U.S. patent application number 12/757425 was filed with the patent office on 2011-06-30 for synergistic stimulation of the immune system using immunostimulatory oligonucleotides and/or immunomer compounds in conjunction with cytokines and/or chemotherapeutic agents or radiation therapy.
This patent application is currently assigned to Idera Pharmaceuticals, Inc.. Invention is credited to Sudhir Agrawal, Ekambar Kandimalla.
Application Number | 20110158937 12/757425 |
Document ID | / |
Family ID | 34107742 |
Filed Date | 2011-06-30 |
United States Patent
Application |
20110158937 |
Kind Code |
A1 |
Kandimalla; Ekambar ; et
al. |
June 30, 2011 |
SYNERGISTIC STIMULATION OF THE IMMUNE SYSTEM USING
IMMUNOSTIMULATORY OLIGONUCLEOTIDES AND/OR IMMUNOMER COMPOUNDS IN
CONJUNCTION WITH CYTOKINES AND/OR CHEMOTHERAPEUTIC AGENTS OR
RADIATION THERAPY
Abstract
The invention provides optimized methods and compositions for
enhancing the immune response caused by immunostimulatory compounds
used for the treatment of disease such as, but not limited to,
treatment of cancer, autoimmune disorders, asthma, respiratory
allergies, food allergies and infectious diseases in a patient. The
optimized methods according to the invention provide synergy
between the therapeutic effects of immunostimulatory
oligonucleotides and immunomer compounds in accordance with the
invention, and the therapeutic effect of cytokine immunotherapy
and/or chemotherapeutic agents and/or radiation.
Inventors: |
Kandimalla; Ekambar;
(Southboro, MA) ; Agrawal; Sudhir; (Shrewsbury,
MA) |
Assignee: |
Idera Pharmaceuticals, Inc.
|
Family ID: |
34107742 |
Appl. No.: |
12/757425 |
Filed: |
April 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10892550 |
Jul 15, 2004 |
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12757425 |
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60487529 |
Jul 15, 2003 |
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60503242 |
Sep 15, 2003 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61K 38/2013 20130101;
A61P 31/00 20180101; C12N 2320/31 20130101; A61K 2039/55561
20130101; A61P 35/00 20180101; A61K 39/39 20130101; C12N 15/111
20130101; C12N 15/117 20130101; A61P 37/04 20180101; A61N 2005/1098
20130101; A61K 38/2013 20130101; A61P 37/08 20180101; A61K 2300/00
20130101; A61K 2039/55533 20130101; C12N 2310/17 20130101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 37/04 20060101 A61P037/04; A61P 35/00 20060101
A61P035/00; A61P 37/08 20060101 A61P037/08; A61P 31/00 20060101
A61P031/00 |
Claims
1. A method for synergistically stimulating an immune response in a
patient comprising administering to the patient at least one
immunomer compound and IL-2, wherein the immunomer compound
comprises two oligonucleotides linked together at their 3' ends, an
internucleotide linkage, or a functionalized nucleobase or sugar by
a non-nucleotidic linker, wherein at least one of the
oligonucleotides is an immunostimulatory oligonucleotide having an
accessible 5' end and comprising an immunostimulatory dinucleotide
selected from the group consisting of CpG, C*pG, CpG*, and C*pG*,
wherein C is cytidine or 2'-deoxycytidine, C* is 2'-deoxythymidine,
arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine,
2'-O-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other
non-natural pyrimidine nucleoside, G is guanosine or
2'-deoxyguanosine, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, or other non-natural purine
nucleoside, and p is an internucleoside linkage selected from the
group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate.
2. The method of claim 1, further comprising administering an
antigen to the patient.
3. The method of claim 2 wherein said antigen is an antigen
associated with cancer, infectious disease or allergy.
4. The method according to claim 1, wherein administration of said
combination synergistically stimulates the production of
cytokines.
5. The method according to claim 4, wherein the cytokines are one
or more cytokines selected from the group consisting of IL-12 and
IFN-.gamma., IFN-.alpha., IFN-.beta. or combinations thereof.
