adamantane. The term
"oligonucleotide" also
encompasses any other nucleobase containing polymer, including, without
limitation, peptide
nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA),
locked nucleic
acids (LNA), morpholino-backbone oligonucleotides, and oligonucleotides having
backbone
sections with alkyl linkers or amino linkers.
The oligonucleotides of the invention can include naturally occurring
nucleosides,
modified nucleosides, or mixtures thereof. As used herein, the term "modified
nucleoside" is
a nucleoside that includes a modified heterocyclic base, a modified sugar
moiety, or a
combination thereof. In some embodiments, the modified nucleoside is a non-
natural
pyrimidine or purine nucleoside, as herein described. In some embodiments, the
modified
nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside or a 2'-
deoxy-2'-
substituted-arabinoside.
For purposes of the invention, the term "2'-substituted ribonucleoside" or "2'-
substituted arabinosiae' inciudes ribonucleosides or arabinonucieoside in
wiiich tne hydroxyi
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group at the 2' position of the pentose moiety is substituted to produce a 2'-
substituted or 2'-
0-substituted ribonucleoside. Preferably, such substitution is with a lower
alkyl group
containing 1-6 saturated or unsaturated carbon atoms, or with an aryl group
having 6-10
carbon atoms, wherein such alkyl, or aryl group may be unsubstituted or may be
substituted,
e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy,
alkoxy, carboxyl,
carboalkoxy, or amino groups. Examples of 2'-O-substituted ribonucleosides or
2'-O-
substituted-arabinosides include, without limitation 2'-O-
methylribonucleosides or 2'-0-
methylarabinosides and 2'-O-methoxyethylribonucleosides or 2'-0-
methoxyethylarabinosides.
The term "2'-substituted ribonucleoside" or "2'-substituted arabinoside" also
includes
ribonucleosides or arabinonucleosides in which the 2'-hydroxyl group is
replaced with a
lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or
with an amino or
halo group. Examples of such 2'-substituted ribonucleosides or 2'-substituted
arabinosides
include, without limitation, 2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl
ribonucleosides or
arabinosides.
The term "oligonucleotide" includes hybrid and chimeric oligonucleotides. A
"chimeric oligonucleotide" is an oligonucleotide having more than one type of
intemucleoside linkage. One preferred example of such a chimeric
oligonucleotide is a
chimeric oligonucleotide comprising a phosphorothioate, phosphodiester or
phosphorodithioate region and non-ionic linkages such as alkylphosphonate or
alkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Patent Nos.
5,635,377 and
5,366,878).
A "hybrid oligonucleotide" is an oligonucleotide having more than one type of
nucleoside. One preferred example of such a hybrid oligonucleotide comprises a
ribonucleotide or 2'-substituted ribonucleotide region, and a
deoxyribonucleotide region (see,
e.g., Metelev and Agrawal, U.S. Patent No. 5,652,355, 6,346,614 and
6,143,881).
For purposes of the invention, the term "immunostimulatory oligonucleotide"
refers
to an oligonucleotide as described above that induces an immune response when
administered to a vertebrate, such as a fish, fowl, or mammal. As used herein,
the term
"mammal" includes, . WithQtlt limifati(~n rate mirP cate rincs~ hnt~AC _< ~, n
. . . . 3 -o a:." .7 Jti:bs,
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rabbits, non-human primates, and humans. Useful immunostimulatory
oligonucleotides can
be found described in Agrawal et al., WO 98/49288, published November 5, 1998;
WO
01/12804, published February 22,2001; WO 01/55370, published August 2, 2001;
PCT/USO1/13682, filed Apri130, 2001; and PCT/USO1/30137, filed September 26,
2001.
Preferably, the immunostimulatory oligonucleotide comprises at least one
phosphodiester,
phosphorothioate, or phosphorodithioate intemucleoside linkage.
In some embodiments, the immunostimulatory oligonucleotide comprises an
immunostimulatory dinucleotide of formula 5'-Pyr-Pur-3', wherein Pyr is a
natural or
synthetic pyrimidine nucleoside and Pur is a natural or synthetic purine
nucleoside. In some
preferred embodiments, the immunostimulatory oligonucleotide comprises an
immunostimulatory dinucleotide of formula 5'-Pur*-Pur-3', wherein Pur* is a
synthetic
purine nucleoside and Pur is a natural or synthetic purine nucleoside. In
various places the
dinucleotide is expressed as RpG, C*pG or YZ, in which case respectively, R,
C*, or Y
represents a synthetic purine. A particularly preferred synthetic purine is 2-
oxo-7-deaza-8-
methyl-purine. When this synthetic purine is in the Pur* position of the
dinucleotide,
species-specificity (sequence dependence) of the immunostimulatory effect is
overcome and
cytokine profile is improved. As used herein, the term "pyrimidine nucleoside"
refers to a
nucleoside wherein the base component of the nucleoside is a monocyclic
nucleobase.
