U.S. patent application number 12/881475 was filed with the patent office on 2011-08-18 for semi-soft c-class immunostimulatory oligonucleotides.
Invention is credited to Arthur M. Krieg, Ulrike Samulowitz, Eugen Uhlmann, Jorg Vollmer.
Application Number | 20110201672 12/881475 |
Document ID | / |
Family ID | 37507656 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201672 |
Kind Code |
A1 |
Krieg; Arthur M. ; et
al. |
August 18, 2011 |
SEMI-SOFT C-CLASS IMMUNOSTIMULATORY OLIGONUCLEOTIDES
Abstract
The invention relates to specific C-Class semi-soft CpG
immunostimulatory oligonucleotides that are useful for stimulating
an immune response. In particular the oligonucleotides are useful
for treating allergy, such as allergic rhinitis and asthma, cancer
and infectious disease, such as hepatitis B and hepatitis C.
Inventors: |
Krieg; Arthur M.;
(Wellesley, MA) ; Samulowitz; Ulrike; (Langenfeld,
DE) ; Vollmer; Jorg; (Dusseldorf, DE) ;
Uhlmann; Eugen; (Glashuetten, DE) |
Family ID: |
37507656 |
Appl. No.: |
12/881475 |
Filed: |
September 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12336986 |
Dec 17, 2008 |
7795235 |
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12881475 |
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11255100 |
Oct 20, 2005 |
7566703 |
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12336986 |
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60620759 |
Oct 20, 2004 |
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Current U.S.
Class: |
514/44R ;
128/203.12; 128/203.15; 536/22.1 |
Current CPC
Class: |
C12N 2310/315 20130101;
C12N 2310/17 20130101; A61P 31/00 20180101; A61P 31/20 20180101;
A61P 37/00 20180101; A61P 31/14 20180101; C12N 2310/345 20130101;
C07H 21/02 20130101; A61K 39/39 20130101; A61P 11/00 20180101; A61K
2039/55561 20130101; A61P 37/04 20180101; C12N 15/117 20130101;
A61P 29/00 20180101; A61P 35/00 20180101; A61K 9/0073 20130101;
A61P 11/06 20180101; A61K 9/0043 20130101; A61P 37/08 20180101 |
Class at
Publication: |
514/44.R ;
536/22.1; 128/203.12; 128/203.15 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/00 20060101 C07H021/00; A61M 15/00 20060101
A61M015/00; A61P 37/04 20060101 A61P037/04 |
Claims
1.-24. (canceled)
25. An oligonucleotide comprising: 5'
TC_GTC_GTN.sub.1TC_GGCGCN.sub.1GCCG 3' (SEQ ID NO: 27), wherein the
oligonucleotide includes at least 2 stabilized internucleotide
linkages and_represents phosphodiester or phosphodiester-like
internucleotide linkage and wherein N.sub.1 is 0-3 nucleotides in
length, with N referring to any nucleotide.
26. The oligonucleotide of claim 25, wherein N.sub.1 is 3
nucleotides in length.
27. The oligonucleotide of claim 25, wherein N.sub.1 is 0
nucleotides in length.
28. An oligonucleotide comprising: 5' TTC_GTC_GTTTX.sub.1--GTC_GTT
3' (SEQ ID NO: 25), wherein the oligonucleotide includes at least 2
stabilized internucleotide linkages and_represents phosphodiester
or phosphodiester-like internucleotide linkage and wherein X.sub.1
is a pyrimidine.
29. The oligonucleotide of claim 28, wherein X.sub.1 is T.
30. The oligonucleotide of claim 29, wherein the oligonucleotide
comprises 5' T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 3' (SEQ ID NO: 5),
wherein * represents a stabilized internucleotide linkage.
31. The oligonucleotide of claim 29, wherein the oligonucleotide is
5' T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 3' (SEQ ID NO: 5), wherein *
represents a stabilized internucleotide linkage, wherein 5' refers
to the free 5' end of the oligonucleotide and 3' refers to the free
3' end of the oligonucleotide.
32. The oligonucleotide of claim 28, wherein X.sub.1 is C
33. The oligonucleotide of claim 32, wherein the oligonucleotide
comprises 5' T*T*T*C_G*T*C_G*T*T*T*C _G*T*C_G*T*T 3' (SEQ ID NO:
6), wherein * represen stabilized internucleotide linkage.
34. The oligonucleotide of claim 32, wherein the oligonucleotide is
5' T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T 3' (SEQ ID NO: 6), wherein *
represents a stabilized internucleotide linkage, wherein 5' refers
to the free 5' end of the oligonucleotide and 3' refers to the free
3' end of the oligonucleotide.
35. An oligonucleotide comprising: T*C_G*T*C_G*T*C, wherein *
represents a stabilized internucleotide linkage and _ represents
phosphodiester or phosphodiester-like internucleotide linkage.
36. The oligonucleotide of claim 35, wherein the oligonucleotide is
5' T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C 3' (SEQ ID NO.: 22), wherein
5' refers to the free 5' end of the oligonucleotide and 3' refers
to the free 3' end of the oligonucleotide.
37. An oligonucleotide comprising: T*C_G*T*T*C_G*G, wherein *
represents a stabilized internucleotide linkage and _ represents
phosphodiester or phosphodiester-like internucleotide linkage.
38. A pharmaceutical composition comprising an oligonucleotide of
claim 25 and a pharmaceutically acceptable carrier.
39. The pharmaceutical composition of claim 38, further comprising
a nebulizer.
40. The pharmaceutical composition of claim 38, further comprising
an inhaler.
41. The pharmaceutical composition of claim 40 wherein the inhaler
is a metered dose inhaler.
42. The pharmaceutical composition of claim 40, wherein the inhaler
is a powder inhaler.
43. The pharmaceutical composition of claim 38, further comprising
a chemotherapeutic agent.
44. The pharmaceutical composition of claim 38, further comprising
an anti-viral agent.
45. The pharmaceutical composition of claim 38, wherein the
pharmaceutically acceptable carrier is formulated for subcutaneous
administration.
46. The pharmaceutical composition of claim 38, wherein the
pharmaceutically acceptable carrier is formulated for oral
administration.
47. The pharmaceutical composition of claim 38, wherein the
pharmaceutically acceptable carrier is formulated for intranasal
administration.
48. A method for modulating an immune response, comprising
administering to a subject an oligonucleotide of claim 25, in an
effective amount to modulate an immune response.
Description
RELATED APPLICATION
[0001] This application is a divisional of co-pending U.S.
application Ser. No. 11/255100, filed Oct. 20, 2005, which claims
priority to U.S. Provisional Application having Ser. No. 60/620,759
entitled "SEMI-SOFT C-CLASS IMMUNOSTIMULATORY OLIGONUCLEOTIDES"
filed Oct. 20, 2004, the entire contents of which are incorporated
by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates generally to immunostimulatory
oligonucleotides with reduced renal inflammatory effects,
compositions thereof and methods of using the immunostimulatory
oligonucleotides. In particular the immunostimulatory
oligonucleotides are C-class semi-soft oligonucleotides that are
particularly effective in the treatment of allergy and asthma,
cancer and infectious disease.
BACKGROUND OF THE INVENTION
[0003] Bacterial DNA has immune stimulatory effects to activate B
cells and natural killer cells, but vertebrate DNA does not
(Tokunaga, T., et al., 1988. Jpn. J. Cancer Res. 79:682-686;
Tokunaga, T., et al., 1984, JNCl 72:955-962; Messina, J. P., et
al., 1991, J. Immunol. 147:1759-1764; and reviewed in Krieg, 1998,
In: Applied Oligonucleotide Technology, C. A. Stein and A. M.
Krieg, (Eds.), John Wiley and Sons, Inc., New York, N.Y., pp.
431-448). It is now understood that these immune stimulatory
effects of bacterial DNA are a result of the presence of
unmethylated CpG dinucleotides in particular base contexts (CpG
motifs), which are common in bacterial DNA, but methylated and
underrepresented in vertebrate DNA (Krieg et al, 1995 Nature
374:546-549; Krieg, 1999 Biochim Biophys. Acta 93321:1-10). The
immune stimulatory effects of bacterial DNA can be mimicked with
synthetic oligodeoxynucleotides (ODN) containing these CpG motifs.
Such CpG ODN have highly stimulatory effects on human and murine
leukocytes, inducing B cell proliferation; cytokine and
immunoglobulin secretion; natural killer (NK) cell lytic activity
and IFN-.gamma. secretion; and activation of dendritic cells (DCs)
and other antigen presenting cells to express costimulatory
molecules and secrete cytokines, especially the Th1-like cytokines
that are important in promoting the development of Th1-like T cell
responses. These immune stimulatory effects of native
phosphodiester backbone CpG ODN are highly CpG specific in that the
effects are dramatically reduced if the CpG motif is methylated,
changed to a GpC, or otherwise eliminated or altered (Krieg et al,
1995 Nature 374:546-549; Hartmann et al, 1999 Proc. Natl. Acad. Sci
USA 96:9305-10).
[0004] In early studies, it was thought that the immune stimulatory
CpG motif followed the formula
purine-purine-CpG-pyrimidine-pyrimidine (Krieg et al, 1995 Nature
374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423; Hacker et al.,
1998 EMBO J. 17:6230-6240; Lipford et al, 1998 Trends in Microbiol.
6:496-500). However, it is now clear that mouse lymphocytes respond
quite well to phosphodiester CpG motifs that do not follow this
"formula" (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same
is true of human B cells and dendritic cells (Hartmann et al, 1999
Proc. Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin. Invest.
98:1119-1129).
[0005] Several different classes of CpG oligonucleotides has
recently been described. 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
oligonucleotides 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 of
CpG oligonucleotides activates B cells and NK cells and induces
IFN-.alpha.; this class has been termed the C-class. The C-class
CpG oligonucleotides, 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
co-pending U.S. patent application US 10/224,523 filed on Aug. 19,
2002 and related PCT Patent Application PCT/US02/26468 published
under International Publication Number WO 03/015711.
SUMMARY OF THE INVENTION
[0006] It has been discovered that immunostimulatory properties of
specific C-class CpG oligonucleotides with the selective inclusion
of one or more non-stabilized linkages between certain nucleotides
have significant activity and are particularly useful in the
treatment of allergy and asthma. The non-stabilized linkages are
preferably natural linkages, i.e., phosphodiester linkages or
phosphodiester-like linkages. A non-stabilized linkage will
typically, but not necessarily, be relatively susceptible to
nuclease digestion. The immunostimulatory oligonucleotides of the
instant invention include at least one non-stabilized linkage
situated between a 5' C and an adjacent 3' G, wherein both the 5' C
and the 3' G are internal nucleotides.
[0007] The immunostimulatory oligonucleotides of the instant
invention are useful for inducing a Th1-like immune response.
Accordingly, the immunostimulatory oligonucleotides of the instant
invention are useful as adjuvants for vaccination, and they are
useful for treating diseases including cancer, infectious disease,
allergy, and asthma. They are believed to be of particular use in
any condition calling for prolonged or repeated administration of
immunostimulatory oligonucleotide for any purpose, but are
particularly useful in the treatment of asthma and allergic
diseases such as allergic rhinitis.
[0008] The present invention relates in part to immunostimulatory
CpG containing oligonucleotides. In one aspect the invention is an
oligonucleotide having the formula: 5' TC_GX.sub.1C_G
X.sub.2N.sub.1 X.sub.3C_GN.sub.2CG 3'(SEQ ID NO: 26). The
oligonucleotide includes at least 2 stabilized internucleotide
linkages. "_" represents phosphodiester or phosphodiester-like
internucleotide linkage. N.sub.1 is 0-3 nucleotides in length,
N.sub.2 is 0-9 nucleotides in length with N referring to any
nucleotide. X.sub.1, X.sub.2, and X.sub.3 are any nucleotide. In
some embodiments X.sub.1, X.sub.2, and X.sub.3 are T.
[0009] In some embodiments the oligonucleotide may comprise 5'
TC_GTC_GTN.sub.1TC_GGCGCN.sub.1GCCG 3'(SEQ ID NO: 27). In one
embodiment the oligonucleotide may comprise 5'
T*C_G*T*C_G*T*N.sub.1*T*C_G*G*C*G*CN.sub.1G*C*C*G 3'(SEQ ID NO:
27). In some embodiments N.sub.1 is 3 or 2 nucleotides in length.
In other embodiments N.sub.1 is 0 nucleotides in length.
[0010] The immunostimulatory oligonucleotide may comprise 5'
T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3'(SEQ ID NO: 2),
wherein * represents a stabilized internucleotide linkage.
Optionally, when specifically stated, 5' may refer to the free 5'
end of the oligonucleotide and 3' may refer to the free 3' end of
the oligonucleotide.
[0011] In other embodiments the immunostimulatory oligonucleotide
may comprise 5' T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 3'(SEQ ID NO:
3), wherein * represents a stabilized internucleotide linkage.
Optionally, when specifically stated, 5' may refer to the free 5'
end of the oligonucleotide and 3' may refer to the free 3' end of
the oligonucleotide.
[0012] In another aspect, the immunostimulatory oligonucleotide has
the following formula TC_G X.sub.1C_G X.sub.2C_G X.sub.3TC_GGCGC_G
N.sub.33'(SEQ ID NO: 28).
[0013] N.sub.3 is 1-5 nucleotides in length with N referring to any
nucleotide. In one embodiment N.sub.3 is 5 nucleotides. X.sub.1,
X.sub.2, and X.sub.3 are any nucleotide. In some embodiments
X.sub.1 and X.sub.3 are T.
[0014] In one embodiment the oligonucleotide may comprise 5'
TC_GTC_GAC_GATC_GGCGC_GCGCCG3' (SEQ ID NO: 4), wherein the
oligonucleotide includes at least 2 stabilized internucleotide
linkages and _ represents phosphodiester or phosphodiester-like
internucleotide linkage. In one embodiment the oligonucleotide may
comprise 5' T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 3' (SEQ
ID NO: 4). Optionally, when specifically stated, 5' may refer to
the free 5' end of the oligonucleotide and 3' may refer to the free
3' end of the oligonucleotide.
[0015] According to another aspect of the invention an
immunostimulatory oligonucleotide having the following formula: 5'
TTC_GX.sub.2C_GN.sub.1X.sub.1--GX.sub.3C_GTT 3' (SEQ ID NO: 24) is
provided. The oligonucleotide includes at least 2 stabilized
internucleotide linkages and _ represents phosphodiester or
phosphodiester-like internucleotide linkage. N.sub.1 is 1-3
nucleotides in length with N referring to any nucleotide. X.sub.1
is a pyrimidine. X.sub.2 and X.sub.3 are any nucleotide. In some
embodiments X.sub.2 and X.sub.3 are T.
[0016] In one embodiment the oligonucleotide may comprise 5'
TTC_GTC_GTTTX.sub.1--GTC_GTT 3' (SEQ ID NO: 25). In another
embodiment the oligonucleotide may comprise 5'
T*T*C_G*T*C_G*T*T*T*X.sub.1--G*T*C_G*T*T 3' (SEQ ID NO: 25). In
some embodiments X.sub.1 is T or C.
[0017] The oligonucleotide may comprise 5'
T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 3' (SEQ ID NO: 5), wherein *
represents a stabilized internucleotide linkage. Optionally, when
specifically stated, 5' may refer to the free 5' end of the
oligonucleotide and 3' may refer to the free 3' end of the
oligonucleotide.
[0018] The oligonucleotide may comprise 5'
T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T 3'(SEQ ID NO: 6), wherein *
represents a stabilized internucleotide linkage. Optionally, when
specifically stated, 5' may refer to the free 5' end of the
oligonucleotide and 3' may refer to the free 3' end of the
oligonucleotide.
[0019] In some aspects of the invention the oligonucleotide has one
of the following formulas TCGTCGTTCGGCGCGCCG (SEQ ID NO: 3),
TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID NO: 2), TCGTCGACGATCGGCGCGCGCCG
(SEQ ID NO: 4), TTCGTCGTTTTGTCGTT. (SEQ ID NO: 5), or
TTTCGTCGTTTCGTCGTT. (SEQ ID NO: 6)
[0020] In other aspects of the invention the oligonucleotide has
one of the following formulas TCGTCGTC, CGTCGTCG, GTCGTCGT,
TCGTCGTT, CGTCGTTC, GTCGTTCG, TCGTTCGG, CGTTCGGC, GTTCGGCG,
TTCGGCGC, TCGGCGCG, CGGCGCGC, GGCGCGCG, GCGCGCGC, CGCGCGCC, or
GCGCGCCG.
[0021] In other aspects of the invention the oligonucleotide has
one of the following formulas T*C_G*T*C_G*T*C, C_G*T*C_G*T*C_G,
G*T*C_G*T*C_G*T, T*C_G*T*C_G*T*T, C_G*T*C_G*T*T*C, G*T*C_G*T*T*C_G,
T*C_G*T*T*C_G*G, C_G*T*T*C_G*G*C, G*T*T*C_G*G*C*G, T*T*C_G*G*C*G*C,
T*C_G*G*C*G*C_G, C_G*G*C*G*C_G*C, G*G*C*G*C_G*C*G, G*C*G*C_G*C*G*C,
C*G*C_G*C*G*C*C, or G*C_G*C*G*C*C*G.
[0022] In other aspects of the invention an oligonucleotide
comprising: T*C_G*T*C_G*T*C, wherein * represents a stabilized
internucleotide linkage and _ represents phosphodiester or
phosphodiester-like internucleotide linkage is provided. Optionally
the oligonucleotide may be 5'
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C 3' (SEQ ID NO.: 21), 5'
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C 3' (SEQ ID NO.: 22), or 5'
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C 3' (SEQ ID NO.: 23),
wherein 5' refers to the free 5' end of the oligonucleotide and 3'
refers to the free 3' end of the oligonucleotide.
[0023] In other aspects an oligonucleotide comprising:
T*C_G*T*T*C_G*G, wherein * represents a stabilized internucleotide
linkage and _ represents phosphodiester or phosphodiester-like
internucleotide linkage is provided. Optionally the oligonucleotide
may be 5' C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID
NO.: 15), 5' G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID
NO.: 16), 5' T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID
NO.: 17), 5' C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID NO.:
18), 5' G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID NO.: 19), or
5' T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 3' (SEQ ID NO.: 20), wherein
5' refers to the free 5' end of the oligonucleotide and 3' refers
to the free 3' end of the oligonucleotide.
[0024] A pharmaceutical composition comprising an oligonucleotide
of the invention and a pharmaceutically acceptable carrier is
provided.
[0025] In some embodiments the composition is formulated in a
nebulizer or an inhaler. The inhaler may be a metered dose inhaler.
Alternatively the inhaler is a powder inhaler.
[0026] In other embodiments the pharmaceutical composition may
include a chemotherapeutic agent. In yet other embodiments the
composition may include an anti-viral agent.
[0027] The pharmaceutical composition may optionally include a
pharmaceutically acceptable carrier formulated for subcutaneous
administration, oral administration or intranasal
administration.
[0028] In one embodiment the oligonucleotide is in a pharmaceutical
composition optionally comprising a pharmaceutically acceptable
carrier. In some embodiments the oligonucleotide is formulated as
an aerosol.
[0029] In one embodiment the oligonucleotide further comprises an
adjuvant or a cytokine.
[0030] In one embodiment the oligonucleotide further comprises an
antigen, wherein the oligonucleotide is a vaccine adjuvant.
[0031] In one embodiment the antigen is selected from the group
consisting of: a viral antigen, a bacterial antigen, a fungal
antigen, a parasitic antigen, and a tumor antigen. In one
embodiment the antigen is encoded by a nucleic acid vector. In one
embodiment the antigen is a peptide antigen. In one embodiment the
antigen is covalently linked to the oligonucleotide or
immunostimulatory nucleic acid molecule. In another embodiment the
antigen is not covalently linked to the oligonucleotide or
immunostimulatory nucleic acid molecule.
[0032] In one embodiment the phosphodiester or phosphodiester-like
linkage is phosphodiester. In one embodiment the
phosphodiester-like linkage is boranophosphonate or
diastereomerically pure Rp phosphorothioate.
[0033] In one embodiment the stabilized backbone comprises a
plurality of internucleotide linkages selected from the group
consisting of: phosphorothioate, phosphorodithioate,
methylphosphonate, methylphosphorothioate, and any combination
thereof. In one embodiment the stabilized backbone comprises a
plurality of phosphorothioate internucleotide linkages.
[0034] In one embodiment the immunostimulatory nucleic acid
molecule is 4-100 nucleotides long.
[0035] In other aspects the invention is a method for treating
asthma by administering to a subject having or at risk of having
asthma an oligonucleotide of the invention in an effective amount
to treat asthma.
[0036] In yet other aspects the invention is a method for treating
allergy by administering to a subject having or at risk of having
allergy an oligonucleotide of the invention in an effective amount
to treat allergy. In one embodiment the subject has allergic
rhinitis. In one embodiment the oligonucleotide is administered to
a mucosal surface. In other embodiments the oligonucleotide is
administered in an aerosol formulation. Optionally the
oligonucleotide is administered intranasally.
[0037] A method for inducing cytokine production is provided
according to another aspect of the invention. The method is
performed by administering to a subject an immunostimulatory CpG
oligonucleotide described herein in an effective amount to induce a
cytokine selected from the group consisting of IL-6, IL-8, IL-12,
IL-18, TNF, IFN-.alpha., chemokines, and IFN-.gamma..