Similarly, the term "purine nucleoside" refers to a nucleoside wherein the
base component of
the nucleoside is a bicyclic nucleobase. For purposes of the invention, a
"synthetic"
pyrimidine or purine nucleoside includes a non-naturally occurring pyrimidine
or purine
base, a non-naturally occurring sugar moiety, or a combination thereof.
Preferred pyrimidine nucleosides according to the invention have the structure
(1):
D
D'
X A'
isi
(I)
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wherein:
D is a hydrogen bond donor;
D' is selected from the group consisting of hydrogen, hydrogen bond donor,
hydrogen
bond acceptor, hydrophilic group, hydrophobic group, electron withdrawing
group and
electron donating group;
A is a hydrogen bond acceptor or a hydrophilic group;
A' is selected from the group consisting of hydrogen bond acceptor,
hydrophilic
group, hydrophobic group, electron withdrawing group and electron donating
group;
X is carbon or nitrogen; and
S' is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
Preferably, the sugar ring is derivatized with a phosphate moiety, modified
phosphate
moiety, or other linker moiety suitable for linking the pyrimidine nucleoside
to another
nucleoside or nucleoside analog.
Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH
and
-OH. Preferred hydrogen bond acceptors include, without limitation, C=O, C=S,
and the ring
nitrogen atoms of an aromatic heterocycle, e.g., N3 of cytosine.
In some embodiments, the base moiety in (1) is a non-naturally occurring
pyrimidine
base. Examples of preferred non-naturally occurring pyrimidine bases include,
without
limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine,
preferably N4-
ethylcytosine, and 4-thiouracil. However, in some embodiments 5-bromocytosine
is
specifically excluded.
In some embodiments, the sugar moiety S' in (1) is a non-naturally occurring
sugar
moiety. For purposes of the present invention, a "naturally occurring sugar
moiety" is a
sugar moiety that occurs naturally as part of nucleic acid, e.g., ribose and
2'-deoxyribose, and
a "non-naturally occurring sugar moiety" is any sugar that does not occur
naturally as part of
a nucleic acid, but which can be used in the backbone for an oligonucleotide,
e.g, hexose.
AraVinose and arabinose derivatives are examples ofpreierred sugar moieties.
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Preferred purine nucleoside analogs according to the invention have the
structure (II):
A
L D
X /
'
I N D
St (rl)
wherein:
D is a hydrogen bond donor;
D' is selected from the group consisting of hydrogen, hydrogen bond donor, and
hydrophilic group;
A is a hydrogen bond acceptor or a hydrophilic group;
X is carbon or nitrogen;
each L is independently an atom selected from the group consisting of C, 0, N
and S;
and
S' is a pentose or hexose sugar ring, or a non-naturally occurring sugar.
Preferably, the sugar ring is derivatized with a phosphate moiety, modified
phosphate
moiety, or other linker moiety suitable for linking the pyrimidine nucleoside
to another
nucleoside or nucleoside analog.
Preferred hydrogen bond donors include, without limitation, -NH-, -NH2, -SH
and
-OH. Preferred hydrogen bond acceptors include, without limitation, C=O, C=S, -
NO2 and
the ring nitrogen atoms of an aromatic heterocycle, e.g., Nl of guanine.
In some embodiments, the base moiety in (II) is a non-naturally occurring
purine
base. Examples of preferred non-naturally occurring purine bases include,
without
limitation, 2-amino-6-thiopurine and 2-amino-6-oxo-7-deazapurine. In some
embodiments,
the sugar moiety S' in (II) is a naturally occurring sugar moiety, as
described above for
structure (1).
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In preferred embodiments, the immunostimulatory dinucleotide is selected from
the
group consisting of CpG, C*pG, CpG*, and C*pG*, wherein the base of C is
cytosine, the
base of C* is 2'-thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4-thiouracil
or other non-
natural pyrimidine, or 2-oxo-7-deaza-8-methylpurine, wherein when the base is
2-oxo-7-
deaza-8-methyl-purine, it is preferably covalently bound to the 1'-position of
a pentose via
the 1 position of the base; the base of G is guanosine, the base of G* is 2-
amino-6-oxo-7-
deazapurine, 2-oxo-7-deaza-8-methylpurine, 6-thioguanine, 6-oxopurine, or
other non-
natural purine nucleoside, and p is an intemucleoside linkage selected from
the group
consisting of phosphodiester, phosphorothioate, and phosphorodithioate. In
certain preferred
embodiments, the immunostimulatory dinucleotide is not CpG.
The imxnunostimulatory oligonucleotides may include immunostimulatory moieties
on one or both sides of the immunostimulatory dinucleotide. Thus, in some
embodiments,
the immunostimulatory oligonucleotide comprises an immunostimulatory domain of
structure (III):
5' Nn-N1-Y-Z N1 Nn-3' (III)
wherein:
the base of Y is cytosine, thymine, 5-hydroxycytosine, N4-alkyl-cytosine, 4-
thiouracil or other non-natural pyrimidine nucleoside, or 2-oxo-7-deaza-8
methyl
purine, wherein when the base is 2-oxo-7-deaza-8-methyl-purine, it is
preferably
covalently bound to the 1'-position of a pentose via the 1 position of the
base;
the base of Z is guanine, 2-amino-6-oxo-7-deazapurine, 2-oxo-7deaza-8-
methylpurine, 2-amino-6-thio-purine, 6-oxopurine or other non-natural purine
nucleoside;
N1 and Nn, independent at each occurrence, is preferably a naturally occurring
or a
synthetic nucleoside or an immunostimulatory moiety selected from the group
consisting of
abasic nucleosides, arabinonucleosides, 2'-deoxyuridine, a-
deoxyribonucleosides,
(3-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester or
modified
intemucleoside linkage to the adjacent nucleoside on the 3' side, the modified
intemucleotide
linkage being selected from, without limitation, a linker having a length of
from about 2
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angstroms to about 200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol)
linker, 2-
aminobutyl-l,3-propanediol linker, glyceryl linker, 2'-5' internucleoside
linkage, and
phosphorothioate, phosphorodithioate, or methylphosphonate intemucleoside
linkage;
provided that at least one N1 or Nn is optionally an immunostimulatory moiety;
wherein n is a number from 0 to 30; and
wherein the 3'end, an intemucleoside linker, or a derivatized nucleobase or
sugar is
linked directly or via a non-nucleotidic linker to another oligonucleotide,
which may or may
not be immunostimulatory.
In some preferred embodiments, YZ is arabinocytidine or 2'-deoxy-2'-
substituted
arabinocytidine and arabinoguanosine or 2'deoxy-2'-substituted
arabinoguanosine. Preferred
immunostimulatory moieties include natural phosphodiester backbones and
modifications in
the phosphate backbones, including, without limitation, methylphosphonates,
methylphosphonothioates, phosphotriesters, phosphothiotriesters,
phosphorothioates,
phosphorodithioates, triester prodrugs, sulfones, sulfonamides, sulfamates,
formacetal, N-
methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate,
phosphoramidates, especially primary amino-phosphoramidates, N3
phosphoraniidates and
N5 phosphoramidates, and stereospecific linkages (e.g., (Rp)- or (Sp)-
phosphorothioate,
alkylphosphonate, or phosphotriester linkages).
Preferred immunostimulatory moieties according to the invention further
include
nucleosides having sugar modifications, including, without limitation, 2'-
substituted pentose
sugars including, without limitation, 2'-O-methylribose, 2'-O-
methoxyethylribose, 2'-O-
propargylribose, and 2'-deoxy-2'-fluororibose; 3'-substituted pentose sugars,
including,
without limitation, 3'-O-methylribose; 1',2'-dideoxyribose; arabinose;
substituted arabinose
sugars, including, without limitation, 1'-methylarabinose, 3'-
hydroxymethylarabinose,
4'-hydroxymethylarabinose, 3'-hydroxyarabinose and 2'-substituted arabinose
sugars;
hexose sugars, including, without limitation, 1,5-anhydrohexitol; and alpha-
anomers. In
embodiments in which the modified sugar is a 3'-deoxyribonucleoside or a 3'-O-
substituted
ribonucleoside, the immunostimulatory moiety is attached to the adjacent
nucleoside by way
of a 2'-5' internucleoside linkage.
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Preferred immunostimulatory moieties according to the invention further
include
oligonucleotides having other carbohydrate backbone modifications and
replacements,
including peptide nucleic acids (PNA), peptide nucleic acids with phosphate
groups
(PHONA), locked nucleic acids (LNA), morpholino backbone oligonucleotides, and
oligonucleotides having backbone linker sections having a length of from about
2 angstroms
to about 200 angstroms, including without limitation, alkyl linkers or amino
linkers. The
alkyl linker may be branched or unbranched, substituted or unsubstituted, and
chirally pure
or a racemic mixture. Most preferably, such alkyl linkers have from about 2 to
about 18
carbon atoms. In some preferred embodiments such alkyl linkers have from about
3 to about
9 carbon atoms. Some alkyl linkers include one or more functional groups
selected from the
group consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide,
ester, urea, and
thioether. Some such functionalized alkyl linkers are poly(ethylene glycol)
linkers of
formula -O-(CH2-CHa-O-)n (n = 1-9). Some other functionalized alkyl linkers
are peptides or
amino acids.
Preferred immunostimulatory moieties according to the invention further
include
DNA isoforms, including, without limitation, (3-L-deoxyribonucleosides and
a-deoxyribonucleosides. Preferred immunostimulatory moieties according to the
invention
incorporate 3' modifications, and further include nucleosides having unnatural
intemucleoside linkage positions, including, without limitation, 2'-5', 2'-2',
3'-3' and 5'-5'
linkages.
Preferred immunostimulatory moieties according to the invention further
include
nucleosides having modified heterocyclic bases, including, without limitation,
5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine, preferably
N4-ethylcytosine, 4-thiouracil, 6-thioguanine, 7-deazaguanine, inosine,
nitropyrrole,
C5-propynylpyrimidine, and diaminopurines, including, without limitation,
2,6-diaminopurine.