[0038] In another aspect the invention is a composition of the CpG
immunostimulatory oligonucleotides described herein in combination
with an antigen or other therapeutic compound, such as an
anti-microbial agent. The anti-microbial agent may be, for
instance, an anti-viral agent, an anti-parasitic agent, an
anti-bacterial agent or an anti-fungal agent.
[0039] A composition of a sustained release device including the
CpG immunostimulatory oligonucleotides described herein is provided
according to another aspect of the invention.
[0040] The composition may optionally include a pharmaceutical
carrier and/or be formulated in a delivery device. In some
embodiments the delivery device is selected from the group
consisting of cationic lipids, cell permeating proteins, and
sustained release devices. In one embodiment the sustained release
device is a biodegradable polymer or a microparticle.
[0041] According to another aspect of the invention a method of
stimulating an immune response is provided. The method involves
administering a CpG immunostimulatory oligonucleotide to a subject
in an amount effective to induce an immune response in the subject.
Preferably the CpG immunostimulatory oligonucleotide is
administered orally, locally, in a sustained release device,
mucosally, systemically, parenterally, or intramuscularly. When the
CpG immunostimulatory oligonucleotide is administered to the
mucosal surface it may be delivered in an amount effective for
inducing a mucosal immune response or a systemic immune response.
In preferred embodiments the mucosal surface is selected from the
group consisting of an oral, nasal, rectal, vaginal, and ocular
surface.
[0042] In some embodiments the method includes exposing the subject
to an antigen wherein the immune response is an antigen-specific
immune response. In some embodiments the antigen is selected from
the group consisting of a tumor antigen, a viral antigen, a
bacterial antigen, a parasitic antigen and a peptide antigen.
[0043] CpG immunostimulatory oligonucleotides are capable of
provoking a broad spectrum of immune response. For instance these
CpG immunostimulatory oligonucleotides can be used to redirect a
Th2 to a Th1 immune response. CpG immunostimulatory
oligonucleotides may also be used to activate an immune cell, such
as a lymphocyte (e.g., B and T cells), a dendritic cell, and an NK
cell. The activation can be performed in vivo, in vitro, or ex
vivo, i.e., by isolating an immune cell from the subject,
contacting the immune cell with an effective amount to activate the
immune cell of the CpG immunostimulatory oligonucleotide and
re-administering the activated immune cell to the subject. In some
embodiments the dendritic cell presents a cancer antigen. The
dendritic cell can be exposed to the cancer antigen ex vivo.
[0044] The immune response produced by CpG immunostimulatory
oligonucleotides may also result in induction of cytokine
production, e.g., production of IL-6, IL-8, IL-12, IL-18, TNF,
IFN-.alpha., chemokines, and IFN-.gamma..
[0045] In still another embodiment, the CpG immunostimulatory
oligonucleotides are useful for treating cancer. The CpG
immunostimulatory oligonucleotides are also useful according to
other aspects of the invention in preventing cancer (e.g., reducing
a risk of developing cancer) in a subject at risk of developing a
cancer. The cancer may be selected from the group consisting of
biliary tract cancer, breast cancer, cervical cancer,
choriocarcinoma, colon cancer, endometrial cancer, gastric cancer,
intraepithelial neoplasms, lymphomas, liver cancer, lung cancer
(e.g. small cell and non-small cell), melanoma, neuroblastomas,
oral cancer, ovarian cancer, pancreatic cancer, prostate cancer,
rectal cancer, sarcomas, thyroid cancer, and renal cancer, as well
as other carcinomas and sarcomas. In some important embodiments,
the cancer is selected from the group consisting of bone cancer,
brain and CNS cancer, connective tissue cancer, esophageal cancer,
eye cancer, Hodgkin's lymphoma, larynx cancer, oral cavity cancer,
skin cancer, and testicular cancer.
[0046] CpG immunostimulatory oligonucleotides may also be used for
increasing the responsiveness of a cancer cell to a cancer therapy
(e.g., an anti-cancer therapy), optionally when the CpG
immunostimulatory oligonucleotide is administered in conjunction
with an anti-cancer therapy. The anti-cancer therapy may be a
chemotherapy, a vaccine (e.g., an in vitro primed dendritic cell
vaccine or a cancer antigen vaccine) or an antibody based therapy.
This latter therapy may also involve administering an antibody
specific for a cell surface antigen of, for example, a cancer cell,
wherein the immune response results in antibody dependent cellular
cytotoxicity (ADCC). In one embodiment, the antibody may be
selected from the group consisting of Ributaxin, Herceptin,
Quadramet, Panorex, IDEC-Y2B8, BEC2, C225, Oncolym, SMART M195,
ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22,
OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2,
MELIMMUNE-1, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H,
GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior
egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART 1D10 Ab,
SMART ABL 364 Ab and ImmuRAIT-CEA.
[0047] Thus, according to some aspects of the invention, a subject
having cancer or at risk of having a cancer is administered a CpG
immunostimulatory oligonucleotide and an anti-cancer therapy. In
some embodiments, the anti-cancer therapy is selected from the
group consisting of a chemotherapeutic agent, an immunotherapeutic
agent and a cancer vaccine. In some embodiments the cancer
medicament is taxol or a combination of carboplatin and
paclitaxel.
[0048] In still another embodiment of the methods directed to
preventing or treating cancer, the subject may be further
administered interferon-.alpha..
[0049] The invention in other aspects relates to methods for
preventing disease in a subject. The method involves administering
to the subject a CpG immunostimulatory oligonucleotide on a regular
basis to promote immune system responsiveness to prevent disease in
the subject. Examples of diseases or conditions sought to be
prevented using the prophylactic methods of the invention include
microbial infections (e.g., sexually transmitted diseases) and
anaphylactic shock from food allergies.
[0050] In other aspects, the invention is a method for inducing an
innate immune response by administering to the subject a CpG
immunostimulatory oligonucleotide in an amount effective for
activating an innate immune response.
[0051] According to another aspect of the invention a method for
treating or preventing a viral or retroviral infection is provided.
The method involves administering to a subject having or at risk of
having a viral or retroviral infection, an effective amount for
treating or preventing the viral or retroviral infection of any of
the compositions of the invention. In some embodiments the virus is
caused by a hepatitis virus e.g., hepatitis B, hepatitis C, HIV,
herpes virus, or papillomavirus.
[0052] A method for treating or preventing a bacterial infection is
provided according to another aspect of the invention. The method
involves administering to a subject having or at risk of having a
bacterial infection, an effective amount for treating or preventing
the bacterial infection of any of the compositions of the
invention. In one embodiment the bacterial infection is due to an
intracellular bacteria.
[0053] In another aspect the invention is a method for treating or
preventing a parasite infection by administering to a subject
having or at risk of having a parasite infection, an effective
amount for treating or preventing the parasite infection of any of
the compositions of the invention. In one embodiment the parasite
infection is due to an intracellular parasite. In another
embodiment the parasite infection is due to a non-helminthic
parasite.
[0054] In some embodiments the subject is a human and in other
embodiments the subject is a non-human vertebrate selected from the
group consisting of a dog, cat, horse, cow, pig, turkey, goat,
fish, monkey, chicken, rat, mouse, and sheep.
[0055] In another aspect the invention relates to a method for
inducing a TH1 immune response by administering to a subject any of
the compositions of the invention in an effective amount to produce
a TH1 immune response.
[0056] In another aspect, the invention relates to a method for
treating autoimmune disease by administering to a subject having or
at risk of having an autoimmune disease an effective amount for
treating or preventing the autoimmune disease of any of the
compositions of the invention.
[0057] In other embodiments the oligonucleotide is delivered to the
subject in an effective amount to induce cytokine expression.
Optionally the cytokine is selected from the group consisting of
IL-6, TNF.alpha., IFN.alpha., IFN.gamma. and IP-10. In other
embodiments the oligonucleotide is delivered to the subject in an
effective amount to shift the immune response to a Th1 biased
response form a Th2 biased response.
[0058] The invention in some aspects is a method for treating
airway remodeling, comprising: administering to a subject an
oligonucleotide comprising a CG dinucleotide, in an effective
amount to treat airway remodeling in the subject. In one embodiment
the subject has asthma, chronic obstructive pulmonary disease, or
is a smoker. In other embodiments the subject is free of symptoms
of asthma.
[0059] Use of an oligonucleotide of the invention for stimulating
an immune response is also provided as an aspect of the
invention.
[0060] Use of an oligonucleotide of the invention in the
manufacture of a medicament of for stimulating an immune response
and performing any of the methods of the invention is also
provided.
[0061] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1 is a series of graphs depicting IFN-alpha induction
by human PBMC treated with CpG ODN.
[0063] FIG. 2 is a series of graphs depicting the effects of the
CpG oligodeoxynucleotide SEQ ID NO. 7 against antigen-induced
increase in nasal resistance in guinea pigs administered in a dose
of (A) 1 mg/kg and (B) (0.03-0.3 mg/kg). Results are mean.+-.s.e.m.
((A)=n=5-8; (B)=n=14-15). *P<0.05 compared with
antigen-challenged group treated with test vehicle (t test).
[0064] FIG. 3 is a series of graphs depicting the effects of the
CpG oligodeoxynucleotide SEQ ID NO. 7 on antigen-induced sneezing
(3A) and nasal rubbing (3B) in a mouse model of allergic rhinitis.
Results are mean.+-.s.e.m. (n=10). *P<0.05 compared with
vehicle-treated group (Mann-Whitney test).
[0065] FIG. 4 is a graph depicting a titration of influenza virus
and determination of time course of infection. Cell numbers in
bronchoalveolar lavage fluid. Results are mean.+-.s.e.m. (n=5).
Mice infected with 500 EID.sub.50 were sacrificed after 6 days
because of weight loss.
[0066] FIG. 5 is a graph depicting the protective effects of CpG
ODNs on virus load in the lung. Virus load assayed by enzyme
immunoassay. Results are mean.+-.s.e.m. (n=5-10). *P<0.05
compared with group B (Kruskal-Wallis test, Dunn's post-test).
n.d.=no data.
[0067] FIG. 6 is a series of graphs depicting the protective
effects of CpG ODNs on virus-induced airways inflammation measuring
total leukocytes (6A), total neutrophils (6B) and total mononuclear
cells (6C). Cell numbers in bronchoalveolar lavage fluid. Results
are mean.+-.s.e.m. (n=10). *P<0.05 compared with group B
(Kruskal-Wallis test, Dunn's post-test).
[0068] FIG. 7 is a series of graphs depicting the protective
effects of CpG ODNs on cell numbers in antigen-induced airways
inflammation in eosinophils (7A) and neutrophils (7B). Results are
mean.+-.s.e.m. (n=10-14). *P<0.05 compared with antigen
challenged, vehicle-treated group (Kruskal-Wallis multiple
comparison test, Dunn's post test).
[0069] FIG. 8 is a series of graphs depicting the protective
effects of CpG ODNs on CD3.sup.+ (8A) and CD3.sup.+CD4.sup.+ (8B)
cell numbers in antigen-induced airways inflammation. Results are
mean.+-.s.e.m. (n=8). *P<0.05 compared with antigen challenged,
vehicle-treated group (Kruskal-Wallis multiple comparison test,
Dunn's post test).
[0070] FIG. 9 is a series of graphs depicting cell numbers in
bronchoalveolar lavage fluid 8 hours after dosing with 0.1 mg/kg
ODN (9A); 15 hours after dosing with 0.1 mg/kg ODN (9B); 8 hours
after dosing with 1 mg/kg ODN (9C); and 15 hours after dosing with
1 mg/kg ODN (9D). Results are mean.+-.s.e.m (n=10)
[0071] FIG. 10 is a series of graphs depicting concentrations of
IFN alpha in bronchoalveolar lavage fluid 8 hours after dosing with
0.1 mg/kg ODN (10A); 15 hours after dosing with 0.1 mg/kg ODN
(10B); 8 hours after dosing with 1 mg/kg ODN (10C); and 15 hours
after dosing with 1 mg/kg ODN (10D).
[0072] FIG. 11 is a series of graphs depicting concentrations of
IFN gamma in bronchoalveolar lavage fluid 8 hours after dosing with
0.1 mg/kg ODN (110A); 15 hours after dosing with 0.1 mg/kg ODN
(11B); 8 hours after dosing with 1 mg/kg ODN (11C); and 15 hours
after dosing with 1 mg/kg ODN (11D).
[0073] FIG. 12 is a series of graphs depicting concentrations of
IP-10 in bronchoalveolar lavage fluid 8 hours after dosing with 0.1
mg/kg ODN (12A); 15 hours after dosing with 0.1 mg/kg ODN (12B); 8
hours after dosing with 1 mg/kg ODN (12C); and 15 hours after
dosing with 1 mg/kg ODN (12D).
[0074] FIG. 13 is a series of graphs depicting concentrations of
IL-12p40 in bronchoalveolar lavage fluid 8 hours after dosing with
0.1 mg/kg ODN (13A); 15 hours after dosing with 0.1 mg/kg ODN
(13B); 8 hours after dosing with 1 mg/kg ODN (13C); and 15 hours
after dosing with 1 mg/kg ODN (13D).
[0075] FIG. 14 is a series of graphs depicting concentrations of
IL-6 in bronchoalveolar lavage fluid 8 hours after dosing with 0.1
mg/kg ODN (14A); 15 hours after dosing with 0.1 mg/kg ODN (14B); 8
hours after dosing with 1 mg/kg ODN (14C); and 15 hours after
dosing with 1 mg/kg ODN (14D).
[0076] FIG. 15 is a series of graphs depicting concentrations of
TNFalpha in bronchoalveolar lavage fluid 8 hours after dosing with
0.1 mg/kg ODN (15A); 15 hours after dosing with 0.1 mg/kg ODN
(15B); 8 hours after dosing with 1 mg/kg ODN (15C); and 15 hours
after dosing with 1 mg/kg ODN (15D).
[0077] FIG. 16 is a series of graphs depicting concentrations of
IFN gamma in serum 8 hours after dosing with 0.1 mg/kg ODN (16A);
15 hours after dosing with 0.1 mg/kg ODN (16B); 8 hours after
dosing with 1 mg/kg ODN (16C); and 15 hours after dosing with 1
mg/kg ODN (16D).
[0078] FIG. 17 is a series of graphs depicting concentrations of
IL-6 in serum 8 hours after dosing with 0.1 mg/kg ODN (17A); 15
hours after dosing with 0.1 mg/kg ODN (17B); 8 hours after dosing
with 1 mg/kg ODN (17C); and 15 hours after dosing with 1 mg/kg ODN
(17D).
[0079] FIG. 18 is a series of graphs depicting concentrations of
TNF alpha in serum 8 hours after dosing with 0.1 mg/kg ODN (18A);
15 hours after dosing with 0.1 mg/kg ODN (18B); 8 hours after
dosing with 1 mg/kg ODN (18C); and 15 hours after dosing with 1
mg/kg ODN (18D).
[0080] FIG. 19 is a series of graphs depicting the effects of CpG
oligodeoxynucleotides ODN SEQ ID NO: 2 and ODN SEQ ID NO: 7 on
antigen-induced IgE (19A) and IgG2a (19B) production in the mouse.
Results are mean.+-.s.e.m. (n=10). *P<0.05 compared with antigen
sensitized, vehicle-treated group (Kruskal-Wallis test with Dunn's
post test).
[0081] FIG. 20 is a series of graphs depicting Effects of ODN SEQ
ID NO: 2 against influenza-induced exacerbation of allergic airway
inflammation in mice, depicting total leukocytes (20A), eosinophils
(20B), neutrophils (20C), mononuclear cells (20D), and body weight
(20E).
[0082] FIG. 21 is a guinea pig AHR Protocol used herein.
[0083] FIG. 22 is a set of graphs depicting the effect of SEQ ID
NO:7 on Airway resistance and lung compliance. For each animal, a
dose-response curve was obtained, and airway reactivity was
quantified as the area under the curve. Guinea pigs were given
intranasally either carrier (saline), OVA alone, or concentrations
of SEQ ID NO:7 of 10 .mu.l/kg, 30 .mu.l/kg, 100 .mu.l/kg, or 300
.mu.l/kg, i.t. The data demonstrate that SEQ ID NO:7 caused a
dose-dependent reduction in AUC-resistance. FIGS. 22A and 22B
depict histamine release as a function of increased airway
resistance (22A) or decrease in lung compliance (22B). FIGS. 22C
and 22D are bar graphs depicting the increase in airway resistance
(22C) or decrease in lung compliance (22D) in response to treatment
with saline, OVA or SEQ ID NO 7 at the indicated dosages.
[0084] FIG. 23 is a Summary of Statistical Analysis used herein
(FIG. 22) for airway resistance (23A) and lung compliance
(23B).
[0085] FIG. 24 is a set of graphs depicting the effect of SEQ ID
NO:2 on Airway resistance and lung compliance. For each animal, a
dose-response curve was obtained, and airway reactivity was
quantified as the area under the curve. Guinea pigs were given
intranasally either carrier (saline), OVA alone, or concentrations
of SEQ ID NO:2 of 10 .mu.l/kg, 30 .mu.l/kg, 100 .mu.l/kg, or 300
.mu.l/kg, i.t. The data demonstrate that SEQ ID NO:2 caused a
dose-dependent reduction in AUC-resistance. FIGS. 24A and 24B
depict histamine release as a function of increased airway
resistance (24A) or decrease in lung compliance (24B). FIGS. 24C
and 24D are bar graphs depicting the increase in airway resistance
(24C) or decrease in lung compliance (24D) in response to treatment
with saline, OVA or SEQ ID NO 2 at the indicated dosages.
[0086] FIG. 25 is a Summary of Statistical Analysis used herein
(FIG. 24) for airway resistance (25A) and lung compliance (25B)
[0087] FIG. 26 is a summary of the graphs in FIGS. 22 and 24. FIGS.
26A and 26B correspond to FIGS. 22C and 22D. FIGS. 26C and 26D
correspond to FIGS. 24C and 24D.
[0088] FIG. 27 is a set of graphs depicting levels of IL-10 (pg/ml)
secreted from human PBMC (3 donors) following exposure of these
cells to the oligonucleotides listed by SEQ ID No. along the bottom
X-axis of the graph (data points from the 3 donors are depicted by
a .tangle-solidup., .box-solid. and x) for 48 hours. The test
oligonucleotides shown in FIG. 27 include SEQ ID NOs: 10, 9, 13,
14, 1, and 2. The concentration of oligonucleotide used to produce
a particular data point is depicted along the X-axis (.mu.M).
Supernatants were harvested and IL-10 measured by ELISA. Given are
the mean cytokine amounts of all donors.
[0089] FIG. 28 is a set of graphs depicting levels of TNF-alpha
(28A), interferon-gamma (28B), and IL-6 (28C) (.mu.M) secreted from
human PBMC following exposure of these cells to the
oligonucleotides listed by SEQ ID NO. in the key of the graph. Each
data point is the calculated mean cytokine value of three donors.
The PBMC were incubated with the indicated ODN concentrations.
Supernatants were harvested and cytokines measured by ELISA. Given
are the calculated mean cytokine amounts of all donors.
[0090] FIG. 29 is a set of graphs depicting levels of TNF-alpha
(pg/ml) secreted from human PBMC following exposure of these cells
to the oligonucleotides for 16 hours listed by the SEQ ID NO along
the bottom X-axis of the graph. The oligonucleotides shown in FIG.
29 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ to ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
Supernatants were harvested and IL-6 measured by ELISA. Given are
the mean cytokine amounts of all donors.
[0091] FIG. 30 is a set of graphs depicting levels of IL-6 (pg/ml)
secreted from human PBMC following exposure of these cells to the
oligonucleotides for 24 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 30
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated
CpG.
[0092] FIG. 31 is a set of graphs depicting levels of IFN-gamma
(pg/ml) secreted from human PBMC following exposure of these cells
to the oligonucleotides for 48 hours listed by the SEQ ID NO along
the bottom X-axis of the graph. The oligonucleotides shown in FIG.
31 include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
Supernatants were harvested and IFN-gamma measured by ELISA. Given
are the mean cytokine amounts of all donors.
[0093] FIG. 32 is a set of graphs depicting levels of CD69
expression (MFI) on NK cells as an indicator of NK cell activation
(32A) and CD80 (32B) and CD86 (32C) expression on B cells following
exposure of these cells to the oligonucleotides for 24 or 48 hours
listed by SEQ ID NO. in the key of the graph. Each data point is
the mean fluorescence intensity of three donors. The cells were
incubated with the indicated ODN concentrations for 24, or 48
hours. Cells were stained and analyzed by flow cytometry.
[0094] FIG. 33 is a set of graphs depicting levels of levels of
CD69 expression on NK cells as an indicator of NK cell activation
following exposure of these cells to the oligonucleotides for 24
hours listed by the SEQ ID NO along the bottom X-axis of the to
graph. The oligonucleotides shown in FIG. 33 include SEQ ID NO: 9,
13, 10, 14, 2, 1, 11, 12, and 3. The concentration of
oligonucleotide used to produce a particular data point is depicted
along the X-axis (.mu.M). The data shown represents the values of
three donors. Below the SEQ ID NOs is a designation referring to
the class of ODN. Css=C-class semi soft, C=C-class, B=B-class,
non-CpG=an ODN without an unmethylated CpG. The cells were stained
with antibodies to CD3, CD56, and CD69 and analyzed by flow
cytometry. The data presented is the mean fluorescence
intensity.
[0095] FIG. 34 is a set of graphs depicting CD86 expression on
human PBMC following exposure of these cells to the
oligonucleotides for 48 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 34
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD86, CD80, CD19, and
CD14 and analyzed by flow cytometry. The data presented is the mean
fluorescence intensity.