By way of specific illustration and not by way of limitation, for example, in
the
immunostimulatory domain of structure (III), a methylphosphonate
intemucleoside linkage
at position N1 or Nn is an immunostimulatory moiety, a linker having a length
of from about
2 angstroms to about 200 angstroms, C2-C18 alkvvl linker at position X1 is an
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immunostimulatory moiety, and a(3-L-deoxyribonucleoside at position Xl is an
immunostimulatory moiety. See Table 1 below for representative positions and
structures of
immunostimulatory moieties. It is to be understood that reference to a linker
as the
immunostimulatory moiety at a specified position means that the nucleoside
residue at that
position is substituted at its 3'-hydroxyl with the indicated linker, thereby
creating a modified
intemucleoside linkage between that nucleoside residue and the adjacent
nucleoside on the 3'
side. Similarly, reference to a modified intemucleoside linkage as the
immunostimulatory
moiety at a specified position means that the nucleoside residue at that
position is linked to
the adjacent nucleoside on the 3' side by way of the recited linkage.
Table 1
Position TYPICAL IMMUNOSTIMULATORY MOIETIES
N1 Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside, 2'-
deoxyuridine, (3-L-deoxyribonucleoside C2-C 18 alkyl linker, poly(ethylene
glycol) linkage, 2-aminobutyl-1,3-propanediol linker (amino linker), 2'-5'
intemucleoside linkage, methylphosphonate internucleoside linkage
Nn Naturally-occurring nucleosides, abasic nucleoside, arabinonucleosides, 2'-
deoxyuridine, 2'-O-substituted ribonucleoside, 2'-5' internucleoside linkage,
methylphosphonate intemucleoside linkage, provided that N1 and N2 cannot
both be abasic linkages
Table 2 shows representative positions and structures of immunostimulatory
moieties
within an immunostimulatory oligonucleotide having an upstream potentiation
domain. As
used herein, the term "Spacer 9" refers to a poly(ethylene glycol) linker of
formula
-O-(CH2CH2-O)n , wherein n is 3. The term "Spacer 18" refers to a
poly(ethylene glycol)
linker of formula -O-(CHaCH2-O)n , wherein n is 6. As used herein, the term
"C2-C 18 alkyl
linker refers to a linker of formula -O-(CH2)q O-, where q is an integer from
2 to 18.
Accordingly, the terms "C3-linker" and "C3-alkyl linker" refer to a linker of
formula
-O-(CH2)3-0-. For each of Spacer 9, Spacer 18, and C2-C18 alkyl linker, the
linker is
connected to the adjacent nucleosides by way of phosphodiester,
phosphorothioate, or
phosphorodithioate linkages.
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Table 2
Position TYPICAL IMIVIUNOSTIMULATORY MOIETY
5' N2 Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol linker
5' Nl Naturally-occurring nucleosides, (3-L-deoxyribonucleoside, C2-C18 alkyl
linker, poly(ethylene glycol), abasic linker, 2-aminobutyl-1,3-propanediol
linker
3' N1 Naturally-occurring nucleosides, 1',2'-dideoxyribose, 2'-O-methyl-
ribonucleoside, C2-C 18 alkyl linker, Spacer 9, Spacer 18
3' N2 Naturally-occurring nucleosides, 1',2'-dideoxyribose, 3'-
deoxyribonucleoside, (3-L-deoxyribonucleoside, 2'-O-propargyl-
ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18,
methylphosphonate intemucleoside linkage
3' N 3 Naturally-occurring nucleosides, 1',2'-dideoxyribose, C2-C18 alkyl
linker, Spacer 9, Spacer 18, methylphosphonate intemucleoside linkage,
2'-5' intemucleoside linkage, d(G)n, polyl-polyC
3'N 2+ 3'N 3 1',2'-dideoxyribose, (3-L-deoxyribonucleoside, C2-C18 alkyl
linker,
d(G)n, polyl-polyC
3'N3+ 3' N 4 2'-O-methoxyethyl-ribonucleoside, methylphosphonate
intemucleoside
linkage, d(G)n, polyl-polyC
3'N5+ 3' N 6 1',2'-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyl-polyC
5'N1+ 3' N 3 1',2'-dideoxyribose, d(G)n, polyI-polyC
Table 3 shows representative positions and structures of immunostimulatory
moieties
within an immunostimulatory oligonucleotide having a downstream potentiation
domain.