[0096] FIG. 35 is a set of graphs depicting CD86 expression on
human PBMC following exposure of these cells to the
oligonucleotides for 48 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 35
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD86, CD80, CD19, and
CD14 and analyzed by flow cytometry. The data presented is the mean
fluorescence intensity.
[0097] FIG. 36 is a set of graphs depicting levels of CD86
expression on plasmacytoid dendritic cells (36A) and CD80 (36B) and
CD86 (36C) expression on monocytes following exposure of these
cells to the oligonucleotides listed by SEQ ID NO. in the key of
the graph. Each data point is the a calculated mean fluorescence
intensity of three donors. The cells were incubated with the
indicated ODN concentrations for 48 hours. Cells were stained and
analyzed by flow cytometry.
[0098] FIG. 37 is a set of graphs depicting levels of CD86
expression on monocytes following exposure of the cells to the
oligonucleotides for 48 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 37
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD86, CD80, CD19, and
CD14 and analyzed by flow cytometry. The data presented is the mean
fluorescence intensity.
[0099] FIG. 38 is a set of graphs depicting CD80 expression on
monocytes following exposure of these cells to the oligonucleotides
for 48 hours listed by the SEQ ID NO along the bottom X-axis of the
graph. The oligonucleotides shown in FIG. 38 include SEQ ID NO: 9,
13, 10, 14, 2, 1, 11, 12, and 3. The concentration of
oligonucleotide used to produce a particular data point is depicted
along the X-axis (.mu.M). The data shown represents the values of
three donors. Below the SEQ ID NOs is a designation referring to
the class of ODN. Css=C-class semi soft, C=C-class, B=B-class,
non-CpG=an ODN without an unmethylated CpG. The cells were stained
with antibodies to CD86, CD80, CD19, and CD14 and analyzed by flow
cytometry. The data presented is the mean fluorescence
intensity.
[0100] FIG. 39 is a set of graphs depicting CD80 expression on
plasmacytoid dendritic cells following exposure of these cells to
the oligonucleotides for 48 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 37
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD86, CD11c, CD123, and
HLA-DR and analyzed by flow cytometry. The data presented is the
mean fluorescence intensity.
[0101] FIG. 40 is a set of graphs depicting levels of expression of
intracellular IP-10 in B cells (40B) and monocytes (40A) following
exposure of these cells to the oligonucleotides for 24 hours listed
by SEQ ID NO. in the key of the graph. Each data point is the a
calculated mean fluorescence intensity of three donors. The cells
were incubated with the indicated ODN concentrations for 24 hours.
Cells were stained and analyzed by flow cytometry.
[0102] FIG. 41 is a set of graphs depicting levels of expression of
intracellular IP-10 in monocytes following exposure of the cells to
the oligonucleotides for 24 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 41
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD14, CD19, and IP-10 and
analyzed by flow cytometry. The data presented is the mean
fluorescence intensity.
[0103] FIG. 42 is a set of graphs depicting levels of expression of
intracellular IP-10 in B cells following exposure of these cells to
the oligonucleotides for 24 hours listed by the SEQ ID NO along the
bottom X-axis of the graph. The oligonucleotides shown in FIG. 42
include SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and 3. The
concentration of oligonucleotide used to produce a particular data
point is depicted along the X-axis (.mu.M). The data shown
represents the values of three donors. Below the SEQ ID NOs is a
designation referring to the class of ODN. Css=C-class semi soft,
C=C-class, B=B-class, non-CpG=an ODN without an unmethylated CpG.
The cells were stained with antibodies to CD14, CD19, and IP-10 and
analyzed by flow cytometry. The data presented is the mean
fluorescence intensity.
[0104] FIG. 43 is a set of graphs depicting a comparison of the
abilities of SEQ ID NO.: 2 and fragments thereof (SEQ ID NO 15-17)
to induce cytokine secretion from mouse splenocytes. The cytokines
analyzed include IFN.alpha. (43A), IFN.gamma. (43B), IP-10 (43C),
IL-6 (43D), IL-10 (43E), and TNF.alpha. (43F).
[0105] FIG. 44 is a set of graphs depicting a comparison of the
abilities of SEQ ID NO.: 2 and fragments thereof (SEQ ID NO 18-20)
to induce cytokine secretion from mouse splenocytes. The cytokines
analyzed include IFN.alpha. (44A), IFN.gamma. (44B), IP-10 (44C),
IL-6 (44D), IL-10 (44E), and TNF.alpha. (44F).
[0106] FIG. 45 is a set of graphs depicting a comparison of the
abilities of SEQ ID NO.: 2 and fragments thereof (SEQ ID NO 21-23)
to induce cytokine secretion from mouse splenocytes. The cytokines
analyzed include IFN.alpha. (45A), IFN.gamma. (45B), IP-10 (45C),
IL-6 (45D), IL-10 (45E), and TNF.alpha. (45F).
DETAILED DESCRIPTION
[0107] A sub-set of C-class semi-soft immunostimulatory
oligonucleotides are provided according to the invention. The
immunostimulatory oligonucleotides of the invention described
herein, in some embodiments have improved properties including
similar or enhanced potency, reduced systemic exposure to the
kidney, liver and spleen, and may have reduced reactogenicity at
injection sites. Although applicant is not bound by a mechanism, it
is believed that these improved properties are associated with the
strategic placement within the immunostimulatory oligonucleotides
of phosphodiester or phosphodiester-like "internucleotide
linkages". The term "internucleotide linkage" as used herein refers
to the covalent backbone linkage joining two adjacent nucleotides
in a nucleic acid molecule. The covalent backbone linkage will
typically be a modified or unmodified phosphate linkage, but other
modifications are possible. Thus a linear oligonucleotide that is n
nucleotides long has a total of n-1 internucleotide linkages. These
covalent backbone linkages can be modified or unmodified in the
immunostimulatory oligonucleotides according to the teachings of
the invention.
[0108] 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.
[0109] The invention is based at least in some aspects on the
surprising discovery that the specific C-class semi-soft
oligonucleotides described herein have important immunostimulatory
activity and are preferably useful in the treatment of allergy and
asthma. These molecules have at least the same or in many cases
possess greater immunostimulatory activity, in many instances, than
corresponding fully stabilized immunostimulatory oligonucleotides
having the same nucleotide sequence.
[0110] 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,
preferably CG) 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 at lower effective concentations and
have lower effective doses than conventional fully stabilized
immunostimulatory oligonucleotides in order to achieve a desired
biological effect.
[0111] Whereas fully stabilized immunostimulatory oligonucleotides
can exhibit dose-response maxima, semi-soft oligonucleotides of the
instant invention appear to have monotonically increasing
dose-response curves (as assayed by TLR9 stimulation) extending
into higher concentrations beyond the optimal concentration for
corresponding fully stabilized immunostimulatory oligonucleotides.
Thus it is believed that semi-soft oligonuncleotides of the instant
invention may induce greater immunostimulation than fully
stabilized immunostimulatory oligonucleotides.
[0112] Whereas fully stabilized immunostimulatory oligonucleotides
less than 20 nucleotides long can have modest immunostimulatory
activity compared with longer (e.g., 24 nucleotides long) fully
stabilized oligonucleotides, semi-soft oligonucleotides as short as
16 nucleotides long have been discovered to have immunostimulatory
activity at least equal to immunostimulatory activity of fully
stabilized oligonucleotides over 20 nucleotides long.
[0113] In some instances where a 6-mer phosphorothioate
oligonucleotide appeared to lack immunostimulatory activity,
substitution of even one phosphodiester internal CG internucleotide
linkage for a phosphorothioate linkage was found to yield a
corresponding 6-mer with immunostimulatory activity.
[0114] Thus the size (i.e., the number of nucleotide residues along
the length of the oligonucleotide) of the immunostimulatory
oligonucleotide may also contribute to the stimulatory activity of
the oligonucleotide. For facilitating uptake into cells
immunostimulatory oligonucleotides may have a minimum length of 6
nucleotide residues. Oligonucleotides 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 or 10 and 40 nucleotides
long. In certain preferred embodiments according to the instant
invention, the immunostimulatory oligonucleotides are between 6 and
19 or 6 and 24 nucleotides long.
[0115] It is also believed that the foregoing properties of
semi-soft oligonucleotides generally increase with increasing
"dose" of phosphodiester or phosphodiester-like internucleotide
linkages involving internal CG dinucleotides. Thus it is believed,
for example, that generally for a given oligonucleotide sequence
with five internal CG dinucleotides, an oligonucleotide with five
internal phosphodiester or phosphodiester-like CG internucleotide
linkages is more immunostimulatory than an oligonucleotide with
four internal phosphodiester or phosphodiester-like CG
internucleotide linkages, which in turn is more immunostimulatory
than an oligonucleotide with three internal phosphodiester or
phosphodiester-like CG internucleotide linkages, which in turn is
more immunostimulatory than an oligonucleotide with two internal
phosphodiester or phosphodiester-like CG internucleotide linkages,
which in turn is more immunostimulatory than an oligonucleotide
with one internal phosphodiester or phosphodiester-like CG
internucleotide linkage. Importantly, inclusion of even one
internal phosphodiester or phosphodiester-like CG internucleotide
linkage is believed to be advantageous over no internal
phosphodiester or phosphodiester-like CG internucleotide linkage.
In addition to the number of phosphodiester or phosphodiester-like
internucleotide linkages, the position along the length of the
oligonucleotide can also affect potency.
[0116] The immunostimulatory oligonucleotides of the present
invention are generally protected from rapid degradation in the
serum. The immunostimulatory oligonucleotides of the present
invention are also generally protected from rapid degradation in
most tissues, with the exception of particular tissues with
specific or excessive nuclease activity that are capable of
degrading the immunostimulatory oligonucleotides. This results in
the reduction of immunostimulatory oligonucleotides in those
particular tissues, the accumulation of which could otherwise lead
to undesirable effects from long-term therapy utilizing
degradation-resistant oligonucleotides. The oligonucleotides of the
instant invention 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. Yet other
stabilized ends, including but not limited to those described
further below, are meant to be encompassed by the invention.
[0117] As described above, the oligonucleotides of the instant
invention include phosphodiester or phosphodiester-like linkages
within and optionally adjacent to internal CG dinucleotides. Such
CG dinucleotides are frequently part of immunostimulatory to
motifs. It is not necessary, however, that an oligonucleotide
contain phosphodiester or phosphodiester-like linkages within every
immunostimulatory motif. Additional phosphodiester or
phosphodiester-like linkages may also be maintained for even more
rapid renal digestion of these otherwise "stabilized
oligonucleotides".
[0118] A phosphodiester internucleotide linkage is the type of
linkage characteristic of nucleic acids found in nature. 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.
[0119] 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.
Pate. 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. 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.
[0120] The immunostimulatory oligonucleotide molecules of the
instant invention have 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 to 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.
[0121] 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.
[0122] 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 to 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.
[0123] The oligonucleotides 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. Oligonucleotides which contain diol, such as
tetraethyleneglycol or hexaethyleneglycol, at either or both
termini have also been shown to be substantially resistant to
nuclease degradation.
[0124] The oligonucleotides of the present invention are nucleic
acids that contain specific sequences found to elicit an immune
response. These specific sequences that elicit an immune response
are referred to as "immunostimulatory motifs", and the
oligonucleotides that contain immunostimulatory motifs are referred
to as "immunostimulatory nucleic acid molecules" and, equivalently,
"immunostimulatory nucleic acids" or "immunostimulatory
oligonucleotides". The immunostimulatory oligonucleotides of the
invention thus include at least one immunostimulatory motif. In a
preferred embodiment the immunostimulatory motif is an "internal
immunostimulatory motif". The term "internal immunostimulatory
motif" refers to the position of the motif sequence within a longer
nucleic acid sequence, which is longer in length than the motif
sequence by at least one nucleotide linked to both the 5' and 3'
ends of the immunostimulatory motif sequence.
[0125] The immunostimulatory oligonucleotides include
immunostimulatory motifs which are "CpG dinucleotides". A CpG
dinucleotide can be methylated or unmethylated. An
immunostimulatory oligonucleotide 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 oligonucleotide is a CpG oligonucleotide.
CpG oligonucleotides 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
oligonucleotide containing at least one methylated CpG dinucleotide
is an oligonucleotide 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.
[0126] It has recently been described that there are different
classes of CpG oligonucleotides. 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
oligonucleotides 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 oligonucleotides typically have
stabilized poly-G sequences at 5' and 3' ends and a central
palindromic phosphodiester CpG dinucleotide-containing sequence of
at least 6 nucleotides. See, for example, published patent
application PCT/US00/26527 (WO 01/22990).
[0127] Yet another class of CpG oligonucleotides activates B cells
and NK cells and induces IFN-.alpha.; this class has been termed
the C-class. The C-class CpG oligonucleotides, 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 co-pending U.S. patent application
Ser. No. 10/224,523, filed Aug. 19, 2002 and published as
US2003/0148976 and US 10/978,283 filed on Oct. 29, 2004, with a
related PCT application published as WO2005/042018 the entire
contents of which are incorporated herein by reference.
[0128] C-class oligonucleotides are also referred to as type C CpG
ODN. In certain embodiments the C CpG ODN involve a combination of
motifs wherein one motif is a CG-rich palindrome or a neutralizing
motif, and another motif is a stimulatory motif, e.g., a CpG motif
or the sequence TCGTCG.
[0129] The C CpG ODN may have the formula: 5' X.sub.1DCGHX.sub.2
3'. X.sub.1 and X.sub.2 are independently any sequence 0 to 10
nucleotides long. D is a nucleotide other than C. C is cytosine. G
is guanine. H is a nucleotide other than G. The nucleic acid
sequence also includes a nucleic acid sequence selected from the
group consisting of P and N positioned immediately 5' to X.sub.1 or
immediately 3' to X.sub.2 N is a B-cell neutralizing sequence which
begins with a CGG trinucleotide and is at least 10 nucleotides
long. P is a GC-rich palindrome containing sequence at least 10
nucleotides long.
[0130] In some embodiments the immunostimulatory nucleic acid is 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', or 5'DCGHPX.sub.3
3'. X.sub.3 is any sequence 0 to 10 nucleotides long. In other
embodiments the immunostimulatory nucleic acid is 5' DCGHP 3'.
[0131] Optionally D and/or H are thymine (T).
[0132] In other embodiments H is T and X.sub.2 is CG, CGT, CGTT,
CGTTT, or CGTTTT.
[0133] H is T and X.sub.2 is CG or CGTTTT according to other
embodiments.
[0134] According to other embodiments C is unmethylated.
[0135] N includes at least four CG dinucleotides and no more than
two CCG trinucleotides in some embodiments.
[0136] Optionally P includes at least one Inosine.
[0137] The nucleic acid may also include a poly-T sequence at the
5' end or the 3' end.
[0138] Alternatively the C CpG ODN may have the formula: 5'
N.sub.1PyGN.sub.2P 3'. G is guanine. N.sub.1 is any sequence 1 to 6
nucleotides long. In some embodiments N.sub.1 is at least 50%
pyrimidines and preferably at least 50% T. In other embodiments
N.sub.1 includes at least one CG motif, at least one TCG motif, at
least one CI motif, at least one TCI motif, at least one IG motif,
or at least one TIG motif. N.sub.1 is TCGG or TCGH in other
embodiments. H is a nucleotide other than G.
[0139] Py is a pyrimidine. In some embodiments Py is an
unmethylated C.
[0140] N.sub.2 is any sequence 0 to 30 nucleotides long. In some
embodiments N.sub.2 is at least 50% pyrimidines or is at least 50%
T. In other embodiments N.sub.2 does not includes any poly G or
poly A motifs.
[0141] P is a GC-rich palindrome containing sequence at least 10
nucleotides long. In some embodiments P is completely palindromic.
In other embodiments P is a palindrome having between 1 and 3
consecutive intervening nucleotides. Optionally the intervening
nucleotides may be TG. In other embodiments P includes at least 3,
4, or 5 C and at least 3, 4, or 5 G nucleotides. According to other
embodiments P includes at least one Inosine.
[0142] In one embodiment the GC-rich palindrome has a base content
of at least two-thirds G and C. In another embodiment the GC-rich
palindrome has a base content of at least 81 percent G and C. In
some embodiments the GC-rich palindrome is at least 12 nucleotides
long. The GC-rich palindrome may be made up exclusively of C and G.
In some embodiments the GC-rich palindrome can include at least one
nucleotide that is neither C nor G.
[0143] 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 some embodiments the GC-rich palindrome includes at
least four CG dinucleotides. In certain preferred embodiments the
GC-rich palindrome has a central CG dinucleotide.
[0144] In certain embodiments the GC-rich palindrome is
CGGCGCGCGCCG (SEQ ID NO: 58), CGGCGGCCGCCG (SEQ ID NO: 59),
CGACGATCGTCG (SEQ ID NO: 60) or CGACGTACGTCG (SEQ ID NO: 61).
[0145] In certain embodiments the GC-rich palindrome is
CGCGCGCGCGCG (SEQ ID NO:62), GCGCGCGCGCGC (SEQ ID NO: 63),
CCCCCCGGGGGG (SEQ ID NO: 64), GGGGGGCCCCCC (SEQ ID NO: 65),
CCCCCGGGGG (SEQ ID NO: 66) or GGGGGCCCCC (SEQ ID NO: 67).
[0146] In some 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.
[0147] An immunostimulatory nucleic acid of 13-100 nucleotides in
length is provided according to other aspects of the invention. The
nucleic acid has the formula: 5' N.sub.1PyG/IN.sub.2P 3'. G/I
refers to single nucleotide which is either a G or an I. G is
guanine and I is Inosine.
[0148] N.sub.1 is any sequence 1 to 6 nucleotides long. Py is a
pyrimidine. N.sub.2 is any sequence 0 to 30 nucleotides long.
[0149] P is a palindrome containing sequence at least 10
nucleotides long. In some embodiments P is a GC-rich palindrome. In
other embodiments P is an IC-rich palindrome.
[0150] N.sub.1PyIN.sub.2 in some embodiments is TCITCITTTT (SEQ ID
NO: 62).
[0151] A class of oligonucleotides referred to herein as modified
C-class oligonucleotides are characteristically monomeric in
solution. It is believed that these nucleic acid molecules can form
intramolecular duplex structures in vitro, rendering them stable
against nuclease digestion. It is also believed that these same
nucleic acid molecules can form intermolecular duplex and possibly
even higher order structures within the environment of the
intraendosomal compartment, where they are believed to exert their
biological activity.
[0152] Modified C-class oligonucleotides have 3 general formulas.
Formula I
Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N (X.sub.2Y.sub.2R.sub.2).sub.k
Z.sub.2].sub.p (S.sub.1).sub.q N' (N.sub.n) . . .
(N.sub.2)(N.sub.1) S.sub.2 (N.sub.1#)(N.sub.2#) . . . (N.sub.n#)
Z.sub.3 (Formula I)
wherein each of Z.sub.1, Z.sub.2, and Z.sub.3 is independently any
sequence 0 to 12 nucleotides long which optionally includes a
non-nucleotidic linker or abasic dSpacer; each of X.sub.1 and
X.sub.2 is independently a thymidine, deoxyuridine, deoxyadenosine
or a 5-substituted deoxyuridine; each of Y.sub.1 and Y.sub.2 is
independently a cytosine (C) or a modified cytosine; each of
R.sub.1 and R.sub.2 is independently a guanine (G) or a modified
guanine; each of N and N' is independently any sequence 0 to 12
nucleotides long which optionally includes a non-nucleotidic linker
or abasic dSpacer; S.sub.1 is a non-nucleotidic linker, an abasic
linker (dSpacers), triethylene glycol units or hexaethylene glycol
units, which optionally provides for 2'5'-, 5'5'-, 3'3'-, 2'2'-, or
2'3'-internucleoside linkages; S.sub.2 is any non-palindromic
sequence 1 to 10 nucleotides long or a non-nucleotidic linker, an
abasic linker (dSpacers), triethylene glycol units or hexaethylene
glycol units; each of N.sub.1, N.sub.2, . . . N.sub.n, and
N.sub.1#, N.sub.2#, . . . N.sub.n# is any nucleotide or modified
nucleotide wherein N.sub.1 base-pairs with N.sub.1#, N.sub.2
base-pairs with N.sub.2#, . . . and N.sub.n base-pairs with
N.sub.n#; k is an integer from 0 to 5; n is an integer from 2 to
16; p is an integer from 1 to 6; and q is an integer from 0 to 10,
and wherein when (N.sub.n) . . . (N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#) . . . (N.sub.n#) is 10 to 42 nucleotides long,
S.sub.2 is 4 to 10 nucleotides long, S.sub.2 comprises a
non-nucleotidic linker, an abasic linker (dSpacers), triethylene
glycol units or hexaethylene glycol units, and/or (N.sub.n) . . .
(N.sub.2)(N.sub.1) S.sub.2 (N.sub.1#)(N.sub.2#) . . . (N.sub.n#)
has a GC content that is less than 2/3.
[0153] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n# is chosen from C, G, or
modifications thereof, wherein C base-pairs with G.
[0154] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n# is chosen from T, A, or
modifications thereof, and T base-pairs with A.
[0155] In these and other embodiments each of C, G, A, and T can
refer to deoxynucleotides with corresponding bases cytosine,
guanine, adenine, and thymine.
[0156] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n# is chosen from C, T, A, G,
or modifications thereof, and C base-pairs with G, T base-pairs
with G, A base-pairs with T, and A base-pairs with G.
[0157] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n# is chosen from unmodified or
modified nucleotides which form Watson-Crick basepairs.
[0158] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n# is chosen from unmodified or
modified nucleotides which form non-Watson-Crick basepairs.
[0159] In one embodiment the immunostimulatory nucleic acid
molecule includes a partially stabilized backbone with at least one
phosphodiester bond.
[0160] In one embodiment the immunostimulatory nucleic acid
molecule includes a backbone with at least one stabilized
internucleotide linkage.
[0161] In one embodiment internucleotide linkages of the
oligonucleotide are all phosphorothioate linkages.
[0162] In one embodiment the immunostimulatory nucleic acid
molecule includes a partially stabilized backbone with a
phosphodiester bond joining at least one of Y.sub.1R.sub.1 or
Y.sub.2R.sub.2.
[0163] In one embodiment Y.sub.1 is C.
[0164] In one embodiment R.sub.1 is G.
[0165] In one embodiment Y.sub.1 is C and R.sub.1 is G.
[0166] In one embodiment X.sub.1 or X.sub.2 is T.
[0167] In one embodiment X.sub.1 is T, X.sub.2 is T, Y.sub.1 is C,
R.sub.1 is G, and k is 1.
[0168] In one embodiment X.sub.1 is T, X.sub.2 is T, Y.sub.1 is C,
R.sub.1 is G, k is 1, p is 1, N and N' and Z.sub.3 each contain
zero nucleotides, and Z.sub.2 is TTTT or d(UUUU).
[0169] In one embodiment S.sub.2 is a non-nucleotidic linker.
[0170] In one embodiment S.sub.2 contains at least one abasic
dSpacer residue.
[0171] In one embodiment the oligonucleotide includes at least one
branched non-nucleoside linkage.
[0172] In one embodiment the immunostimulatory nucleic acid
molecule includes at least one doubler unit, at least one trebler
unit, or at least one doubler unit and at least one trebler
unit.
[0173] In one embodiment S.sub.1 is a doubler unit or a trebler
unit.
[0174] In one embodiment the oligonucleotide includes at least one
2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'-internucleoside linkage.
[0175] In one aspect the invention provides an immunostimulatory
nucleic acid molecule of Formula II
Z.sub.1 (N.sub.1)(N.sub.n-1) . . . (N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#) . . . (N.sub.n-1#)(N.sub.n#) (S.sub.1).sub.1
Z.sub.3 [(X.sub.1Y.sub.R.sub.1) N (X.sub.2Y.sub.2R.sub.2).sub.k
Z.sub.2].sub.p (Formula II)
wherein each of Z.sub.1, Z.sub.2, and Z.sub.3 is independently any
sequence 0 to 12 nucleotides long which optionally includes a
non-nucleotidic linker or abasic dSpacer; each of X.sub.1 and
X.sub.2 is independently a thymidine, deoxyuridine, deoxyadenosine
or a 5-substituted deoxyuridine; each of Y.sub.1 and Y.sub.2 is
independently a cytosine (C) or a modified cytosine; each of
R.sub.1 and R.sub.2 is independently a guanine (G) or a modified
guanine; N is any sequence 0 to 12 nucleotides long which
optionally includes a non-nucleotidic linker or abasic dSpacer;
S.sub.1 is a non-nucleotidic linker, an abasic linker (dSpacers),
triethylene glycol units or hexaethylene glycol units, which
optionally provides for 2'5'-, 5'5'-, 3'3'-, 2'2'-, or
2'3'-internucleoside linkages; S.sub.2 is any non-palindromic
sequence 1 to 10 nucleotides long or a non-nucleotidic linker, an
abasic linker (dSpacers), triethylene glycol units or hexaethylene
glycol units; each of N.sub.1, N.sub.2, . . . N.sub.n-1, N.sub.n,
and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is any
nucleotide or modified nucleotide wherein N.sub.1 base-pairs with
N.sub.1#, N.sub.2 base-pairs with N.sub.2#, . . . N.sub.n-1
base-pairs with N.sub.n-1#, and N.sub.n base-pairs with N.sub.n#; k
is an integer from 0 to 5; n is an integer from 2 to 16; p is an
integer from 1 to 6; and q is an integer from 0 to 10, and wherein
when (N.sub.n) . . . (N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#) . . . (N.sub.n#) is 10 to 42 nucleotides long,
S.sub.2 is 4 to 10 nucleotides long, S.sub.2 comprises a
non-nucleotidic linker, an abasic linker (dSpacers), triethylene
glycol units or hexaethylene glycol units, and/or (N.sub.n) . . .
(N.sub.2)(N.sub.1) S.sub.2 (N.sub.1#)(N.sub.2#) . . . (N.sub.n#)
has a GC content that is less than 2/3.
[0176] In one embodiment Z.sub.1 (N.sub.n)(N.sub.n-1) is TYR, where
Y is a cytosine or a modified cytosine and R is a guanine or a
modified guanine.
[0177] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n-1,
N.sub.n, and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is
chosen from C, G, or modifications thereof, wherein C base-pairs
with G.
[0178] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n-1,
N.sub.n, and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is
chosen from T, A, or modifications thereof, and T base-pairs with
A.
[0179] In these and other embodiments each of C, G, A, and T can
refer to deoxynucleotides with corresponding bases cytosine,
guanine, adenine, and thymine.
[0180] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n-1,
N.sub.n, and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is
chosen from C, T, A, G, or modifications thereof, and C base-pairs
with G, T base-pairs with G, A base-pairs with T, and A base-pairs
with G.
[0181] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n-1,
N.sub.n, and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is
chosen from unmodified or modified nucleotides which form
Watson-Crick basepairs.
[0182] In one embodiment each of N.sub.1, N.sub.2, . . . N.sub.n-1,
N.sub.n, and N.sub.1#, N.sub.2#, . . . N.sub.n-1#, N.sub.n# is
chosen from unmodified or modified nucleotides which form
non-Watson-Crick basepairs.
[0183] In one embodiment the immunostimulatory nucleic acid
molecule includes a partially stabilized backbone with at least one
phosphodiester bond.
[0184] In one embodiment the immunostimulatory nucleic acid
molecule includes a backbone with at least one stabilized
internucleotide linkage.
[0185] In one embodiment internucleotide linkages of the
oligonucleotide are all phosphorothioate linkages.
[0186] In one embodiment the immunostimulatory nucleic acid
molecule includes a partially stabilized backbone with a
phosphodiester bond joining at least one of Y.sub.1R.sub.1 or
Y.sub.2R.sub.2.
[0187] In one embodiment Y.sub.1 is C.
[0188] In one embodiment R.sub.1 is G.
[0189] In one embodiment Y.sub.1 is C and R.sub.1 is G.
[0190] In one embodiment X.sub.1 or X.sub.2 is T.
[0191] In one embodiment X.sub.1 is T, X.sub.2 is T, Y.sub.1 is C,
R.sub.1 is G, and k is 1.
[0192] In one embodiment X.sub.1 is T, X.sub.2 is T, Y.sub.1 is C,
R.sub.1 is G, k is 1, p is 1, N and N' and Z.sub.3 each contain
zero nucleotides, and Z.sub.2 is TTTT or d(UUUU).
[0193] In one embodiment S.sub.2 is a non-nucleotidic linker.
[0194] In one embodiment S.sub.2 contains at least one abasic
dSpacer residue.
[0195] In one embodiment the oligonucleotide includes at least one
branched non-nucleoside linkage.
[0196] In one embodiment the immunostimulatory nucleic acid
molecule includes at least one doubler unit, at least one trebler
unit, or at least one doubler unit and at least one trebler
unit.
[0197] In one embodiment S.sub.1 is a doubler unit or a trebler
unit.
[0198] In one embodiment the oligonucleotide includes at least one
2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'-internucleoside linkage.
[0199] In one aspect the invention provides an immunostimulatory
nucleic acid molecule of Formula III
(U).sub.m Z.sub.3 (S.sub.3) (Formula III)
wherein U is Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1).sub.q N'
(N.sub.n) . . . (N.sub.3)(N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#)(N.sub.3#) . . . (N.sub.n#); each of Z.sub.1,
Z.sub.2, and Z.sub.3 is independently any sequence 0 to 12
nucleotides long which optionally includes a non-nucleotidic linker
or abasic dSpacer; each of X.sub.1 and X.sub.2 is independently a
thymidine, deoxyuridine, deoxyadenosine or a 5-substituted
deoxyuridine; each of Y.sub.1 and Y.sub.2 is independently a
cytosine or a modified cytosine; each of R.sub.1 and R.sub.2 is
independently a guanine or a modified guanine; each of N and N' is
independently any sequence 0 to 12 nucleotides long which
optionally includes a non-nucleotidic linker or abasic dSpacer;
S.sub.1 is a non-nucleotidic linker, an abasic linker (dSpacers),
triethylene glycol units or hexaethylene glycol units, which
optionally provides for 2'5'-, 5'5'-, 3'3'-, 2'2'-, or
2'3'-internucleoside linkages; S.sub.2 is any non-palindromic
sequence 1 to 10 nucleotides long or a non-nucleotidic linker, an
abasic linker (dSpacers), triethylene glycol units or hexaethylene
glycol units; S.sub.3 is a direct or indirect 2'5'-, 5'5'-, 3'3'-,
2'2'-, or 2'3'-internucleoside linkage or a non-nucleotidic linker,
said non-nucleotidic linker including abasic linkers (dSpacers),
triethylene glycol units, or hexaethylene glycol units facilitating
a 2'5'-, 5'5'-, 3'3'-, 2'2'-, or 2'3'-linkage of m sequence parts;
each of N.sub.1, N.sub.2, . . . N.sub.n, and N.sub.1#, N.sub.2#, .
. . N.sub.n# is any nucleotide or modified nucleotide wherein
N.sub.1 base-pairs with N.sub.1#, N.sub.2 base-pairs with N.sub.2#,
N.sub.3 base-pairs with N.sub.3#, . . . and N.sub.n base-pairs with
N.sub.n#; k is an integer from 0 to 5; m is an integer from 2 to
10; n is an integer from 2 to 16; p is an integer from 1 to 6; and
q is an integer from 0 to 10.
[0200] In certain embodiments Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1).sub.q is a
non-palindromic sequence.
[0201] In certain embodiments Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1).sub.q is
TCGTCGTTTT (SEQ ID NO:29), TCGTCGTTDD (SEQ ID NO:30), TCGA, TCGAC,
TCGACGTC, or TCGACGTCG, wherein D is dSpacer.
[0202] In certain embodiments Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1).sub.q is a
palindromic sequence.
[0203] In certain embodiments Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1).sub.q is
TCGACGTCGA (SEQ ID NO:31) or TCGTCGACGA (SEQ ID NO:32).
[0204] In certain embodiments Z.sub.1 [(X.sub.1Y.sub.1R.sub.1) N
(X.sub.2Y.sub.2R.sub.2).sub.k Z.sub.2].sub.p (S.sub.1)q is
TCGCGACGTT (SEQ ID NO:33) or TCGCGTCGTT (SEQ ID NO:34).
[0205] In one embodiment (N.sub.n) . . . (N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#) . . . (N.sub.n#) Z.sub.3 includes a sequence
AGCGAAGCT, CAATATTTATTG (SEQ ID NO:35), CCGTTTTGTGG (SEQ ID NO:36),
CGGCGCCGTGCCG (SEQ ID NO:37), CGGCGCCGTTGCCG (SEQ ID NO:38),
CGGCGDDCGCCG (SEQ ID NO:39), CGGCGDDDTGCCG (SEQ ID NO:40),
CGGCGGDDCCGCCG (SEQ ID NO:41), CGGCGTCGCCGCCG (SEQ ID NO:42),
CGTCGACGGGACGGG (SEQ ID NO:43), CGTCGACGTGACGGG (SEQ ID NO:44),
GAGAGTTGGGCTCTC (SEQ ID NO:45), GTCGAGGAGGT (SEQ ID NO:46),
TAATADDTATTA (SEQ ID NO:47), TAATATCCATTA (SEQ ID NO:48), or
TAATATTTATTA (SEQ ID NO:49), wherein D is dSpacer.
[0206] In one embodiment (N.sub.n) . . . (N.sub.2)(N.sub.1) S.sub.2
(N.sub.1#)(N.sub.2#) . . . (N.sub.n#) includes a sequence
GGCGCGCTGCCG (SEQ ID NO:50).
[0207] In one embodiment the 5' end of the nucleic acid begins with
an immunostimulatory motif chosen from (TCG).sub.nN and
RDCGY.sub.1Y.sub.2N. T is thymine, C is unmethylated cytosine, G is
guanine, R is a purine, D is not C, each of Y.sub.1 and Y.sub.2
independently is a pyrimidine, n is an integer between 1 and 4,
inclusive, and N is any sequence 0-12 bases long.
[0208] The 3' end of the nucleic acid terminates in an inverted
repeat capable of forming a hairpin or stem-loop structure. The
term "terminates" refers to a structure at or near the 3' end.
Thus, the end of the near palindrome may be positioned at the
actual 3' end of the molecule or alternatively the 3' end may
include 1 or more additional nucleotides that are not part of the
inverted repeat structure. Preferably the 3' end of the molecule
includes 3 or fewer nucleotides that do not form part of the
inverted repeat structure.
[0209] In one embodiment an "inverted repeat capable of forming a
hairpin or stem-loop structure" as used herein refers to a sequence
of nucleotides that forms a GC-rich stem or hairpin that is 2 to 10
consecutive base pairs long, and includes at least one unmatched or
mismatched base. In individual embodiments the GC-rich stem is 2,
3, 4, 5, 6, 7, 8, 9, or 10 consecutive base pairs long. In some
embodiments the GC-rich stem includes at least 2, 3, or 4 G-C base
pairs.
[0210] In one embodiment an "inverted repeat capable of forming a
hairpin or stem-loop structure" as used herein refers to a sequence
of nucleotides that forms an AT-rich stem or hairpin that is 2 to
10 consecutive base pairs long, and includes at least one unmatched
or mismatched base. In individual embodiments the AT-rich stem is
2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive base pairs long. In some
embodiments the AT-rich stem includes at least 2, 3, or 4 A-T base
pairs.
[0211] In some instances the at least one unmatched or mismatched
base bridges the ends of the stem or hairpin. This may allow the
formation of the secondary structure by providing a flexible point
in the molecule for the stems to base pair and form a hairpin.
Alternatively the unmatched or mismatched base(s) may be within the
stem. Preferably if the mismatched base is within the stem, then
the stem is at least 3 basepairs long. The unmatched or mismatched
bases(s) may be any nucleotide. In some embodiments the unmatched
or mismatched base is a T. Unmatched nucleotides at the end of
double-strands are also known as overhanging nucleotides or
dangling ends which can significantly stabilize duplex formation or
hairpin formation. Freier S M et al. (1983) Effects of 3' dangling
end stacking on the stability of GGCC and CCGG double helixes.
Biochemistry 22:6198-206.
[0212] The nucleic acid also includes a partially stabilized
backbone including at least one phosphodiester 5'-CpG-3'
linkage.
[0213] In some instances the double-stranded part of the molecule
may also contain unnatural (non-standard) basepairs (e.g.,
diaminopyridine paired with xanthosine). Lutz M J et al. (1998)
Recognition of a non-standard base pair by thermostable DNA
polymerases. Bioorg Med Chem Lett 8:1149-52.
[0214] The formulas define subsets of the class of CpG
oligonucleotides which demonstrated excellent immune stimulating
properties. In the formulas 5' refers to the free 5' end of the
oligonucleotide and 3' refers to the free 3' end of the
oligonucleotide.
[0215] The oligonucleotides may have one or more accessible 5' or
3' ends. In some embodiments a 3' end can be linked to another 3'
end. Since the importance of the 5' and 3' motifs has been
discovered and described herein, it is also possible to create
modified oligonucleotides having two such 5' or 3' 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. Such a structure might have a formula such as
5'-RDCGY.sub.1Y.sub.2N--NY.sub.2Y.sub.1GCDR-5' (wherein D
represents not C; SEQ ID NO:51) or 5'-(TCG).sub.nN--N(GCT).sub.n-5'
(SEQ ID NO:52). The 3'3'- or 5'5'-linkage may be a phosphodiester,
phosphorothioate, or any other modified internucleoside bridge.
Methods for accomplishing such linkages are known in the art. For
instance, such linkages have been described in Seliger H et al.
(1991) Oligonucleotide analogs with terminal 3'-3'- and
5'-5'-internucleotidic linkages as antisense inhibitors of viral
gene expression, Nucleosides & Nucleotides 10:469-77 and Jiang
Z et al. (1999) Pseudo-cyclic oligonucleotides: in vitro and in
vivo properties, Bioorg Med Chem 7:2727-35.
[0216] In some embodiments the oligonucleotide has one of the
following structures:
TABLE-US-00001 TCGTCGTTTTA, (SEQ ID NO: 53) CGGCGCCGTGCCG, (SEQ ID
NO: 54) CGGCGTCGTGCCG, (SEQ ID NO: 55) TCGTCGTTTTACGGCGCCGTGCCG,
(SEQ ID NO: 56) TCGTCGTTTTACGGCGTCGTGCCG, (SEQ ID NO: 57)
[0217] The invention in one aspect involves the finding that a
specific sub-class of C-class CpG immunostimulatory
oligonucleotides having a chimeric backbone is highly effective in
mediating immune stimulatory effects. These CpG oligonucleotides
are useful therapeutically and prophylactically for stimulating the
immune system to treat cancer, infectious diseases, allergy,
asthma, autoimmune disease, and other disorders and to help protect
against opportunistic infections following cancer chemotherapy. The
strong yet balanced, cellular and humoral immune responses that
result from CpG stimulation reflect the body's own natural defense
system against invading pathogens and cancerous cells.
[0218] The invention involves, in one aspect, the discovery that a
subset of CpG immunostimulatory oligonucleotides have improved
immune stimulatory properties and reduced renal inflammatory
effect. In some instances, renal inflammation has been observed in
subjects that have been administered a completely phosphorothioate
oligonucleotide. It is believed that the chimeric oligonucleotides
described herein produce less renal inflammation than fully
phosphorothioate oligonucleotides. Additionally these
oligonucleotides are highly effective in stimulating an immune
response. Thus, the phosphodiester region of the molecule did not
reduce it's affectivity.
[0219] The preferred CpG immunostimulatory oligonucleotides fall
within one of the following 7 general formulas:
[0220] 5' TTC_GX.sub.2C_GN.sub.1X.sub.1--GX.sub.3C_GTT 3' (SEQ ID
NO.: 24) where N.sub.1 is 1-3 nucleotides in length with N
referring to any nucleotide, X.sub.1 is a pyrimidine, X.sub.2 and
X.sub.3 are any nucleotide.
[0221] 5' TTC_GTC_GTTTX.sub.1--GTC_GTT 3' (SEQ ID NO.: 25), wherein
X.sub.1 is a pyrimidine.
[0222] 5' T*T*C_G*T*C_G*T*T*T*X.sub.1--G*T*C_G*T*T 3' (SEQ ID NO.:
25), wherein X.sub.1 is a pyrimidine.
[0223] 5' TC_GX.sub.1C_G X.sub.2N.sub.1 X.sub.3C_GN.sub.2CG 3' (SEQ
ID NO.: 26), wherein N.sub.1 is 0-3 nucleotides in length, N.sub.2
is 0-9 nucleotides in length with N referring to any nucleotide and
X.sub.1, X.sub.2, and X.sub.3 are any nucleotide.
[0224] 5' TC_GTC_GTN.sub.1TC_GGCGCN.sub.1GCCG 3' (SEQ ID NO.: 27),
wherein N.sub.1 is 0-3 nucleotides in length.
[0225] 5' T*C_G*T*C_G*T*N.sub.1*T*C_G*G*C*G*CN.sub.1G*C*C*G 3' (SEQ
ID NO.: 27), wherein N.sub.1 is 0-3 nucleotides in length.
[0226] 5' TC_G X.sub.1C_G X.sub.2C_G X.sub.3TC_GGCGC_G N.sub.33'
(SEQ ID NO: 28), wherein N.sub.3 is 1-5 nucleotides in length with
N referring to any nucleotide and X.sub.1, X.sub.2, and X.sub.3 are
any nucleotide.
[0227] Optionally, when specified in the formula, 5' refers to the
free 5' end of the oligonucleotide and 3' refers to the free 3' end
of the oligonucleotide.
[0228] The symbol * used in the formulas refers to the presence of
a stabilized internucleotide linkage. The symbol _ in these
structures refers to the presence of a phosphodiester
internucleotide linkage. The internucleotide linkages not marked
with an * may be stabilized or unstabilized, as long as the
oligonucleotide includes at least 2-3 phosphodiester or
phosphodiester like internucleotide linkages. In some embodiments
it is preferred that the oligonucleotides include 3-6
phosphodiester or phosphodiester like linkages. In some cases the
linkages between the CG motifs are phosphodiester and in other
cases they are phosphorothioate or other stabilized linkages.
[0229] In some embodiment the oligonucleotide has one of the
following structures:
TABLE-US-00002 (SEQ ID NO: 2)
T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 3)
T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G (SEQ ID NO: 4)
TC_GTC_GAC_GATC_GGCGC_GCGCCG (SEQ ID NO: 4)
T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G (SEQ ID NO: 5)
T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T (SEQ ID NO: 6)
T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T (SEQ ID NO: 3)
TCGTCGTTCGGCGCGCCG (SEQ ID NO: 2) TCGTCGTCGTTCGGCGCGCGCCG (SEQ ID
NO: 4) TCGTCGACGATCGGCGCGCGCCG (SEQ ID NO: 5) TTCGTCGTTTTGTCGTT
(SEQ ID NO: 6) TTTCGTCGTTTCGTCGTT TCGTCGTC CGTCGTCG GTCGTCGT
TCGTCGTT CGTCGTTC GTCGTTCG TCGTTCGG CGTTCGGC GTTCGGCG TTCGGCGC
TCGGCGCG CGGCGCGC GGCGCGCG GCGCGCGC CGCGCGCC GCGCGCCG.