Table 3
Position TYPICAL IMMUNOSTIMULATORY MOIETY
5' N2 methylphosphonate internucleoside linkage
5' Nl methylphosphonate internucleoside linkage
3' N1 1',2'-dideoxyribose, methylphosphonate internucleoside linkage, 2'-O-
methyl
3' N2 1',2'-dideoxyribose, (3-L-deoxyribonucleoside, C2-C18 alkyl linker,
Spacer 9, Spacer 18, 2-aminobutyl-l,3-propanediol linker,
methyl hos honate internucleoside linkage, 2'-O-methyl
3' N3 3'-deoxyribonucleoside, 3'-0-substituted ribonucleoside,
2'-0 - ro arg l-ribonucleoside
3'N2 + 3' N3 1',2'-dideoxyribose, (3-L-deoxyribonucleoside
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The immunostimulatory oligonucleotides according to the invention comprise at
least
two oligonucleotides linked at their 3' ends or intemucleoside linkage or a
functionalized
nucleobase or sugar via a non-nucleotidic linker. For purposes of the
invention, a "non-
nucleotidic linker" is any moiety that can be linked to the oligonucleotides
by way of
covalent or non-covalent linkages. Preferably such linker is from about 2
angstroms to about
200 angstroms in length. Several examples of preferred linkers are set forth
below. Non-
covalent linkages include, but are not limited to, electrostatic interaction,
hydrophobic
interactions, 7c-stacking interactions, and hydrogen bonding. The term "non-
nucleotidic
linker" is not meant to refer to an intemucleoside linkage, as described
above, e.g., a
phosphodiester, phosphorothioate, or phosphorodithioate functional group, that
directly
connects the 3'-hydroxyl groups of two nucleosides. For purposes of this
invention, such a
direct 3'-3' linkage (no linker involved) is considered to be a "nucleotidic
linkage."
In some embodiments, the non-nucleotidic linker is a metal, including, without
limitation, gold particles. In some other embodiments, the non-nucleotidic
linker is a soluble
or insoluble biodegradable polymer bead.
In yet other embodiments, the non-nucleotidic linker is an organic moiety
having
functional groups that permit attachment to the oligonucleotide. Such
attachment preferably
is by any stable covalent linkage. As a non-limiting example, the linker may
be attached to
any suitable position on the nucleoside, as illustrated in Figure 5. In some
preferred
embodiments, the linker is attached to the 3'-hydroxyl. In such embodiments,
the linker
preferably comprises a hydroxyl functional group, which preferably is attached
to the 3'-
hydroxyl by means of a phosphodiester, phosphorothioate, phosphorodithioate or
non-
phosphate-based linkages.
In some embodiments, the non-nucleotidic linker is a biomolecule, including,
without
limitation, polypeptides, antibodies, lipids, antigens, allergens, and
oligosaccharides. In
some other embodiments, the non-nucleotidic linker is a small molecule. For
purposes of the
invention, a small molecule is an organic moiety having a molecular weight of
less than
1,000 Da. In some embodiments, the small molecule has a molecular weight of
less than 750
Da.
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In some embodiments, the small molecule is an aliphatic or aromatic
hydrocarbon,
either of which optionally can include, either in the linear chain connecting
the
oligonucleotides or appended to it, one or more functional groups selected
from the group
consisting of hydroxy, amino, thiol, thioether, ether, amide, thioamide,
ester, urea, and
thiourea. The small molecule can be cyclic or acyclic. Examples of small
molecule linkers
include, but are not limited to, amino acids, carbohydrates, cyclodextrins,
adamantane,
cholesterol, haptens and antibiotics. However, for purposes of describing the
non-nucleotidic
linker, the term "small molecule" is not intended to include a nucleoside.
In some embodiments, the small molecule linker is glycerol or a glycerol
homolog of
the formula HO-(CH2)o CH(OH)-(CHa)p-OH, wherein o andp independently are
integers
from 1 to about 6, from 1 to about 4, or from 1 to about 3. In some other
embodiments, the
small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some
such
derivatives have the formula HO-(CHa)m C(O)NH-CH2-CH(OH)-CHZ-NHC(O)-(CH2)m OH,
wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about
6, or from 2 to
about 4.
Some non-nucleotidic linkers according to the invention permit attachment of
more
than two oligonucleotides. For example, the small molecule linker glycerol has
three
hydroxyl groups to which oligonucleotides may be covalently attached. Some
immunostimulatory oligonucleotides according to the invention, therefore,
comprise more
than two oligonucleotides linked at their 3' ends to a non-nucleotidic linker.
The immunostimulatory oligonucleotides of the invention may conveniently be
synthesized using an automated synthesizer and phosphoramidite approach as
schematically
depicted in Figures 3 and 4, and further described in the Examples. In some
embodiments,
the immunostimulatory oligonucleotides are synthesized by a linear synthesis
approach (see
Figure 3). As used herein, the term "linear synthesis" refers to a synthesis
that starts at one
end of the immunostimulatory oligonucleotide and progresses linearly to the
other end.
Linear synthesis permits incorporation of either identical or un-identical (in
terms of length,
base composition and/or chemical modifications incorporated) monomeric units
into the
immunostimulatory oligonucleotides.
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An alternative mode of synthesis is "parallel synthesis", in which synthesis
proceeds
outward from a central linker moiety (see Figure 4). A solid support attached
linker can be
used for parallel synthesis, as is described in U.S. Patent No. 5,912,332.