T*C_G*T*C_G*T*C C_G*T*C_G*T*C_G G*T*C_G*T*C_G*T T*C_G*T*C_G*T*T
C_G*T*C_G*T*T*C G*T*C_G*T*T*C_G T*C_G*T*T*C_G*G C_G*T*T*C_G*G*C
G*T*T*C_G*G*C*G T*T*C_G*G*C*G*C T*C_G*G*C*G*C_G C_G*G*C*G*C_G*C
G*G*C*G*C_G*C*G G*C*G*C_G*C*G*C C*G*C_G*C*G*C*C G*C_G*C*G*C*C*G
[0230] 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 oligonucleotides 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
oligonucleotides may include a 2'-O-alkylated ribose group. In
addition, modified oligonucleotides may include sugars such as
arabinose or 2'-fluoroarabinose instead of ribose. Thus the
oligonucleotides 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).
[0231] 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.
[0232] 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.
[0233] For example, the invention relates to an oligonucleotide
which may comprise one or more modifications and wherein each
modification is independently selected from: [0234] 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, [0235] b) the replacement of phosphodiester
bridge located at the 3' and/or the 5' end of a nucleotide by a
dephospho bridge, [0236] c) the replacement of a sugar phosphate
unit from the sugar phosphate backbone by another unit, [0237] d)
the replacement of a .beta.-D-ribose unit by a modified sugar unit,
and [0238] e) the replacement of a natural nucleotide base by a
modified nucleotide base.
[0239] More detailed examples for the chemical modification of an
oligonucleotide are as follows.
[0240] 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.
[0241] 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,
methylenedimethylhydrazo, dimethylenesulfone and/or silyl
groups.
[0242] 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 EP 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.
[0243] 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)alkyll-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) Hely Chim Acta 76:481).
[0244] 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.
[0245] 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.
[0246] 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.
[0247] Additionally, 3'3'-linked oligonucleotides 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.
[0248] It recently has been reported that CpG oligonucleotides
appear to exert their immunostimulatory effect through interaction
with Toll-like receptor 9 (TLR9). Hemmi H et al. (2000) Nature
408:740-5. TLR9 signaling activity thus can be measured in response
to CpG oligonucleotide or other immunostimulatory oligonucleotide
by measuring NF-.kappa.B, NF-.kappa.B-related signals, and suitable
events and intermediates upstream or downstream of NF-.kappa.B.
[0249] 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); and 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.
[0250] 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 oligonucleotides,
combinations of phosphodiester and phosphorothioate
oligonucleotide, methylphosphonate, methylphosphorothioate,
phosphorodithioate, p-ethoxy, and combinations thereof.
[0251] 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).
[0252] 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. Oligonucleotides which contain diol,
such as tetraethyleneglycol or hexaethyleneglycol, at either or
both termini have also been shown to be substantially resistant to
nuclease degradation.
[0253] It has been discovered according to the invention that the
subsets of CpG immunostimulatory oligonucleotides have dramatic
immune stimulatory effects on human cells such as PBMC cells,
suggesting that these CpG immunostimulatory oligonucleotides are
effective therapeutic agents for human vaccination, cancer
immunotherapy, asthma immunotherapy, general enhancement of immune
function, enhancement of hematopoietic recovery following radiation
or chemotherapy, autoimmune disease and other immune modulatory
applications. It has also been demonstrated that the subsets of CpG
immunostimulatory oligonucleotides are useful in vivo for the
treatment of asthma and allergic rhinitis.
[0254] A subject having an allergy is a subject that has or is at
risk of developing an allergic reaction in response to an allergen.
An allergy refers to acquired hypersensitivity to a substance
(allergen). Allergic conditions include but are not limited to
eczema, allergic rhinitis or coryza, hay fever, conjunctivitis,
bronchial asthma, urticaria (hives) and food allergies, and other
atopic conditions.
[0255] Allergies are generally caused by IgE antibody generation
against harmless allergens. The cytokines that are induced by
systemic or mucosal administration of CpG immunostimulatory
oligonucleotides are predominantly of a class called Th1 (examples
are IL-12, IP-10, IFN-.alpha. and IFN-.gamma.) and these induce
both humoral and cellular immune responses. The other major type of
immune response, which is associated with the production of IL-4
and IL-5 cytokines, is termed a Th2 immune response. In general, it
appears that allergic diseases are mediated by Th2 type immune
responses. Based on the ability of the CpG immunostimulatory
oligonucleotides to shift the immune response in a subject from a
predominant Th2 (which is associated with production of IgE
antibodies and allergy) to a balanced Th2/Th1 response (which is
protective against allergic reactions), an effective dose for
inducing an immune response of a CpG immunostimulatory
oligonucleotide as a stand alone therapy without allergen or in
combination with allergen can be administered to a subject to treat
or prevent asthma and allergy.
[0256] Thus, the CpG immunostimulatory oligonucleotides have
significant therapeutic utility in the treatment of allergic
conditions such as asthma and allergic rhinitis. Th2 cytokines,
especially IL-4 and IL-5 are elevated in the airways of asthmatic
subjects. These cytokines promote important aspects of the
asthmatic inflammatory response, including IgE isotope switching,
eosinophil chemotaxis and activation and mast cell growth. Th1
cytokines, especially IFN-.gamma. and IL-12, can suppress the
formation of Th2 clones and production of Th2 cytokines. Asthma
refers to a disorder of the respiratory system characterized by
inflammation, narrowing of the airways and increased reactivity of
the airways to inhaled agents. Asthma is frequently, although not
exclusively associated with atopic or allergic symptoms.
[0257] Asthma may be exacerbated by viral infections. The
combination of asthma and viral infections significantly worsens
the symptoms in a subject. The oligonucleotides used herein provide
significant benefits for the treatment of viral induced
exacerbation of asthma. Several examples of such therapy are
presented below.
[0258] Allergic rhinitis is a disorder resulting in inflammation of
the nasal mucosa caused by allergens such as pollen or dust. The
term includes rhinitis medicamentosa, rhinitis sicca, and atrophic
rhinitis. There are two general types of allergic rhinitis,
seasonal and perennial. Seasonal allergic rhinitis is normally
referred to as hay fever and is usually caused by mould or pollen.
Perennial allergic rhinitis is usually caused by an inherent
sensitivity to one or more types of allergen. This condition
generally continues throughout the year or for as long as the
patient is exposed to the allergen. Both types of allergic rhinitis
involve a type 1 (IgE-mediated) hypersensitivity that leads to
inflammation. This inflammation is thought to be caused by an
excessive degranulation of mast cells and of blood-borne basophils
in response to certain allergens. This leads to increased IgE
levels and the concomitant release of inflammatory mediators, such
as histamine, and of chemotactic factors, such as cytokines,
prostaglandins and leukotrienes, that result in a localized
inflammatory reaction.
[0259] The immunostimulatory oligonucleotides may be administered
as stand alone therapy without an additional anti-allergy/asthma
medicament or therapy or in combination with such a therapy or
medicament. Typical anti allergy/asthma medicaments and therapies
include the use of intranasal vasoconstrictors, intranasal and
systemic antihistamines, intranasal glucocorticoids, mast cell
stabilizers, such as cromolyn compounds, and oral
decongestants.
[0260] An allergen refers to a substance (antigen) that can induce
an allergic or asthmatic response in a susceptible subject. The
list of allergens is enormous and can include pollens, insect
venoms, animal dander dust, fungal spores and drugs (e.g.
penicillin). Examples of natural, animal and plant allergens
include but are not limited to proteins specific to the following
genuses: Canine (Canis familiaris); Dermatophagoides (e.g.
Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia
(Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium
multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria
(Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula
(Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa);
Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago
lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria
judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis
multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus
arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus
sabinoides, Juniperus virginiana, Juniperus communis and Juniperus
ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.
Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana);
Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale);
Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis
glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis
or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus
lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum
(e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum
(e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea);
Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum
halepensis); and Bromus (e.g. Bromus inermis).
[0261] The oligonucleotides are also useful for redirecting an
immune response from a Th2 immune response to a Th1 immune
response. This results in the production of a relatively balanced
Th1/Th2 environment. Redirection of an immune response from a Th2
to a Th1 immune response can be assessed by measuring the levels of
cytokines produced in response to the nucleic acid (e.g., by
inducing monocytic cells and other cells to produce Th1 cytokines,
including IL-12, IFN-.gamma. and GM-CSF). The redirection or
rebalance of the immune response from a Th2 to a Th1 response is
particularly useful for the treatment or prevention of asthma. For
instance, an effective amount for treating asthma can be that
amount; useful for redirecting a Th2 type of immune response that
is associated with asthma to a Th1 type of response or a balanced
Th1/Th2 environment. Th2 cytokines, especially IL-4 and IL-5 are
elevated in the airways of asthmatic subjects. The CpG
immunostimulatory oligonucleotides of the invention cause an
increase in Th1 cytokines which helps to rebalance the immune
system, preventing or reducing the adverse effects associated with
a predominately Th2 immune response.
[0262] The CpG immunostimulatory oligonucleotides are also useful
in some aspects of the invention as a vaccine for the treatment of
a subject at risk of developing an infection with an infectious
organism or a cancer in which a specific cancer antigen has been
identified, in addition to allergy or asthma. The CpG
immunostimulatory oligonucleotides can also be given without the
antigen or allergen for protection against infection, allergy or
cancer, and in this case repeated doses may allow longer term
protection. A subject at risk as used herein is a subject who has
any risk of exposure to an infection causing pathogen or a cancer
or an allergen or a risk of developing cancer. For instance, a
subject at risk may be a subject who is planning to travel to an
area where a particular type of infectious agent is found or it may
be a subject who through lifestyle or medical procedures is exposed
to bodily fluids which may contain infectious organisms or directly
to the organism or even any subject living in an area where an
infectious organism or an allergen has been identified. Subjects at
risk of developing infection also include general populations to
which a medical agency recommends vaccination with a particular
infectious organism antigen. If the antigen is an allergen and the
subject develops allergic responses to that particular antigen and
the subject may be exposed to the antigen, i.e., during pollen
season, then that subject is at risk of exposure to the antigen. A
subject at risk of developing an allergy to asthma includes those
subjects that have been identified as having an allergy or asthma
but that don't have the active disease during the CpG
immunostimulatory oligonucleotide treatment as well as subjects
that are considered to be at risk of developing these diseases
because of genetic or environmental factors.
[0263] A subject at risk of developing a cancer is one who has a
high probability of developing cancer. These subjects include, for
instance, subjects having a genetic abnormality, the presence of
which has been demonstrated to have a correlative relation to a
higher likelihood of developing a cancer and subjects exposed to
cancer causing agents such as tobacco, asbestos, or other chemical
toxins, or a subject who has previously been treated for cancer and
is in apparent remission. When a subject at risk of developing a
cancer is treated with an antigen specific for the type of cancer
to which the subject is at risk of developing and a CpG
immunostimulatory oligonucleotide, the subject may be able to kill
the cancer cells as they develop. If a tumor begins to form in the
subject, the subject will develop a specific immune response
against the tumor antigen.
[0264] In addition to the use of the CpG immunostimulatory
oligonucleotides for prophylactic treatment, the invention also
encompasses the use of the CpG immunostimulatory oligonucleotides
for the treatment of a subject having an infection, an allergy,
asthma, or a cancer.
[0265] A subject having an infection is a subject that has been
exposed to an infectious pathogen and has acute or chronic
detectable levels of the pathogen in the body. The CpG
immunostimulatory oligonucleotides can be used with or without an
antigen to mount an antigen specific systemic or mucosal immune
response that is capable of reducing the level of or eradicating
the infectious pathogen. An infectious disease, as used herein, is
a disease arising from the presence of a foreign microorganism in
the body. It is particularly important to develop effective vaccine
strategies and treatments to protect the body's mucosal surfaces,
which are the primary site of pathogenic entry.
[0266] A subject having a cancer is a subject that has detectable
cancerous cells. The cancer may be a malignant or non-malignant
cancer. Cancers or tumors include but are not limited to biliary
tract cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; lymphomas; liver
cancer; lung cancer (e.g. small cell and non-small cell); melanoma;
neuroblastomas; oral cancer; ovarian cancer; pancreas cancer;
prostate cancer; rectal cancer; sarcomas; skin cancer; testicular
cancer; thyroid cancer; and renal cancer, as well as other
carcinomas and sarcomas. In one embodiment the cancer is hairy cell
leukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,
multiple myeloma, follicular lymphoma, malignant melanoma, squamous
cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder
cell carcinoma, or colon carcinoma.
[0267] A subject shall mean a human or vertebrate animal including
but not limited to a dog, cat, horse, cow, pig, sheep, goat,
turkey, chicken, primate, e.g., monkey, and fish (aquaculture
species), e.g. salmon. Thus, the invention can also be used to
treat cancer and tumors, infections, and allergy/asthma in non
human subjects. Cancer is one of the leading causes of death in
companion animals (i.e., cats and dogs).
[0268] As used herein, the term treat, treated, or treating when
used with respect to an disorder such as an infectious disease,
cancer, allergy, or asthma refers to a prophylactic treatment which
increases the resistance of a subject to development of the disease
(e.g., to infection with a pathogen) or, in other words, decreases
the likelihood that the subject will develop the disease (e.g.,
become infected with the pathogen) as well as a treatment after the
subject has developed the disease in order to fight the disease
(e.g., reduce or eliminate the infection) or prevent the disease
from becoming worse.
[0269] In the instances when the CpG oligonucleotide is
administered with an antigen, the subject may be exposed to the
antigen. As used herein, the term exposed to refers to either the
active step of contacting the subject with an antigen or the
passive exposure of the subject to the antigen in vivo. Methods for
the active exposure of a subject to an antigen are well-known in
the art. In general, an antigen is administered directly to the
subject by any means such as intravenous, intramuscular, oral,
transdermal, mucosal, intranasal, intratracheal, or subcutaneous
administration. The antigen can be administered systemically or
locally. Methods for administering the antigen and the CpG
immunostimulatory oligonucleotide are described in more detail
below. A subject is passively exposed to an antigen if an antigen
becomes available for exposure to the immune cells in the body. A
subject may be passively exposed to an antigen, for instance, by
entry of a foreign pathogen into the body or by the development of
a tumor cell expressing a foreign antigen on its surface.
[0270] The methods in which a subject is passively exposed to an
antigen can be particularly dependent on timing of administration
of the CpG immunostimulatory oligonucleotide. For instance, in a
subject at risk of developing a cancer or an infectious disease or
an allergic or asthmatic response, the subject may be administered
the CpG immunostimulatory oligonucleotide on a regular basis when
that risk is greatest, i.e., during allergy season or after
exposure to a cancer causing agent. Additionally the CpG
immunostimulatory oligonucleotide may be administered to travelers
before they travel to foreign lands where they are at risk of
exposure to infectious agents. Likewise the CpG immunostimulatory
oligonucleotide may be administered to soldiers or civilians at
risk of exposure to biowarfare to induce a systemic or mucosal
immune response to the antigen when and if the subject is exposed
to it.
[0271] An antigen as used herein is a molecule capable of provoking
an immune response. Antigens include but are not limited to cells,
cell extracts, proteins, polypeptides, peptides, polysaccharides,
polysaccharide conjugates, peptide and non-peptide mimics of
polysaccharides and other molecules, small molecules, lipids,
glycolipids, carbohydrates, viruses and viral extracts and
muticellular organisms such as parasites and allergens. The term
antigen broadly includes any type of molecule which is recognized
by a host immune system as being foreign. Antigens include but are
not limited to cancer antigens, microbial antigens, and
allergens.
[0272] A cancer antigen as used herein is a compound, such as a
peptide or protein, associated with a tumor or cancer cell surface
and which is capable of provoking an immune response when expressed
on the surface of an antigen presenting cell in the context of an
MHC molecule. Cancer antigens can be prepared from cancer cells
either by preparing crude extracts of cancer cells, for example, as
described in Cohen, et al., 1994, Cancer Research, 54:1055, by
partially purifying the antigens, by recombinant technology, or by
de novo synthesis of known antigens. Cancer antigens include but
are not limited to antigens that are recombinantly expressed, an
immunogenic portion of, or a whole tumor or cancer. Such antigens
can be isolated or prepared recombinantly or by any other means
known in the art.
[0273] A microbial antigen as used herein is an antigen of a
microorganism and includes but is not limited to virus, bacteria,
parasites, and fungi. Such antigens include the intact
microorganism as well as natural isolates and fragments or
derivatives thereof and also synthetic compounds which are
identical to or similar to natural microorganism antigens and
induce an immune response specific for that microorganism. A
compound is similar to a natural microorganism antigen if it
induces an immune response (humoral and/or cellular) to a natural
microorganism antigen. Such antigens are used routinely in the art
and are well known to those of ordinary skill in the art.
[0274] The term substantially purified as used herein refers to a
polypeptide which is substantially free of other proteins, lipids,
carbohydrates or other materials with which it is naturally
associated. One skilled in the art can purify viral or bacterial
polypeptides using standard techniques for protein purification.
The substantially pure polypeptide will often yield a single major
band on a non-reducing polyacrylamide gel. In the case of partially
glycosylated polypeptides or those that have several start codons,
there may be several bands on a non-reducing polyacrylamide gel,
but these will form a distinctive pattern for that polypeptide. The
purity of the viral or bacterial polypeptide can also be determined
by amino-terminal amino acid sequence analysis. Other types of
antigens not encoded by a nucleic acid vector such as
polysaccharides, small molecule, mimics etc are included within the
invention.
[0275] The oligonucleotides of the invention may be administered to
a subject with an anti-microbial agent. An anti-microbial agent, as
used herein, refers to a naturally-occurring or synthetic compound
which is capable of killing or inhibiting infectious
microorganisms. The type of anti-microbial agent useful according
to the invention will depend upon the type of microorganism with
which the subject is infected or at risk of becoming infected.
Anti-microbial agents include but are not limited to anti-bacterial
agents, anti-viral agents, anti-fungal agents and anti-parasitic
agents. Phrases such as "anti-infective agent", "anti-bacterial
agent", "anti-viral agent", "anti-fungal agent", "anti-parasitic
agent" and "parasiticide" have well-established meanings to those
of ordinary skill in the art and are defined in standard medical
texts. Briefly, anti-bacterial agents kill or inhibit bacteria, and
include antibiotics as well as other synthetic or natural compounds
having similar functions. Antibiotics are low molecular weight
molecules which are produced as secondary metabolites by cells,
such as microorganisms. In general, antibiotics interfere with one
or more bacterial functions or structures which are specific for
the microorganism and which are not present in host cells.
Anti-viral agents can be isolated from natural sources or
synthesized and are useful for killing or inhibiting viruses.
Anti-fungal agents are used to treat superficial fungal infections
as well as opportunistic and primary systemic fungal infections.
Anti-parasite agents kill or inhibit parasites.
[0276] Examples of anti-parasitic agents, also referred to as
parasiticides useful for human administration include but are not
limited to albendazole, amphotericin B, benznidazole, bithionol,
chloroquine HCl, chloroquine phosphate, clindamycin,
dehydroemetine, diethylcarbamazine, diloxanide furoate,
eflornithine, furazolidaone, glucocorticoids, halofantrine,
iodoquinol, ivermectin, mebendazole, mefloquine, meglumine
antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,
nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,
piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel
pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,
quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin,
stibogluconate sodium (sodium antimony gluconate), suramin,
tetracycline, doxycycline, thiabendazole, tinidazole,
trimethroprim-sulfamethoxazole, and tryparsamide some of which are
used alone or in combination with others.
[0277] Antibacterial agents kill or inhibit the growth or function
of bacteria. A large class of antibacterial agents is antibiotics.
Antibiotics, which are effective for killing or inhibiting a wide
range of bacteria, are referred to as broad spectrum antibiotics.
Other types of antibiotics are predominantly effective against the
bacteria of the class gram-positive or gram-negative. These types
of antibiotics are referred to as narrow spectrum antibiotics.
Other antibiotics which are effective against a single organism or
disease and not against other types of bacteria, are referred to as
limited spectrum antibiotics. Antibacterial agents are sometimes
classified based on their primary mode of action. In general,
antibacterial agents are cell wall synthesis inhibitors, cell
membrane inhibitors, protein synthesis inhibitors, nucleic acid
synthesis or functional inhibitors, and competitive inhibitors.
[0278] 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 (e.g. interferon), replication of viral RNA or DNA
(e.g. nucleotide analogues), maturation of new virus proteins (e.g.
protease inhibitors), and budding and release of the virus.
[0279] 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. Nucleotide analogues include, but are
not limited to, acyclovir (used for the treatment of herpes simplex
virus and varicella-zoster virus), gancyclovir (useful for the
treatment of cytomegalovirus), idoxuridine, ribavirin (useful for
the treatment of respiratory syncitial virus), dideoxyinosine,
dideoxycytidine, zidovudine (azidothymidine), imiquimod, and
resimiquimod.
[0280] The interferons are cytokines which are secreted by
virus-infected cells as well as immune cells. The interferons
function by binding to specific receptors on cells adjacent to the
infected cells, causing the change in the cell which protects it
from infection by the virus. .alpha. and .beta.-interferon also
induce the expression of Class I and Class II MHC molecules on the
surface of infected cells, resulting in increased antigen
presentation for host immune cell recognition. .alpha. and
.beta.-interferons are available as recombinant forms and have been
used for the treatment of chronic hepatitis B and C infection. At
the dosages which are effective for anti-viral therapy, interferons
have severe side effects such as fever, malaise and weight
loss.
[0281] Anti-viral agents useful in the invention include but are
not limited to immunoglobulins, amantadine, interferons, nucleotide
analogues, and protease inhibitors. Specific examples of
anti-virals include but are not limited to Acemannan; Acyclovir;
Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox;
Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate;
Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride;
Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril;
Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine
Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscarnet
Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium;
Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine
Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;
Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;
Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine;
Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride;
Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate;
Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
[0282] Anti-fungal agents are useful for the treatment and
prevention of infective fungi. Anti-fungal agents are sometimes
classified by their mechanism of action. Some anti-fungal agents
function as cell wall inhibitors by inhibiting glucose synthase.
These include, but are not limited to, basiungin/ECB. Other
anti-fungal agents function by destabilizing membrane integrity.
These include, but are not limited to, immidazoles, such as
clotrimazole, sertaconzole, fluconazole, itraconazole,
ketoconazole, miconazole, and voriconacole, as well as FK 463,
amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,
butenafine, and terbinafine. Other anti-fungal agents function by
breaking down chitin (e.g. chitinase) or immunosuppression (501
cream).
[0283] CpG immunostimulatory oligonucleotides can be combined with
other therapeutic agents such as adjuvants to enhance immune
responses. The CpG immunostimulatory oligonucleotide and other
therapeutic agent may be administered simultaneously or
sequentially. When the other therapeutic agents are administered
simultaneously they can be administered in the same or separate
formulations, but are administered at the same time. The other
therapeutic agents are administered sequentially with one another
and with CpG immunostimulatory oligonucleotide, when the
administration of the other therapeutic agents and the CpG
immunostimulatory oligonucleotide is temporally separated. The
separation in time between the administration of these compounds
may be a matter of minutes or it may be longer. Other therapeutic
agents include but are not limited to adjuvants, cytokines,
antibodies, antigens, etc.
[0284] The compositions of the invention may also be administered
with non-nucleic acid adjuvants. A non-nucleic acid adjuvant is any
molecule or compound except for the CpG immunostimulatory
oligonucleotides described herein which can stimulate the humoral
and/or cellular immune response. Non-nucleic acid adjuvants
include, for instance, adjuvants that create a depo effect, immune
stimulating adjuvants, and adjuvants that create a depo effect and
stimulate the immune system.
[0285] The CpG immunostimulatory oligonucleotides are also useful
as mucosal adjuvants. It has previously been discovered that both
systemic and mucosal immunity are induced by mucosal delivery of
CpG oligonucleotides. Thus, the oligonucleotides may be
administered in combination with other mucosal adjuvants.
[0286] Immune responses can also be induced or augmented by the
co-administration or co-linear expression of cytokines (Bueler
& Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997;
Iwasaki et al., 1997; Kim et al., 1997) or B-7 co-stimulatory
molecules (Iwasaki et al., 1997; Tsuji et al., 1997) with the CpG
immunostimulatory oligonucleotides. The term cytokine is used as a
generic name for a diverse group of soluble proteins and peptides
which act as humoral regulators at nano- to picomolar
concentrations and which, either under normal or pathological
conditions, modulate the functional activities of individual cells
and tissues. These proteins also mediate interactions between cells
directly and regulate processes taking place in the extracellular
environment. Examples of cytokines include, but are not limited to
IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18,
granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), interferon-.gamma.
(.gamma.-IFN), IFN-.alpha., tumor necrosis factor (TNF),
TGF-.beta., FLT-3 ligand, and CD40 ligand.
[0287] The oligonucleotides are also useful for improving survival,
differentiation, activation and maturation of dendritic cells. The
CpG immunostimulatory oligonucleotides have the unique capability
to promote cell survival, differentiation, activation and
maturation of dendritic cells.
[0288] CpG immunostimulatory oligonucleotides also increase natural
killer cell lytic activity and antibody dependent cellular
cytotoxicity (ADCC). ADCC can be performed using a CpG
immunostimulatory oligonucleotide in combination with an antibody
specific for a cellular target, such as a cancer cell. When the CpG
immunostimulatory oligonucleotide is administered to a subject in
conjunction with the antibody the subject's immune system is
induced to kill the tumor cell. The antibodies useful in the ADCC
procedure include antibodies which interact with a cell in the
body. Many such antibodies specific for cellular targets have been
described in the art and many are commercially available.
[0289] The CpG immunostimulatory oligonucleotides may also be
administered in conjunction with an anti-cancer therapy.
Anti-cancer therapies include cancer medicaments, radiation and
surgical procedures. As used herein, a "cancer medicament" refers
to a agent which is administered to a subject for the purpose of
treating a cancer. As used herein, "treating cancer" includes
preventing the development of a cancer, reducing the symptoms of
cancer, and/or inhibiting the growth of an established cancer. In
other aspects, the cancer medicament is administered to a subject
at risk of developing a cancer for the purpose of reducing the risk
of developing the cancer. Various types of medicaments for the
treatment of cancer are described herein. For the purpose of this
specification, cancer medicaments are classified as
chemotherapeutic agents, immunotherapeutic agents, cancer vaccines,
hormone therapy, and biological response modifiers.
[0290] In one embodiment, the cancer medicament is a
chemotherapeutic agent selected from the group consisting of
methotrexate, vincristine, adriamycin, cisplatin, non-sugar
containing chloroethylnitrosoureas, 5-fluorouracil, mitomycin C,
bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine
GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566,
RAS famesyl transferase inhibitor, famesyl transferase inhibitor,
MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,
Hycamtin/Topotecan, PKC412, Valspodar/PSC833,
Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070,
BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853,
ZD0101, IS1641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP
845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,
Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,
Temodal/Temozolomide, Evacet/liposomal doxorubicin,
Yewtaxan/Placlitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,
Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,
SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609
(754)/RAS oncogene inhibitor, BMS-182751/oral platinum,
UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU
enhancer, Campto/Levamisole, Camptosar/Irinotecan,
Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel,
Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin,
Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU
79553/Bis-Naphtalimide, LU 103793/Dolastain, Caetyx/liposomal
doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, lodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCI, Dactinomycin,
Daunorubicin HCl, Estramustine phosphate sodium, Etoposide
(VP16-213), Floxuridine, Fluorouracil (5-FU), Flutamide,
Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a,
Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue),
Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),
Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl,
Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen
citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine
(m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM),
Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal
bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin),
Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.
In an important embodiment, the cancer medicament is taxol. In
another embodiment the cancer medicament is a combination of
carboplatin and paclitaxel.
[0291] In another embodiment, the cancer medicament is an
immunotherapeutic agent selected from the group consisting of
Ributaxin, Herceptin, Quadramet, Panorex, IDEC-Y2B8, BEC2, C225,
Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6,
MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax,
MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-1, CEACIDE, Pretarget,
NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA
676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2,
MDX-260, ANA Ab, SMART 1D10 Ab, SMART ABL 364 Ab and
ImmuRAIT-CEA.
[0292] Additionally, the methods of the invention are intended to
embrace the use of more than one cancer medicament along with the
CpG immunostimulatory oligonucleotides. As an example, where
appropriate, the CpG immunostimulatory oligonucleotides may be
administered with both a chemotherapeutic agent and an
immunotherapeutic agent. Alternatively, the cancer medicament may
embrace an immunotherapeutic agent and a cancer vaccine, or a
chemotherapeutic agent and a cancer vaccine, or a chemotherapeutic
agent, an immunotherapeutic agent and a cancer vaccine all
administered to one subject for the purpose of treating a subject
having a cancer or at risk of developing a cancer.
[0293] The use of CpG immunostimulatory oligonucleotides in
conjunction with immunotherapeutic agents such as monoclonal
antibodies is able to increase long-term survival through a number
of mechanisms including significant enhancement of ADCC (as
discussed above), activation of natural killer (NK) cells and an
increase in IFN.alpha. levels. The oligonucleotides when used in
combination with monoclonal antibodies serve to reduce the dose of
the antibody required to achieve a biological result.
[0294] As used herein, the terms "cancer antigen" and "tumor
antigen" are used interchangeably to refer to antigens which are
differentially expressed by cancer cells and can thereby be
exploited in order to target cancer cells. Cancer antigens are
antigens which can potentially stimulate apparently tumor-specific
immune responses. Some of these antigens are encoded, although not
necessarily expressed, by normal cells. These antigens can be
characterized as those which are normally silent (i.e., not
expressed) in normal cells, those that are expressed only at
certain stages of differentiation and those that are temporally
expressed such as embryonic and fetal antigens. Other cancer
antigens are encoded by mutant cellular genes, such as oncogenes
(e.g., activated ras oncogene), suppressor genes (e.g., mutant
p53), fusion proteins resulting from internal deletions or
chromosomal translocations. Still other cancer antigens can be
encoded by viral genes such as those carried on RNA and DNA tumor
viruses.
[0295] The CpG immunostimulatory oligonucleotides are also useful
for treating and preventing autoimmune disease. Autoimmune disease
is a class of diseases in which a subject's own antibodies react
with host tissue or in which immune effector T cells are
autoreactive to endogenous self peptides and cause destruction of
tissue. Thus an immune response is mounted against a subject's own
antigens, referred to as self antigens. Autoimmune diseases include
but are not limited to rheumatoid arthritis, Crohn's disease,
multiple sclerosis, systemic lupus erythematosus (SLE), autoimmune
encephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,
Goodpasture's syndrome, pemphigus (e.g., pemphigus vulgaris),
Grave's disease, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, scleroderma with anti-collagen
antibodies, mixed connective tissue disease, polymyositis,
pernicious anemia, idiopathic Addison's disease,
autoimmune-associated infertility, glomerulonephritis (e.g.,
crescentic glomerulonephritis, proliferative glomerulonephritis),
bullous pemphigoid, Sjogren's syndrome, insulin resistance, and
autoimmune diabetes mellitus.
[0296] A "self-antigen" as used herein refers to an antigen of a
normal host tissue. Normal host tissue does not include cancer
cells. Thus an immune response mounted against a self-antigen, in
the context of an autoimmune disease, is an undesirable immune
response and contributes to destruction and damage of normal
tissue, whereas an immune response mounted against a cancer antigen
is a desirable immune response and contributes to the destruction
of the tumor or cancer. Thus, in some aspects of the invention
aimed at treating autoimmune disorders it is not recommended that
the CpG immunostimulatory oligonucleotides be administered with
self antigens, particularly those that are the targets of the
autoimmune disorder.
[0297] In other instances, the CpG immunostimulatory
oligonucleotides may be delivered with low doses of self-antigens.
A number of animal studies have demonstrated that mucosal
administration of low doses of antigen can result in a state of
immune hyporesponsiveness or "tolerance." The active mechanism
appears to be a cytokine-mediated immune deviation away from a Th1
towards a predominantly Th2 and Th3 (i.e., TGF-.beta. dominated)
response. The active suppression with low dose antigen delivery can
also suppress an unrelated immune response (bystander suppression)
which is of considerable interest in the therapy of autoimmune
diseases, for example, rheumatoid arthritis and SLE. Bystander
suppression involves the secretion of Th1-counter-regulatory,
suppressor cytokines in the local environment where proinflammatory
and Th1 cytokines are released in either an antigen-specific or
antigen-nonspecific manner. "Tolerance" as used herein is used to
refer to this phenomenon. Indeed, oral tolerance has been effective
in the treatment of a number of autoimmune diseases in animals
including: experimental autoimmune encephalomyelitis (EAE),
experimental autoimmune myasthenia gravis, collagen-induced
arthritis (CIA), and insulin-dependent diabetes mellitus. In these
models, the prevention and suppression of autoimmune disease is
associated with a shift in antigen-specific humoral and cellular
responses from a Th1 to Th2/Th3 response.
[0298] The invention also includes a method for inducing antigen
non-specific innate immune activation and broad spectrum resistance
to infectious challenge using the CpG immunostimulatory
oligonucleotides. The term antigen non-specific innate immune
activation as used herein refers to the activation of immune cells
other than B cells and for instance can include the activation of
NK cells, T cells or other immune cells that can respond in an
antigen independent fashion or some combination of these cells. A
broad spectrum resistance to infectious challenge is induced
because the immune cells are in active form and are primed to
respond to any invading compound or microorganism. The cells do not
have to be specifically primed against a particular antigen. This
is particularly useful in biowarfare, and the other circumstances
described above such as travelers.
[0299] The CpG immunostimulatory oligonucleotides may be directly
administered to the subject or may be administered in conjunction
with a nucleic acid delivery complex. A nucleic acid delivery
complex shall mean a nucleic acid molecule associated with (e.g.
ionically or covalently bound to; or encapsulated within) a
targeting means (e.g. a molecule that results in higher affinity
binding to target cell. Examples of nucleic acid delivery complexes
include nucleic acids associated with a sterol (e.g. cholesterol),
a lipid (e.g. a cationic lipid, virosome or liposome), or a target
cell specific binding agent (e.g. a ligand recognized by target
cell specific receptor). Preferred complexes may be sufficiently
stable in vivo to prevent significant uncoupling prior to
internalization by the target cell. However, the complex can be
cleavable under appropriate conditions within the cell so that the
oligonucleotide is released in a functional form.
[0300] Delivery vehicles or delivery devices for delivering antigen
and oligonucleotides to surfaces have been described. The CpG
immunostimulatory oligonucleotide and/or the antigen and/or other
therapeutics may be administered alone (e.g., in saline or buffer)
or using any delivery vehicles known in the art.
[0301] The term effective amount of a CpG immunostimulatory
oligonucleotide refers to the amount necessary or sufficient to
realize a desired biologic effect. For example, an effective amount
of a CpG immunostimulatory oligonucleotide administered with an
antigen for inducing mucosal immunity is that amount necessary to
cause the development of IgA in response to an antigen upon
exposure to the antigen, whereas that amount required for inducing
systemic immunity is that amount necessary to cause the development
of IgG in response to an antigen upon exposure to the antigen.
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
prophylactic or therapeutic treatment regimen can be planned which
does not cause substantial toxicity and yet is entirely effective
to treat the particular subject. The effective amount for any
particular application can vary depending on such factors as the
disease or condition being treated, the particular CpG
immunostimulatory oligonucleotide being administered the size of
the subject, or the severity of the disease or condition. One of
ordinary skill in the art can empirically determine the effective
amount of a particular CpG immunostimulatory oligonucleotide and/or
antigen and/or other therapeutic agent without necessitating undue
experimentation.
[0302] Subject doses of the compounds described herein for mucosal
or local delivery typically range from about 0.1 .mu.g to 10 mg per
administration, which depending on the application could be given
daily, weekly, or monthly and any other amount of time there
between. More typically mucosal or local doses range from about 10
.mu.g to 5 mg per administration, and most typically from about 100
.mu.g to 1 mg, with 2-4 administrations being spaced days or weeks
apart. More typically, immune stimulant doses range from 1 .mu.g to
10 mg per administration, and most typically 10 .mu.g to 1 mg, with
daily or weekly administrations. Subject doses of the compounds
described herein for parenteral delivery for the purpose of
inducing an antigen-specific immune response, wherein the compounds
are delivered with an antigen but not another therapeutic agent are
typically 5 to 10,000 times higher than the effective mucosal dose
for vaccine adjuvant or immune stimulant applications, and more
typically 10 to 1,000 times higher, and most typically 20 to 100
times higher. Doses of the compounds described herein for
parenteral delivery for the purpose of inducing an innate immune
response or for increasing ADCC or for inducing an antigen specific
immune response when the CpG immunostimulatory oligonucleotides are
administered in combination with other therapeutic agents or in
specialized delivery vehicles typically range from about 0.1 .mu.g
to 10 mg per administration, which depending on the application
could be given daily, weekly, or monthly and any other amount of
time there between. More typically parenteral doses for these
purposes range from about 10 .mu.g to 5 mg per administration, and
most typically from about 100 .mu.g to 1 mg, with 2-4
administrations being spaced days or weeks apart. In some
embodiments, however, parenteral doses for these purposes may be
used in a range of 5 to 10,000 times higher than the typical doses
described above.
[0303] For any compound described herein the therapeutically
effective amount can be initially determined from animal models. A
therapeutically effective dose can also be determined from human
data for CpG oligonucleotides which have been tested in humans
(human clinical trials have been initiated) and for compounds which
are known to exhibit similar pharmacological activities, such as
other adjuvants, e.g., LT and other antigens for vaccination
purposes. Higher doses may be required for parenteral
administration. The applied dose can be adjusted based on the
relative bioavailability and potency of the administered compound.
Adjusting the dose to achieve maximal efficacy based on the methods
described above and other methods as are well-known in the art is
well within the capabilities of the ordinarily skilled artisan.
[0304] 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.
[0305] For use in therapy, an effective amount of the CpG
immunostimulatory oligonucleotide can be administered to a subject
by any mode that delivers the oligonucleotide to the desired
surface, 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,
intramuscular, intranasal, sublingual, intratracheal, inhalation,
ocular, vaginal, and rectal.
[0306] For oral administration, the compounds (i.e., CpG
immunostimulatory oligonucleotides, antigens and other therapeutic
agents) can be formulated readily by combining the active
compound(s) with pharmaceutically acceptable carriers well known in
the art. Such carriers enable the compounds of the invention to be
formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions and the like, for oral ingestion by a
subject to be treated. Pharmaceutical preparations for oral use can
be obtained as solid excipient, optionally grinding a resulting
mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Optionally the oral formulations
may also be formulated in saline or buffers, i.e. EDTA for
neutralizing internal acid conditions or may be administered
without any carriers.
[0307] Also specifically contemplated are oral dosage forms of the
above component or components. The component or components may be
chemically modified so that oral delivery of the derivative is
efficacious. Generally, the chemical modification contemplated is
the attachment of at least one moiety to the component molecule
itself, where said moiety permits (a) inhibition of proteolysis;
and (b) uptake into the blood stream from the stomach or intestine.
Also desired is the increase in overall stability of the component
or components and increase in circulation time in the body.
Examples of such moieties include: polyethylene glycol, copolymers
of ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In:
Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience,
New York, N.Y., pp. 367-383; Newmark, et al., 1982, J. Appl.
Biochem. 4:185-189. Other polymers that could be used are
poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are polyethylene glycol
moieties.
[0308] For the component the location of release may be the
stomach, the small intestine (the duodenum, the jejunum, or the
ileum), or the large intestine. One skilled in the art has
available formulations which will not dissolve in the stomach, yet
will release the material in the duodenum or elsewhere in the
intestine. Preferably, the release will avoid the deleterious
effects of the stomach environment, either by protection of the
oligonucleotide or by release of the biologically active material
beyond the stomach environment, such as in the intestine.
[0309] To ensure full gastric resistance a coating impermeable to
at least pH 5.0 is essential. Examples of the more common inert
ingredients that are used as enteric coatings are cellulose acetate
trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP),
HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit
L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L,
Eudragit S, and Shellac. These coatings may be used as mixed
films.
[0310] A coating or mixture of coatings can also be used on
tablets, which are not intended for protection against the stomach.
This can include sugar coatings, or coatings which make the tablet
easier to swallow. Capsules may consist of a hard shell (such as
gelatin) for delivery of dry therapeutic i.e. powder; for liquid
forms, a soft gelatin shell may be used. The shell material of
cachets could be thick starch or other edible paper. For pills,
lozenges, molded tablets or tablet triturates, moist massing
techniques can be used.
[0311] The therapeutic can be included in the formulation as fine
multi-particulates in the form of granules or pellets of particle
size about 1 mm The formulation of the material for capsule
administration could also be as a powder, lightly compressed plugs
or even as tablets. The therapeutic could be prepared by
compression.
[0312] Colorants and flavoring agents may all be included. For
example, the oligonucleotide may be formulated (such as by liposome
or microsphere encapsulation) and then further contained within an
edible product, such as a refrigerated beverage containing
colorants and flavoring agents.
[0313] One may dilute or increase the volume of the therapeutic
with an inert material. These diluents could include carbohydrates,
especially mannitol, a-lactose, anhydrous lactose, cellulose,
sucrose, modified dextrans and starch. Certain inorganic salts may
be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially
available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and
Avicell.
[0314] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrates include but are not limited to starch, including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0315] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0316] An anti-frictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0317] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0318] To aid dissolution of the therapeutic into the aqueous
environment a surfactant might be added as a wetting agent.
Surfactants may include anionic detergents such as sodium lauryl
sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethomium chloride. The list of
potential non-ionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the
oligonucleotide or derivative either alone or as a mixture in
different ratios.
[0319] 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.
[0320] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0321] 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.
[0322] Also contemplated herein is pulmonary delivery of the
oligonucleotides (or derivatives thereof). The oligonucleotide is
delivered to the lungs of a mammal while inhaling and traverses
across the lung epithelial lining to the blood stream. Other
reports of inhaled molecules include Adjei et al., 1990,
Pharmaceutical Research, 7:565-569; Adjei et al., 1990,
International Journal of Pharmaceutics, 63:135-144 (leuprolide
acetate); Braquet et al., 1989, Journal of Cardiovascular
Pharmacology, 13(suppl. 5):143-146 (endothelin-1); Hubbard et al.,
1989, Annals of Internal Medicine, Vol. III, pp. 206-212
(a1-antitrypsin); Smith et al., 1989, J. Clin. Invest. 84:1145-1146
(a-1-proteinase); Oswein et al., 1990, "Aerosolization of
Proteins", Proceedings of Symposium on Respiratory Drug Delivery
II, Keystone, Colorado, March, (recombinant human growth hormone);
Debs et al., 1988, J. Immunol. 140:3482-3488 (interferon-g and
tumor necrosis factor alpha) and Platz et al., U.S. Pat. No.