Alternatively, a
universal solid support (such as phosphate attached controlled pore glass)
support can be
used.
Parallel synthesis of immunostimulatory oligonucleotides has several
advantages over
linear synthesis: (1) parallel synthesis permits the incorporation of
identical monomeric units;
(2) unlike in linear synthesis, both (or all) the monomeric units are
synthesized at the same
time, thereby the number of synthetic steps and the time required for the
synthesis is the
same as that of a monomeric unit; and (3) the reduction in synthetic steps
improves purity
and yield of the final immunostimulatory oligonucleotide product.
At the end of the synthesis by either linear synthesis or parallel synthesis
protocols,
the immunostimulatory oligonucleotides may conveniently be deprotected with
concentrated
ammonia solution or as recommended by the phosphoramidite supplier, if a
modified
nucleoside is incorporated. The product immunostimulatory oligonucleotide is
preferably
purified by reversed phase HPLC, detritylated, desalted and dialyzed.
Table 4 shows representative immunostimulatory oligonucleotides according to
the
invention.
Table 4. Examples of Immunostimulatory Oligonucleotides Sequences
SEQ ID NO. Sequences and Modification
1 5'-TCTGTR'GTTCT-X-TCTTGR'TGTCT-5'
2 5'-ACACACCAACT-X-TCAACCACACA-5' (Control)
3 5'-TCTGTR'GTTC1U1-X-U1C1TTGR'TGTCT-5'
4 5'-CTGTR'GTTCTC-X-CTCTTGR'TGTC-5'
5 5'-CTGTR'GTTCU1C1-X-C1U1CTTGR'TGTC-5'
6 5'-CTGTR'GTTC1U1C1-X-C1U1CiTTGR'TGTC-5'
7 5'-TCTGTR'GTTCT-X-CGTTCGAACGT-5'
8 5'-TCTGTR'GACAG-X-GACAGR'TGTCT-5'
9 5'-TCTGTR'GACA1G1-X-G1A1CAGR'TGTCT-5'
UR' 1 l7l,L:T-J "
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11 5'-TCAGTR'GACTG-X-GTCAGR'TGACT-5'
12 5'-TR'GTR'GAR'GAT-X-TAGR'AGR'TGR'T-5'
13 5'-TR'GTR'GTAGTA-X-ATGATGR'TGR'T-5'
14 5'-TR'GAAR'GTTCT-X-TCTTGR'AAGR'T-5'
15 5'-TR'GTAR'GTACT-X-TCATGR'ATGR'T-5'
16 5'-TCRAACRTTCR-X-RCTTRCAARCT-5'
R' = 1-(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
Al/Cl/Gl/U1= 2'-O-
methyl-ribonucleotides; R = 2'-deoxy-7-deazaguanosine; X= glycerol linker.
In a second aspect, the invention provides immunostimulatory oligonucleotide
conjugates comprising an immunostimulatory oligonucleotide, as described
above, and an
antigen conjugated to the immunostimulatory oligonucleotide at a position
other than the
accessible 5' end. In some embodiments, the non-nucleotidic linker comprises
an antigen,
which is conjugated to the oligonucleotide. In some other embodiments, the
antigen is
conjugated to the oligonucleotide at a position other than its 3' end. In some
embodiments,
the antigen produces a vaccine effect.
The antigen is preferably selected from the group consisting of antigens
associated
with a pathogen, antigens associated with a cancer, antigens associated with
an auto-immune
disorder, and antigens associated with other diseases such as, but not limited
to, veterinary or
pediatric diseases. For purposes of the invention, the term "associated with"
means that the
antigen is present when the pathogen, cancer, auto-immune disorder, food
allergy, respiratory
allergy, asthma or other disease is present, but either is not present, or is
present in reduced
amounts, when the pathogen, cancer, auto-immune disorder, food allergy,
respiratory allergy,
or disease is absent.
The immunostimulatory oligonucleotide is covalently linked to the antigen, or
it is
otherwise operatively associated with the antigen. As used herein, the term
"operatively
associated with" refers to any association that maintains the activity of both
immunostimulatory oligonucleotide and antigen. Nonlimiting examples of such
operative
associations include being part of the same liposome or other such delivery
vehicle or
reagent. In embodiments wherein the immunostimulatory oligonucleotide is
covalently
linked to the antigen, such covalent linkage preferably is at any position on
the
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immunostimulatory oligonucleotide other than an accessible 5' end of an
immunostimulatory
oligonucleotide. For example, the antigen may be attached at an
internucleoside linkage or
may be attached to the non-nucleotidic linker. Alternatively, the antigen may
itself be the
non-nucleotidic linker.
In a third aspect, the invention provides pharmaceutical formulations
comprising an
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate
according to the invention and a physiologically acceptable carrier. As used
herein, the term
"physiologically acceptable" refers to a material that does not interfere with
the effectiveness
of the immunostimulatory oligonucleotide and is compatible with a biological
system such as
a cell, cell culture, tissue, or organism. Preferably, the biological system
is a living
organism, such as a vertebrate.