5,284,656 (granulocyte colony stimulating factor). A method and
composition for pulmonary delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong
et al.
[0323] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art.
[0324] Some specific examples of commercially available devices
suitable for the practice of this invention are the Ultravent
nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the
Acorn II nebulizer, manufactured by Marquest Medical Products,
Englewood, Colo.; the Ventolin metered dose inhaler, manufactured
by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler
powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
[0325] All such devices require the use of formulations suitable
for the dispensing of oligonucleotide (or derivative). Typically,
each formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy.
Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is contemplated.
Chemically modified oligonucleotide may also be prepared in
different formulations depending on the type of chemical
modification or the type of device employed.
[0326] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise oligonucleotide dissolved in
water at a concentration of about 0.1 to 25 mg of biologically
active oligonucleotide per mL of solution. The formulation may also
include a buffer and a simple sugar (e.g., for oligonucleotide
stabilization and regulation of osmotic pressure). The nebulizer
formulation may also contain a surfactant, to reduce or prevent
surface induced aggregation of the oligonucleotide caused by
atomization of the solution in forming the aerosol.
[0327] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the
oligonucleotide suspended in a propellant with the aid of a
surfactant. The propellant may be any conventional material
employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin Oleic acid may also be useful as a
surfactant.
[0328] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing
oligonucleotide and may also include a bulking agent, such as
lactose, sorbitol, sucrose, or mannitol in amounts which facilitate
dispersal of the powder from the device, e.g., 50 to 90% by weight
of the formulation. The oligonucleotide should most advantageously
be prepared in particulate form with an average particle size of
less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most
effective delivery to the distal lung.
[0329] Nasal delivery of a pharmaceutical composition of the
present invention is also contemplated. Nasal delivery allows the
passage of a pharmaceutical composition of the present invention to
the blood stream directly after administering the therapeutic
product to the nose, without the necessity for deposition of the
product in the lung. Formulations for nasal delivery include those
with dextran or cyclodextran.
[0330] For nasal administration, a useful device is a small, hard
bottle to which a metered dose sprayer is attached. In one
embodiment, the metered dose is delivered by drawing the
pharmaceutical composition of the present invention solution into a
chamber of defined volume, which chamber has an aperture
dimensioned to aerosolize and aerosol formulation by forming a
spray when a liquid in the chamber is compressed. The chamber is
compressed to administer the pharmaceutical composition of the
present invention. In a specific embodiment, the chamber is a
piston arrangement. Such devices are commercially available.
[0331] Alternatively, a plastic squeeze bottle with an aperture or
opening dimensioned to aerosolize an aerosol formulation by forming
a spray when squeezed. The opening is usually found in the top of
the bottle, and the top is generally tapered to partially fit in
the nasal passages for efficient administration of the aerosol
formulation. Preferably, the nasal inhaler will provide a metered
amount of the aerosol formulation, for administration of a measured
dose of the drug.
[0332] 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.
[0333] 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.
[0334] Alternatively, the active compounds may be in powder form
for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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 249:1527-1533, 1990, which is incorporated herein
by reference.
[0339] The CpG immunostimulatory oligonucleotides and optionally
other therapeutics and/or antigens may be administered per se
(neat) or in the form of a pharmaceutically acceptable salt. When
used in medicine the salts should be pharmaceutically acceptable,
but non-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, sulphuric, nitric, phosphoric,
maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric,
methane sulphonic, formic, malonic, succinic,
naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts
can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or calcium salts of the carboxylic acid
group.
[0340] Suitable buffering agents include: acetic acid and a salt
(1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a
salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
Suitable preservatives include benzalkonium chloride (0.003-0.03%
w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and
thimerosal (0.004-0.02% w/v).
[0341] The pharmaceutical compositions of the invention contain an
effective amount of a CpG immunostimulatory oligonucleotide and
optionally antigens and/or other therapeutic agents optionally
included in a pharmaceutically-acceptable carrier. The term
pharmaceutically-acceptable carrier means one or more compatible
solid or liquid filler, diluents 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.
[0342] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
Oligodeoxynucleotides (ODNs)
[0343] The following ODN are used in the examples.
TABLE-US-00003 SEQ ID-NO. Sequence 1
T*C_G*T*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 2
T*C_G*T*C_G*T*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 3
T*C_G*T*C_G*T*T*C_G*G*C*G*C*G*C*C*G 4
T*C_G*T*C_G*A*C_G*A*T*C_G*G*C*G*C_G*C*G*C*C*G 5
T*T*C_G*T*C_G*T*T*T*T_G*T*C_G*T*T 6
T*T*T*C_G*T*C_G*T*T*T*C_G*T*C_G*T*T 7
T*C_G*T*C_G*T*T*T*T*G*A*C_G*T*T*T*T*G*T*C_G*T*T 8 TCG TCG TTT TGT
CGT TTT GTC GTT (all bonds *) 9
T*C_G*C_G*A*C_G*T*T*C_G*G*C*G*C_G*C*G*C*C*G 10 TCG TCG TTT TGA CGT
TTT GTC GTT (all bonds *) 14 TCCAGGACTTCTCTCAGGTT (all bonds *) 15
C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 16
G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 17
T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 18
C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 19
G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 20
T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C*G 21
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C*C 22
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C 23
T*C-G*T*C-G*T*C-G*T*T*C-G*G*C*G*C-G*C*G*C
Materials and Methods:
Oligodeoxynucleotides (ODNs)
[0344] ODNs were purchased from Biospring (Frankfurt, Germany) or
Sigma-Ark (Darmstadt, Germany), and were controlled for identity
and purity by Coley Pharmaceutical GmbH (Langenfeld, Germany). ODNs
were diluted in phosphate-buffered saline (Sigma, Germany), and
stored at -20.degree. C. All dilutions were carried out using
pyrogen-free reagents. SEQ ID NO. 15-23 ODNs were synthesized by
Trilink Biotechnologies.
Cell Purification
[0345] Peripheral blood buffy coat preparations from healthy male
and female human donors were obtained from the Blood Bank of the
University of Dusseldorf (Germany) and from these, PBMC were
purified by centrifugation over Ficoll-Hypaque (Sigma). The
purified PBMC were either used freshly (for most assays) or were
suspended in freezing medium and stored at -70.degree. C. When
required, aliquots of these cells were thawed, washed and
resuspended in RPMI 1640 culture medium (BioWhittaker, Belgium)
supplemented with 5% (v/v) heat inactivated human AB serum
(BioWhittaker, Belgium) or 10% (v/v) heat inactivated FCS, 2 mM
L-glutamine (BioWhittaker), 100 U/ml penicillin and 100 .mu.g/ml
streptomycin (Invitrogen (Karlsruhe, Germany)).
Cytokine Detection
[0346] Thawed or fresh PBMC were seeded on 48 well flat-bottom
plates, or 96 well round-bottom plates, and incubated with ODN in
the concentrations as indicated in a humidified incubator at
37.degree. C. Culture supernatants were collected and if not used
immediately, were frozen at -20.degree. C. until required. Amounts
of cytokines in the supernatants were assessed using commercially
available ELISA Kits (Diaclone, USA) or in-house ELISAs developed
using commercially available antibodies (from Becton
Dickinson/Pharmingen or PBL).
[0347] Studies for Example 13 were conducted as follows:
[0348] Spleens were removed from 6 mice (male, BALB/c). The
splenocytes from each spleen were separated by pushing gently
through a cell sieve (70 .mu.m pore size), and were then pooled.
Splenocytes were added to wells of culture plates. 1.times.10.sup.7
cells in a volume of 900 .mu.l medium were added to each well.
Medium was RPMI 1640 containing 10% fetal bovine serum. 100 .mu.l
aliquots of CpG ODN solutions in medium were added to each well to
give final concentrations in the wells of 0.01-10 .mu.g/ml. After
incubation for 36 hours (37 C, 5% CO.sub.2 incubator), culture
supernatants were collected and assayed for cytokine concentrations
using either the Luminex multiplex assay (IFN.gamma., IP-10, IL-10,
IL-6, TNF.alpha.) or ELISA (IFN.alpha.).
Induction of Antigen-Induced Increase in Nasal Resistance in Guinea
Pigs
[0349] Guinea pigs (male, Hartley) were sensitized with antigen
(ovalbumin, 5 mg both intraperitoneal and subcutaneous) on study
day 0. A boost sensitization (5 mg, intraperitoneal) was given on
study day 4. Guinea pigs were antigen challenged by exposure to
intranasally-administered antigen twice each week for two
consecutive weeks. The first challenge was on study day 14. SEQ ID
NO:7 (lot number AQE-03J-001-M, 0.03-1 mg/kg in 150 .mu.l/kg
saline) was administered intranasally once each week, two days
before the first antigen challenge of the week. With the exception
of the final challenge on study day 24, antigen challenge was with
ovalbumin (1.5 mg/kg in 150 .mu.l/kg saline). Animals were
pretreated with mepyramine (10 mg/kg, intraperitoneal) 30 minutes
before challenge to protect against histamine-induced anaphylaxis.
On study day 24, guinea pigs were anesthetized to allow measurement
of nasal resistance using a Buxco respiratory mechanics system and
software. The final antigen challenge was then made with ovalbumin
(2.5 mg in 250 .mu.l saline) delivered into the nasopharynx.
Animals were pretreated with mepyramine (3 mg/kg, intraperitoneal)
30 minutes before challenge. Nasal resistance was measured for 40
minutes after challenge.
Induction of Antigen-Induced Increase in Nasal Resistance in
Mice
[0350] Mice (male BALB/c) were sensitized on study days 0 and 7
with antigen (ovalbumin, 100 .mu.g, i.p.) with aluminum hydroxide
adjuvant (Pierce Alum i.p.). Mice were antigen challenged daily by
exposure to intranasally-administered antigen (1 mg in 10 .mu.l
saline). The first challenge was on study day 14. SEQ ID NO:7 was
administered intranasally twice. The first dose was given 2 days
before the first antigen challenge. The second dose was given 7
days later. Alternatively, budesonide (an anti-inflammatory,
synthetic corticosteroid) was administered intranasally daily. The
first dose was given 2 days before the first antigen challenge. On
each day, the budesonide dose was administered intranasally 4 hours
before intranasal antigen challenge. Endpoints were measured on
study day 26 (i.e. 7 days after the second dose of SEQ ID NO: 7.
Nasal tissues were taken for histopathological assessment of
inflammation. Separate mice received a final antigen challenge and
incidences of sneezing and nasal rubbing were counted for a 10
minute period after challenge.
Statistical Analysis
[0351] The Mann-Whitney test was used for comparison of data sets
where the populations being compared were not normal. The Dunnitt
multiple comparisons test was used for comparison of data sets to a
single control.
Cytokine Induction in the Mouse In Vivo
[0352] Mice (male, BALB/c) were dosed with CpG ODNs (0.1 and 1
mg/kg) or control vehicle (saline, 25 .mu.l) by intranasal
instillation. Bronchoalveolar lavage fluid and serum (separated
from blood obtained by cardiac puncture) were collected 8 hours and
15 hours after dosing. Cell numbers in bronchoalveolar lavage fluid
were counted with an Advia automated cell counter. Concentrations
of cytokines and chemokines in bronchoalveolar lavage fluid and
serum were assayed using either ELISA (IFN.alpha., IL-12p40) or the
Luminex cytokine multiplex system (IFN.gamma., IP-10, IL-1.alpha.,
IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13,
IL-15, IL-17, GM-CSF, RANTES, TNF.alpha.). Minimum detectable
concentrations were different for each analyte, but were in the
range 0.3-12 pg/ml.
Influenza Virus Induction of Inflammation in Mouse Airways.
[0353] Influenza virus (influenza type A, subtype H1N1, mouse
adapted strain PR8) was a gift from David Woodhouse, Trudeau
Institute, Saranac Lake, N.Y. As a preliminary study to titrate
influenza dose and determine time-course of infection, BALB/c mice
(female) were infected with influenza virus by intranasal
instillation on study day 0. Mice received 50, 200 or 500
egg-infective doses (EID).sub.50 of virus in 40 .mu.l saline.
Airway inflammation was assessed 1, 3, 6, 9 and 14 days after
infection.
Intranasal Administration of CpG ODN and Measurement of Airway
Inflammation
[0354] Mice received a CpG ODN (0.03, 0.3 or 3 mg/kg) by intranasal
instillation in 25 .mu.l saline. Each mouse was dosed twice, 6 days
and 2 days before infection with influenza virus (200 EID.sub.50,
intranasal). This virus dose was selected from the preliminary
study. Airway inflammation and virus load in the lung were assessed
6 days after virus infection. This time point was selected from the
preliminary study. Cells in airways were recovered by
bronchoalveolar lavage. Total leukocyte counts were made with an
Advia automated cell counter (Adiva, Bayer Diagnostics, Zurich,
Switzerland). Differential cell counts were made by light
microscopy of cytocentrifuge preparations stained with
Wright-Giemza stain. Lungs were removed and homogenized with 300
.mu.l sterile PBS. supernatant was collected and virus load was
assayed using an enzyme immunoassay kit (Takara Biomedical, Shiga,
Japan) used to manufacturer's instructions. This assay utilizes a
monoclonal antibody against influenza A virus nuclear protein as
solid phase, and a polyclonal anti-influenza virus detection
antibody.
Example 1
Effects on IFN-.alpha. Secretion by Human PBMC Treated with CpG
ODN
[0355] Methods: Human peripheral blood mononuclear cells from
either three (SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6) or seven (SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3) donors were incubated with CpG
ODN for 48 hours at concentrations indicated. Interferon-alpha
secretion by human PBMC was measured.
[0356] Results: FIG. 1 demonstrates increased production of
IFN-.alpha. upon incubation with CpG ODN. Data represent the
mean+/-SEM. Note that the absolute levels in pg/ml can not be
compared directly, as PBMC from different donors were used and
results from each donor are variable.
Example 2
Stimulation of TLR9-Transfected Cells In Vitro
[0357] Methods: HEK 293 cells transfected with human TLR9 were
incubated with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, or SEQ ID NO:6 for 16 hours. The signal was determined
by a luciferase readout.
[0358] Results: The results are shown in Table 1. The EC50 was
calculated using Sigma Plot (SigmaPlot 2002 for Windows version
8.0). The maximal stimulation index (max SI) was calculated as the
quotient between the highest value of all concentrations tested for
any ODN and the medium control.
TABLE-US-00004 TABLE 1 ODN EC50 max SI SEQ ID NO: 1 2320 22 SEQ ID
NO: 2 4730 20 SEQ ID NO: 3 2400 16 SEQ ID NO: 4 3200 14 SEQ ID NO:
5 4290 13 SEQ ID NO: 6 4580 11
Example 3
Effects of the CpG Oligodeoxynucleotide SEQ ID NO:7 Against
Antigen-Induced Increase in Nasal Resistance in Guinea Pigs
[0359] Methods: To investigate the effects of the CpG
oligodeoxynucleotide SEQ ID NO:7 against antigen induced increases
in nasal resistance in the guinea pig, guinea pigs were sensitized
with antigen, then antigen-challenged nasally. Nasal resistance was
measured for 40 minutes after challenge.
TABLE-US-00005 TABLE 2 Summary of study protocol ##STR00001##
[0360] Results: FIG. 2 demonstrates that antigen challenge caused a
progressive increase in nasal resistance over 40 minutes that was
significantly suppressed in guinea pigs that had been treated with
SEQ ID NO:7 (0.03-1 mg/kg).
Example 4
Effects of CpG Oligonucleotide SEQ ID NO:7 in a Mouse Model of
Allergic Rhinitis
[0361] Methods: BALB/c mice were used to study the effects of SEQ
ID NO:7 on symptoms of allergic rhinitis. After sensitization and
antigen challenge, nasal tissues were taken for histopathological
assessment of inflammation. Separate mice received a final antigen
challenge and incidences of sneezing and nasal rubbing were counted
for a 10 minute period after challenge.
TABLE-US-00006 TABLE 3 Summary of study protocol:
oligonucleotide-treated mice ##STR00002##
TABLE-US-00007 TABLE 4 Summary of study protocol:
budesonide-treated mice ##STR00003##
[0362] Results: Antigen challenge caused sneezing and nasal
rubbing. As demonstrated in FIG. 3 the incidences of both were
suppressed in mice treated with SEQ ID NO:7.
Example 5
Influenza Virus-Induced Airway Inflammation in Mouse Lung: Effects
of Class C CpG Oligodeoxynucleotides
[0363] Introduction: CpG oligodeoxynucleotides (ODNs) induce
immune-modifying cytokines that should provide anti-asthma effects.
Class C CpG ODNs induce higher titers of IFN.alpha. than previous
class B ODNs. Class C CpG ODNs may offer the additional benefit of
suppressing virus-induced exacerbations of asthma. This study
investigated the ability of three class C CpG ODNs to suppress
virus (influenza) load in mouse lungs and virus-induced airways
inflammation.
[0364] Methods: Influenza virus was used to induce airway
inflammation in BALB/c mice. CpG ODNs were administered
intranasally and the protective effect was measured.
[0365] Results: FIG. 4 shows that in a preliminary study to titrate
influenza dose and determine time-course of infection, influenza
virus caused an accumulation of leukocytes in the airways. Peak
inflammation was achieved after 6-9 days. Mice infected with 500
EID50 of virus showed marked weight loss after 6 days and were
sacrificed.
[0366] FIG. 5 demonstrates the protective effects of CpG ODNs on
virus load and virus-induced airways inflammation. Pretreatment
with CpG ODNs before infection with influenza virus (200 EID50)
reduced virus load in the lung as assayed 6 days after infection.
FIG. 6 demonstrates that infection with influenza virus caused an
accumulation of leukocytes in the airways 6 days later. These were
predominantly neutrophils and mononuclear cells (monocytes,
macrophages and lymphocytes). There were very few eosinophils. Cell
accumulation was significantly suppressed in mice pretreated with
any of the CpG ODNs.
Example 6
Effects of Class C CpG Oligodeoxynucleotides Against
Antigen-Induced Airways Inflammation in the Mouse
[0367] The activity of three class C CpG oligodeoxynucleotides
(ODNs) was compared. The class B CpG ODN SEQ ID NO:7 was included
in the study for comparison.
[0368] Methods: Mice (male BALB/c) were sensitized on study days 0
and 7 with antigen (cockroach, 10 .mu.g, intraperitoneal) with
aluminum hydroxide adjuvant (Pierce Alum, intraperitoneal). Mice
were antigen challenged by exposure to intranas ally-administered
antigen (10 .mu.g in 40 .mu.l saline), twice each week for two
consecutive weeks. The first challenge was on study day 21. CpG
ODNs (1, 10 and 100 .mu.g/kg) were administered intranasally once
each week, two days before the first antigen challenge of the
week.
[0369] Airways inflammation was assessed 48 hours after the last
antigen challenge. Cells in airways were recovered by
bronchoalveolar lavage. Differential cell counts were made by an
Advia automated cell counter. Numbers of T cells (Total CD3+ cells,
and CD3+CD4+ cells) were counted by flow cytometry.
TABLE-US-00008 TABLE 5 Summary of study protocol ##STR00004##
TABLE-US-00009 TABLE 6 CpG ODNs tested SEQ ID NO: 7 Semi-soft class
B Lot A25-0313-L1 SEQ ID NO: 1 Semi-soft class C Lot C44-1209-M1B
SEQ ID NO: 2 Semi-soft class C Lot C44-1209-M2D SEQ ID NO: 3
Semi-soft class C Lot C44-1209-M4B
[0370] Results: Antigen challenge caused accumulations of
eosinophils and T cells in the airways (FIGS. 7 and 8). There was
no accumulation of neutrophils. Each of the CpG ODNs caused a
significant suppression of eosinophil accumulation at the highest
dose tested (100 mg/kg) (FIG. 7). Numbers of T cells were also
lower, although the reductions were not generally statistically
significant (FIG. 8).
Example 7
Cytokine Induction by Class C CpG Oligodeoxynucleotides in the
Mouse In Vivo
[0371] The activities of three class C CpG oligodeoxynucleotides
(ODNs)were compared. The class B CpG ODN SEQ ID NO:7 was included
in the study for comparison.
[0372] Methods: Concentrations of cytokines and chemokines in
bronchoalveolar lavage fluid and serum were assayed as described in
materials and methods.
TABLE-US-00010 TABLE 7 Treatment groups Time points Intranasal for
sample CpG oligodeoxynucleotide doses collection Vehicle 8, 15
hours SEQ ID NO: 7 Semi-soft class B Lot A25-0313-L1 0.1, 1 mg/kg
8, 15 hours ODN SEQ ID NO: 1 Semi-soft class C Lot C44-1209-M1B
0.1, 1 mg/kg 8, 15 hours ODN SEQ ID NO: 2 Semi-soft class C Lot
C44-1209-M2D 0.1, 1 mg/kg 8, 15 hours ODN SEQ ID NO: 3 Semi-soft
class C Lot C44-1209-M4B 0.1, 1 mg/kg 8, 15 hours
[0373] Results: FIG. 9 shows cell numbers in bronchoalveolar lavage
fluid. Intranasal instillation of each of the class C ODNs,
especially at 1 mg/kg, showed a trend to causing a very mild
accumulation of leukocytes in bronchoalveolar lavage fluid.