As used herein, the term "carrier" encompasses any excipient, diluent, filler,
salt,
buffer, stabilizer, solubilizer, lipid, or other material well known in the
art for use in
pharmaceutical formulations. It will be understood that the characteristics of
the carrier,
excipient, or diluent will depend on the route of administration for a
particular application.
The preparation of pharmaceutically acceptable formulations containing these
materials is
described in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, ed. A.
Gennaro,
Mack Publishing Co., Easton, PA, 1990.
In a fourth aspect, the invention provides methods for generating an immune
response
in a vertebrate, such methods comprising administering to the vertebrate an
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate
according to the invention. In some embodiments, the vertebrate is a mammal.
For purposes
of this invention, the term "mammal" is expressly intended to include humans.
In preferred
embodiments, the immunostimulatory oligonucleotide or immunostimulatory
oligonucleotide
conjugate is administered to a vertebrate in need of immunostimulation.
In the methods according to this aspect of the invention, administration of
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate can be
by any suitable route, including, without limitation, parenteral, oral,
sublingual, transdermal,
topical, intranasal, aerosol, intraocular, intratracheal, intrarectal,
vaginal, by gene gun, dermal
patch or in eye drop or mouthwash fnr~,,, A~ma ~s~at;~.r ~f+~._ +~.ra -~..4:
.. 0
__ _. ...~ ...~ :.~.:G6%e.i'v 'vviivu.iiialits Vl
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immunostimulatory oligonucleotides can be carried out using known procedures
at dosages
and for periods of time effective to reduce symptoms or surrogate markers of
the disease.
When administered systemically, the therapeutic composition is preferably
administered at a
sufficient dosage to attain a blood level of immunostimulatory oligonucleotide
from about
0.0001 micromolar to about 10 micromolar. For localized administration, much
lower
concentrations than this may be effective, and much higher concentrations may
be tolerated.
Preferably, a total dosage of immunostimulatory oligonucleotide ranges from
about 0.001 mg
per patient per day to about 200 mg per kg body weight per day. It may be
desirable to
administer simultaneously, or sequentially a therapeutically effective amount
of one or more
of the therapeutic compositions of the invention to an individual as a single
treatment
episode.
In certain preferred embodiments, immunostimulatory oligonucleotide or
immunostimulatory oligonucleotide conjugate according to the invention are
administered in
combination with vaccines, antibodies, cytotoxic agents, allergens,
antibiotics, antisense
oligonucleotides, peptides, proteins, gene therapy vectors, DNA vaccines
and/or adjuvants to
enhance the specificity or magnitude of the immune response. In these
embodiments, the
immunostimulatory oligonucleotides of the invention can variously act as
adjuvants and/or
produce direct immunostimulatory effects.
Either the immunostimulatory oligonucleotide or immunostimulatory
oligonucleotide
conjugate or the vaccine, or both, may optionally be linked to an immunogenic
protein, such
as keyhole limpet hemocyanin (KLH), cholera toxin B subunit, or any other
immunogenic
carrier protein. Any of the plethora of adjuvants may be used including,
without limitation,
Freund's complete adjuvant, KLH, monophosphoryl lipid A (MPL), alum, and
saponins,
including QS-21, imiquimod, R848, or combinations thereof.
For purposes of this aspect of the invention, the term "in combination with"
means in
the course of treating the same disease in the same patient, and includes
administering the
immunostimulatory oligonucleotide and/or the vaccine and/or the adjuvant in
any order,
including simultaneous administration, as well as temporally spaced order of
up to several
days apart. Such combination treatment may also include more than a single
administration
of the immunostimulatory oligonucleotide, andlor independently the vaccine,
and/or
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independently the adjuvant. The administration of the immunostimulatory
oligonucleotide
and/or vaccine and/or adjuvant may be by the same or different routes.
The methods according to this aspect of the invention are useful for model
studies of
the immune system. The methods are also useful for the prophylactic or
therapeutic
treatment of human or animal disease. For example, the methods are useful for
pediatric and
veterinary vaccine applications.
In a fifth aspect, the invention provides methods for therapeutically treating
a patient
having a disease or disorder, such methods comprising administering to the
patient an
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate
according to the invention. In various embodiments, the disease or disorder to
be treated is
cancer, an autoimmune disorder, airway inflammation, inflammatory disorders,
allergy,
asthma or a disease caused by a pathogen. Pathogens include bacteria,
parasites, fungi,
viruses, viroids and prions. Administration is carried out as described for
the fourth aspect of
the invention.
For purposes of the invention, the term "allergy" includes, without
limitation, food
allergies and respiratory allergies. The term "airway inflammation" includes,
without
limitation, asthma. As used herein, the term "autoimmune disorder" refers to
disorders in
which "self' proteins undergo attack by the immune system. Such term includes
autoimmune asthma.
In any of the methods according to this aspect of the invention, the
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate can be
administered in combination with any other agent useful for treating the
disease or condition
that does not diminish the immunostimulatory effect of the immunostimulatory
oligonucleotide. For example, in the treatment of cancer, it is contemplated
that the
immunostimulatory oligonucleotide or immunostimulatory oligonucleotide
conjugate may be
administered in combination with a chemotherapeutic compound.
The examples below are intended to further illustrate certain preferred
embodiments
of the invention, and are not intended to limit the scope of the invention.
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EXAMPLES
Example 1: Synthesis of Oligonucleotides Containing immunostimulatory Moieties
Oligonucleotides were synthesized on a 1 mol to 0.1 mM scale using an
automated
DNA synthesizer (OligoPilot II, AKTA, (Amersham) and/or Expedite 8909 (Applied
Biosystem)), following the linear synthesis or parallel synthesis procedures
outlined in
Figures 3 and 4.
5'-DMT dA, dG, dC and T phosphoramidites were purchased from Proligo (Boulder,
CO). 5'-DMT 7-deaza-dG and araG phosphoramidites were obtained from Chemgenes
(Wilmington, MA). DiDMT-glycerol linker solid support was obtained from
Chemgenes.
1-(2'-deoxy-(3-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite was
obtained from
Glen Research (Sterling, VA), 2'-O-methylribonuncleoside amidites were
obtained from
Promega (Obispo, CA). All oligonucleotides were phosphorothioate backbone
modified.
All nucleoside phosphoramidites were characterized by 31P and 'H NMR spectra.
Modified nucleosides were incorporated at specific sites using normal coupling
cycles
recommended by the supplier. After synthesis, oligonucleotides were
deprotected using
concentrated ammonium hydroxide and purified by reverse phase HPLC,
detritylation,
followed by dialysis. Purified oligonucleotides as sodium salt form were
lyophilized prior to
use. Purity was tested by CGE and MALDI-TOF MS. Endotoxin levels were
determined by
LAL test and were below 1.0 EU/mg.
Example 2: Activity of short-immunostimulatory oligonucleotides in murine
spleen
cell cultures
C57/BL6 spleen cells were cultured with indicated concentrations of compounds.
After 24 hours the supematants were collected and the levels of IL-12 and IL-6
were
determined by ELISA. All immunostimulatory oligonucleotides showed a
concentration-
dependent induction of two typical cytokines, IL-12 and IL-6 (Figures 6-7).
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Example 3: (need protocol for IFN-alpha induction in human pDC)
Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthy
volunteer
blood (CBR Laboratories, Boston, MA) were isolated by Ficoll density gradient
centrifugation method (Histopaque-1077, Sigma). pDCs were isolated from PBMCs
by
positive selection using the BDCA4 cell isolation kits (Miltenyi Biotec)
according to the
manufacturer's instructions. pDCs were plated in 96-well dishes using IX106
cells/ml. The
IMOs dissolved in DPBS (pH 7.4; Mediatech) were added to a final concentration
of 10.0
g/ml to the cell cultures. The cells were then incubated at 37 C for 24 hr
and the
supematants were collected for ELISA assays. The experiments were performed in
triplicate
wells. The levels of IFN-a were measured by sandwich ELISA. The required
reagents,
including cytokine antibodies and standards, were purchased from PharMingen.
Example 4: IFN-alpha induction in human PMBC
Human PBMCs were plated in 48-well plates using 5X106 cells/ml. The IMOs
dissolved in DPBS (pH 7.4; Mediatech) were added to a final concentration of
10.0 g/ml to
the cell cultures. The cells were then incubated at 37 C for 24 hr and the
supernatants were
collected for ELISA assays. The experiments were performed in triplicate
wells. The levels
of IFN-a were measured by sandwich ELISA. The required reagents, including
cytokine
antibodies and standards, were purchased from PharMingen.
Example 5: Human B-cell proliferation
The culture medium used for the assay consisted of RPMI 1640 medium
supplemented with 1.5 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential
amino
acids, 50 M 2-mercaptoethanol, 100 IU/ml penicillin-streptomycin mix and 10%
heat-
inactivated fetal bovine serum. A total of 0.5 X 106 B cells per ml (i.e.1 X
105 /200 i/well)
were stimulated in 96 well flat bottom plates with different concentrations of
test
oligonucleotides in triplicate for a total period of 72 hours. After 66 h,
cells were pulsed with
0.75 Ci of [3H]-thymidine (1Ci = 37 GBq; Perkin Elmer Life Sciences) in 20 l
RPMI 1640
iaedium (ito seluumli) per weii and harvesteo. 8ri iater. ine piwLes were
tiien harvested using a
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cell harvester and radioactive incorporation was determined using standard
liquid
scintillation technique. The results are expressed either as mean cpm +/-SD or
as
proliferation index (cpm treated group/cpm medium control).
EQUIVALENTS
While the foregoing invention has been described in some detail for purposes
of
clarity and understanding, it will be appreciated by one skilled in the art
from a reading of
this disclosure that various changes in form and detail can be made without
departing from
the true scope of the invention and appended claims.
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