[0374] FIGS. 10-15 show cytokine concentrations in bronchoalveolar
lavage fluid. Intranasal instillation of each of the CpG ODNs
induced measurable titers of IFN.alpha., IFN.gamma..quadrature.,
IP-10, IL-12p40, IL-6 and TNF.alpha. in bronchoalveolar lavage
fluid. Titers of the other analytes measured did not reach
detectable concentrations or concentrations above background
(typically<20 pg/ml, data not shown). The class C ODNs were each
more potent than the class B ODN SEQ ID NO:7 as inducers of
IFN.alpha., IFN.gamma..quadrature., IP-10, IL-12p40, IL-6 and
TNF.alpha.. The increased potency of the class C ODNs was
especially apparent at the 0.1 mg/kg dose level. Of particular
interest was the observation that only the class C ODNs were able
to induce any measurable titers of IFN.alpha. and
IFN.gamma..quadrature. after dosing at 0.1 mg/kg (FIG. 10).
[0375] FIGS. 15-18 show cytokine concentrations in serum.
Intranasal instillation of each of the CpG ODNs induced measurable
titers of IFN.gamma., IL-6 and TNF.alpha..quadrature..quadrature.
in serum. Titers of the other analytes measured did not reach
detectable concentrations (typically<20 pg/ml, data not shown).
When compared with the class B CpG SEQ ID NO:7, each of the three
class C ODNs were more potent inducers of the immune-modifying
cytokines IFN.alpha., IFN.gamma., IP-10, IL-12p40, IL-6 and
TNF.alpha..
Example 8
Effects of CpG Oligodeoxynucleotides SEQ ID NO:2 and SEQ ID NO:7 on
Antigen-Induced IgE Production in the Mouse
[0376] Methods: Mice (male BALB/c) were sensitized on study days 0
and 7 with antigen (ovalbumin, 10 .mu.g, i.p.) and aluminum
hydroxide adjuvant (Pierce Alum, i.p.). Mice received SEQ ID NO:2
or SEQ ID NO:7 on study days -2, 0, 5 and 7 (i.e. two days before
each sensitization and on the day of sensitization). Mice were bled
by cardiac puncture on study day 18. Serum was collected by
centrifugation and assayed by ELISA for ovalbumin-specific IgE and
IgG2a.
TABLE-US-00011 TABLE 8 Treatment groups: Sensitization Treatment n
1 None None 5 2 Antigen Vehicle, i.p. 10 3 Antigen SEQ ID NO: 2, 1
.mu.g/kg, i.p. 10 4 Antigen SEQ ID NO: 2, 10 .mu.g/kg, i.p. 10 5
Antigen SEQ ID NO: 2, 100 .mu.g/kg, i.p. 10 6 Antigen SEQ ID NO: 2,
1000 .mu.g/kg, i.p. 10 7 Antigen SEQ ID NO: 7, 1 .mu.g/kg, i.p. 10
8 Antigen SEQ ID NO: 7, 10 .mu.g/kg, i.p. 10 9 Antigen SEQ ID NO:
7, 100 .mu.g/kg, i.p. 10 10 Antigen SEQ ID NO: 7, 1000 .mu.g/kg,
i.p. 10
TABLE-US-00012 TABLE 9 Summary of study protocol ##STR00005##
[0377] Results: Antigen sensitization resulted in serum titers of
antigen (ovalbumin)-specific IgE and IgG2a. The production of IgE
was suppressed in mice treated with either SEQ ID NO:2 or SEQ ID
NO:7, while the production of IgG2a was potentiated (FIG. 19).
[0378] Conclusions: The data of Example 8 demonstrates that SEQ ID
NO:2 and SEQ ID NO:7 suppress Th2-associated production of IgE in
response to antigen sensitization, and potentiate Th1-associated
IgG2a production. The results of this study provide further
evidence that these CpG oligos can suppress a Th2-type response to
antigen exposure in the mouse.
Example 9
Effects of SEQ ID NO:2 Against Exacerbated Airways Inflammation
Induced by Combined Influenza Infection and Antigen Challenge
[0379] Introduction: The class C CpG oligodeoxynucleotide SEQ ID
NO:2 can suppress influenza virus load and virus-induced airways
inflammation in mice. The present study investigated the protective
effects of SEQ ID NO:2 against the exacerbated airways inflammation
induced by combined influenza virus infection and antigen
challenge.
[0380] Methods: Antigen and virus administrations: Mice (male
BALB/c) were sensitized on study days 0 and 7 with antigen
(cockroach, 10 .mu.g, intraperitoneal) with aluminum hydroxide
adjuvant (Pierce Alum). Mice were antigen challenged by exposure to
intranasally-administered antigen (10 .mu.g in 40 .mu.l saline),
twice each week for three consecutive weeks. The first challenge
was on study day 21. Mice were infected with influenza virus
(influenza type A, subtype H1N1, mouse adapted strain PR8, 200
EID.sub.50 in 40 .mu.l saline) by intranasal instillation on study
day 34 (i.e. before the last pair of antigen challenges).
Alternatively, separate groups of mice received antigen challenge
alone or virus infection alone.
[0381] SEQ ID NO:2 (100 .mu.g/kg) was administered intranasally
once each week, two days before the first antigen challenge of the
week. Airways inflammation was assessed 48 hours after the last
antigen challenge. Cells in airways were recovered by
bronchoalveolar lavage. Differential cell counts were made by light
microscopy from cytocentrifuge preparations stained with
Wright-Giemza stain.
TABLE-US-00013 TABLE 10 Summary of study protocol ##STR00006##
[0382] Results: FIG. 20 shows that infection with influenza virus
alone or antigen challenge alone each caused an increase in the
total number of leukocytes in bronchoalveolar lavage fluid. In
virus-infected mice, this cell accumulation included a marked
neutrophilia, whereas in antigen-challenged mice, the accumulation
included a marked eosinophilia. When compared with mice that
received antigen challenge alone, those that were
antigen-challenged and virus-infected showed an exacerbated
accumulation of leukocytes in bronchoalveolar lavage fluid. This
increased accumulation included both neutrophils and mononuclear
cells. However, these mice showed reduced eosinophilia. Other
researchers have similarly shown that influenza infection can
suppress airways eosinophilia in antigen-challenged mice, and have
hypothesized that this is a Th1-mediated effect (e.g. Wohlleben et
al., 2003).
[0383] Treatment with SEQ ID NO:2 (100 .mu.g/kg) did not suppress
the virus-induced neutrophilia (FIG. 20). This negative finding was
expected since, in an earlier study a higher dose of 300 .mu.g/kg
was most desirable to show anti-virus effects. Furthermore, SEQ ID
NO:2 (100 .mu.g/kg) did significantly suppress antigen-induced
eosinophilia. This positive finding was in agreement with earlier
studies.
[0384] SEQ ID NO:2 (100 .mu.g/kg) significantly suppressed the
exacerbated airways inflammation induced in mice that were both
virus-infected and antigen-challenged. The exacerbated
accumulations of neutrophils and mononuclear cells were both
suppressed. In addition to exacerbated airways inflammation, mice
that were both virus-infected and antigen-challenged showed a
marked loss of body weight. This was significantly suppressed in
mice treated with SEQ ID NO:2.
[0385] Conclusions: In both children and adults with existing
asthma, infections with respiratory tract viruses are important
precipitants for airway obstruction and wheezing. The inflammatory
processes involved are complex. However, virus-induced neutrophil
and mononuclear cell recruitment and activation are implicated in
aggravating the airway obstruction that contributes to these asthma
exacerbations (reviewed by Gem and Busse, Nature Immunology, 2002).
The data of Example 9 demonstrate that SEQ ID NO:2 markedly
suppresses the exacerbated accumulations of neutrophils and
mononuclear cells induced in mice by combined virus infection and
antigen challenge.
Example 10
Guinea Pig Studies
Example 10a
Summary of Guinea Pig AHR Protocol
[0386] Male guinea pigs were sensitized on study days 0 and 4 with
antigen (ovalbumin, 0.5 ml, 1% OVA i.p./s.c.) with aluminum
hydroxide adjuvant. Guinea pigs were antigen challenged by exposure
to inhaled ovalbumin aerosol, twice each week for two consecutive
weeks. The first challenge was on study day 13. CpG ODN or vehicle
(saline, 20 .mu.l) were administered intranasally once each week,
two days before the first antigen challenge of the week. Airways
hyperreactivity was assessed 24 hours after the last antigen
challenge by measuring bronchoconstriction (increase in airway
resistance) to intravenous methacholine. For each animal, a
dose-response curve to methacholine was obtained, and airway
reactivity was quantified as the area under the curve. FIG. 21
shows a schematic of the procedure.
Example 10b
Effect of SEQ ID NO:7 on Airway Resistance and Lung Compliance in
Guinea Pigs
[0387] Method: Guinea pigs were sensitized as described in Example
10. The first challenge was on study day 13. Guinea pigs were given
intranasally either carrier (saline), OVA alone, of concentrations
of SEQ ID NO:7 of 10 .mu.l/kg, 30 .mu.l/kg, 100 .mu.l/kg, or 300
.mu.l/kg, i.t.
[0388] Results: FIG. 22 shows that SEQ ID NO:7 caused a
dose-dependent reduction in AUC-resistance.
Example 10c
Statistical Analysis of the Effect of SEQ ID NO:7 on Airway
Resistance and Lung Compliance in Guinea Pigs
[0389] Method: The Dunnett multiple comparisons test was used to
analyze the data from the experiments in Example 10b. The Dunnett
multiple comparisons test allows comparison of all samples to a
single control group.
[0390] Results: FIG. 23 shows that SEQ ID NO:7 caused a
dose-dependent reduction in AUC-resistance.
Example 10d
Effect of SEQ ID NO:2 on Airway Resistance and Lung Compliance in
Guinea Pigs
[0391] Method: Guinea pigs were sensitized as described in Example
10. The first challenge was on study day 13. Guinea pigs were given
intranasally either carrier (saline), OVA alone, of concentrations
of SEQ ID NO:2 of 10 .mu.l/kg, 30 .mu.l/kg, 100 .mu.l/kg, or 300
.mu.l/kg, i.t.
[0392] Results: FIG. 24 shows that SEQ ID NO:2 caused a
dose-dependent reduction in AUC-resistance.
Example 10e
Statistical Analysis of the Effect of SEQ ID NO:2 on Airway
Resistance and Lung Compliance in Guinea Pigs
[0393] Method: The Dunnett multiple comparisons test was used to
analyze the data from the experiments in Example 10d. The Dunnett
multiple comparisons test allows comparison of all samples to a
single control group
[0394] Results: FIG. 25 shows that SEQ ID NO:2 caused a
dose-dependent reduction in AUC-resistance.
Example 11
[0395] Levels of IL-10, TNF-alpha, interferon-gamma, and IL-6
(pg/ml) produced by human PBMC following exposure of these cells to
the CpG oligonucleotides described herein is shown in the attached
FIG. 27-31. The test oligonucleotides shown in FIG. 27 include SEQ
ID NOs: 10, 9, 13, 14, 1, and 2. The concentration of
oligonucleotide used to produce a particular data point is depicted
along the X-axis (.mu.M).
[0396] As demonstrated in FIG. 27 each of the oligonucleotides
tested in the assays were able to produce different levels and
patterns of IL-10 secretion. Of those tested ODN SEQ ID NO 1 and 2
resulted in dramatically higher induction of IL-10.
[0397] FIG. 28 depicts data relating to TNF-alpha,
interferon-gamma, and IL-6 at three representative doses. More
detailed graphs on these cytokines are depicted in FIGS. 29-31 with
additional oligonucleotide dosages.
Example 12
[0398] Levels of B cell, plasmacytoid dendritic cell and monocyte
activation following exposure of these cells to the CpG
oligonucleotides described herein is shown in the attached FIGS.
32-42. The oligonucleotides examined are depicted in the Figures by
SEQ ID NO and included SEQ ID NO: 9, 13, 10, 14, 2, 1, 11, 12, and
3. The concentration of oligonucleotide used to produce a
particular data point is depicted along the X-axis (.mu.M).
[0399] As demonstrated in FIGS. 32, 34, 35, 40, and 42 the CpG
oligonucleotides tested in the assays were able activate B cells,
as represented by the markers tested. In FIGS. 32 and 33 the CpG
oligonucleotides tested in the assays were able activate NK cells,
as represented by the markers tested. In FIGS. 36, 37, 40, and 41
the CpG oligonucleotides tested in the assays were able activate
monocytes, as represented by the markers tested. In FIGS. 36 and 39
the CpG oligonucleotides tested in the assays were able activate
plasmacytoid dendritic cells, as represented by the markers
tested.
[0400] All five ODN having a semi soft backbone that were tested in
the assays showed an increased potency in the assays (IFN-alpha,
IP-10, IL-10) compared to SEQ ID NO. 9 (fully phosphorothioate
backbone). The potency of these semi-soft ODN is also increased in:
Monocyte activation (CD80, CD86 expression), pDC activation (CD86
expression), Intracellular IP-10 (Monocytes and B cells), IL-6
secretion, and B cell activation (CD80, CD86 expression). For
instance, at approximately equivalent or lower concentrations most
of the tested ODN resulted in better induction of the cell surface
markers than the fully phosphorothioate SEQ ID NO. 9.
Example 13
[0401] The aim of this study was to investigate the biological
activity of selected fragments (putative metabolites) of SEQ ID NO:
2. Activity was determined by measuring the ability of each
fragment to induce secretion of TLR9-associated cytokines from
mouse splenocytes in vivo.
[0402] Stimulation of cytokine secretion by fragments of SEQ ID
NO.: 2 is shown in the attached FIGS. 43-45. The oligonucleotides
examined are depicted in the Figures by SEQ ID NO and include SEQ
ID NO: 15-23. The concentration of oligonucleotide used to produce
a particular data point is depicted along the X-axis
(.mu.g/ml).
[0403] As demonstrated in FIGS. 43-45 SEQ ID NO: 2 and the
fragments (putative metabolites, SEQ ID NOs:15-23) tested all
induced the TLR9-associated cytokines IFN.alpha., IFN.gamma.,
IP-10, IL-6, IL-10 and TNF.alpha. from mouse splenocytes in vitro
(FIGS. 43, 44 and 45). This data demonstrates that each of the
fragments retained biological activity.
[0404] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the invention.
Sequence CWU 1
1
68123DNAArtificial sequenceSynthetic oligonucleotide 1tcgtcgacgt
tcggcgcgcg ccg 23223DNAArtificial sequenceSynthetic oligonucleotide
2tcgtcgtcgt tcggcgcgcg ccg 23318DNAArtificial sequenceSynthetic
oligonucleotide 3tcgtcgttcg gcgcgccg 18423DNAArtificial
sequenceSynthetic oligonucleotide 4tcgtcgacga tcggcgcgcg ccg
23517DNAArtificial sequenceSynthetic oligonucleotide 5ttcgtcgttt
tgtcgtt 17618DNAArtificial sequenceSynthetic oligonucleotide
6tttcgtcgtt tcgtcgtt 18724DNAArtificial sequenceSynthetic
oligonucleotide 7tcgtcgtttt gacgttttgt cgtt 24824DNAArtificial
sequenceSynthetic oligonucleotide 8tcgtcgtttt gtcgttttgt cgtt
24922DNAArtificial sequenceSynthetic oligonucleotide 9tcgcgacgtt
cggcgcgcgc cg 221024DNAArtificial sequenceSynthetic oligonucleotide
10tcgtcgtttt gacgttttgt cgtt 241120DNAArtificial sequenceSynthetic
oligonucleotide 11tccaggactt ctctcaggtt 201222DNAArtificial
sequenceSynthetic oligonucleotide 12cgtcgtcgtt cggcgcgcgc cg
221321DNAArtificial sequenceSynthetic oligonucleotide 13gtcgtcgttc
ggcgcgcgcc g 211420DNAArtificial sequenceSynthetic oligonucleotide
14tcgtcgttcg gcgcgcgccg 201522DNAArtificial sequenceSynthetic
oligonucleotide 15cgtcgtcgtt cggcgcgcgc cg 221621DNAArtificial
sequenceSynthetic oligonucleotide 16gtcgtcgttc ggcgcgcgcc g
211720DNAArtificial sequenceSynthetic oligonucleotide 17tcgtcgttcg
gcgcgcgccg 201819DNAArtificial sequenceSynthetic oligonucleotide
18cgtcgttcgg cgcgcgccg 191918DNAArtificial sequenceSynthetic
oligonucleotide 19gtcgttcggc gcgcgccg 182017DNAArtificial
sequenceSynthetic oligonucleotide 20tcgttcggcg cgcgccg
172122DNAArtificial sequenceSynthetic oligonucleotide 21tcgtcgtcgt
tcggcgcgcg cc 222217DNAArtificial sequenceSynthetic oligonucleotide
22tcgtcgtcgt tcggcgc 172321DNAArtificial sequenceSynthetic
oligonucleotide 23tcgtcgtcgt tcggcgcgcg c 212417DNAArtificial
sequenceSynthetic oligonucleotide 24ttcgncgnnn ngncgtt
172517DNAArtificial sequenceSynthetic oligonucleotide 25ttcgtcgttt
ngtcgtt 172624DNAArtificial sequenceSynthetic oligonucleotide
26tcgncgnnnn ncgnnnnnnn nncg 242724DNAArtificial sequenceSynthetic
oligonucleotide 27tcgtcgtnnn tcggcgcnnn gccg 242823DNAArtificial
sequenceSynthetic oligonucleotide 28tcgncgncgn tcggcgcgnn nnn
232910DNAArtificial sequenceSynthetic oligonucleotide 29tcgtcgtttt
10308DNAArtificial sequenceSynthetic oligonucleotide 30tcgtcgtt
83110DNAArtificial sequenceSynthetic oligonucleotide 31tcgacgtcga
103210DNAArtificial sequenceSynthetic oligonucleotide 32tcgtcgacga
103310DNAArtificial sequenceSynthetic oligonucleotide 33tcgcgacgtt
103410DNAArtificial sequenceSynthetic oligonucleotide 34tcgcgtcgtt
103512DNAArtificial sequenceSynthetic oligonucleotide 35caatatttat
tg 123611DNAArtificial sequenceSynthetic oligonucleotide
36ccgttttgtg g 113713DNAArtificial sequenceSynthetic
oligonucleotide 37cggcgccgtg ccg 133814DNAArtificial
sequenceSynthetic oligonucleotide 38cggcgccgtt gccg
143910DNAArtificial sequenceSynthetic oligonucleotide 39cggcgcgccg
104010DNAArtificial sequenceSynthetic oligonucleotide 40cggcgtgccg
104112DNAArtificial sequenceSynthetic oligonucleotide 41cggcggccgc
cg 124214DNAArtificial sequenceSynthetic oligonucleotide
42cggcgtcgcc gccg 144315DNAArtificial sequenceSynthetic
oligonucleotide 43cgtcgacggg acggg 154415DNAArtificial
sequenceSynthetic oligonucleotide 44cgtcgacgtg acggg
154515DNAArtificial sequenceSynthetic oligonucleotide 45gagagttggg
ctctc 154611DNAArtificial sequenceSynthetic oligonucleotide
46gtcgaggagg t 114710DNAArtificial sequenceSynthetic
oligonucleotide 47taatatatta 104812DNAArtificial sequenceSynthetic
oligonucleotide 48taatatccat ta 124912DNAArtificial
sequenceSynthetic oligonucleotide 49taatatttat ta
125012DNAArtificial sequenceSynthetic oligonucleotide 50ggcgcgctgc
cg 125136DNAArtificial sequenceSynthetic oligonucleotide
51nncgnnnnnn nnnnnnnnnn nnnnnnnnnn nngcnn 365248DNAArtificial
sequenceSynthetic oligonucleotide 52tcgtcgtcgt cgnnnnnnnn
nnnnnnnnnn nnnnnngctg ctgctgct 485311DNAArtificial
sequenceSynthetic oligonucleotide 53tcgtcgtttt a
115413DNAArtificial sequenceSynthetic oligonucleotide 54cggcgccgtg
ccg 135513DNAArtificial sequenceSynthetic oligonucleotide
55cggcgtcgtg ccg 135624DNAArtificial sequenceSynthetic
oligonucleotide 56tcgtcgtttt acggcgccgt gccg 245724DNAArtificial
sequenceSynthetic oligonucleotide 57tcgtcgtttt acggcgtcgt gccg
245812DNAArtificial sequenceSynthetic oligonucleotide 58cggcgcgcgc
cg 125912DNAArtificial sequenceSynthetic oligonucleotide
59cggcggccgc cg 126012DNAArtificial sequenceSynthetic
oligonucleotide 60cgacgatcgt cg 126112DNAArtificial
sequenceSynthetic oligonucleotide 61cgacgtacgt cg
126212DNAArtificial sequenceSynthetic oligonucleotide 62cgcgcgcgcg
cg 126312DNAArtificial sequenceSynthetic oligonucleotide
63gcgcgcgcgc gc 126412DNAArtificial sequenceSynthetic
oligonucleotide 64ccccccgggg gg 126512DNAArtificial
sequenceSynthetic oligonucleotide 65ggggggcccc cc
126610DNAArtificial sequenceSynthetic oligonucleotide 66cccccggggg
106710DNAArtificial sequenceSynthetic oligonucleotide 67gggggccccc
106810DNAArtificial sequenceSynthetic oligonucleotide 68tcntcntttt
10
* * * * *