6. The method according to claim 1, wherein the immunostimulatory
oligonucleotide comprises an immunostimulatory domain having the
structure 5'-Nn-N1-Y-Z-N1-Nn-3' (III) wherein: Y is cytidine, 2'
deoxycytidine arabinocytidine, 2'-deoxythymidine,
2'-deoxy-2'-substituted-arabinocytidine,
2'-O-substituted-arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine or other
non-natural pyrimidine nucleoside; Z is guanosine or
2'-deoxyguanosine, 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'deoxyinosine or other
non-natural purine nucleoside, N1, at each occurrence, is
preferably a naturally occurring or a synthetic nucleoside or an
immunostimulatory moiety; Nn, at each occurrence, is a naturally
occurring nucleoside or an immunostimulatory moiety; provided that
at least one N1 or Nn is an immunostimulatory moiety and that the
5' N1 includes a nucleobase; and wherein n is a number from
0-30.
7. The method according to claim 6, wherein the immunostimulatory
moiety is selected from the group consisting of abasic nucleosides,
arabinonucleosides, 2'-deoxyuridine, .alpha.-deoxyribonucleosides,
.beta.-L-deoxyribonucleosides, and nucleosides linked by a modified
internucleoside linkage to the adjacent nucleoside on the 3' side,
the modified internucleotide linkage being selected from the group
consisting of C2-C18 alkyl linker, poly(ethylene glycol) linkage,
2-aminobutyl-1,3-propanediol linker, 2'-5' internucleoside linkage,
methylphosphonate internucleoside linkage; methylphosphonothioates,
phosphotriesters, phosphothiotriesters, phosphorothioates,
phosphorodithioates, triester prodrugs, sulfones, sulfonamides,
sulfamates, formacetal, N-methylhydroxylamine, carbonate,
carbamate, morpholino, boranophosphonate, phosphoramidates,
especially primary amino-phosphoramidates, N3 phosphoramidates and
N5 phosphoramidates, and stereospecific linkages, nucleosides
having sugar modifications, 2'-substituted pentose sugars
including, without limitation, 2'-O-methylribose,
2'-O-methoxyethylribose, 2'-O-propargylribose, and
2'-deoxy-2'-fluororibose; 3'-substituted pentose sugars, including,
without limitation, 3'-O-methylribose; 1',2'-dideoxyribose;
arabinose; substituted arabinose sugars, hexose sugars, and
alpha-anomers, peptide nucleic acids (PNA), peptide nucleic acids
with phosphate groups (PHONA), locked nucleic acids (LNA),
morpholinonucleic acids, and oligonucleotides having backbone
linker sections having a length of from about 2 angstroms to about
200 angstroms, alkyl linkers or amino linkers, DNA isoforms,
.beta.-L-deoxyribonucleosides, .alpha.-deoxyribonucleosides,
nucleosides having unnatural internucleoside linkage positions, and
nucleosides having modified heterocyclic bases.
8. The method according to claim 1 or 6, wherein the other
non-natural pyrimidine nucleoside has the structure (I):
##STR00163## wherein: D is a hydrogen bond donor; D' is selected
from the group consisting of hydrogen, hydrogen bond donor,
hydrogen bond acceptor, hydrophilic group, hydrophobic group,
electron withdrawing group and electron donating group, excluding
bromine; A is a hydrogen bond acceptor or a hydrophilic group; A is
a hydrogen bond acceptor or a hydrophilic group; A' is selected
from the group consisting of hydrogen bond acceptor, hydrophilic
group, hydrophobic group, electron withdrawing group and electron
donating group; X is carbon or nitrogen; and S' is a pentose or
hexose sugar ring or a non-naturally occurring sugar.
9. The method according to claim 8, wherein the sugar ring is
derivatized with a phosphate moiety, modified phosphate moiety, or
other non-nucleotidic linker moiety suitable for linking the
pyrimidine nucleoside to another nucleoside or nucleoside
analog.
10. The method according to claim 8, wherein the hydrogen bond
donors are selected from the group consisting of --NH--,
--NH.sub.2, --SH and --OH.
11. The method according to claim 8, wherein the hydrogen bond
acceptors are selected from the group consisting of C.dbd.O,
C.dbd.S, and the ring nitrogen atoms of an aromatic
heterocycle.
12. The method according to claim 8, wherein the non-naturally
occurring pyrimidine base is selected from the group consisting of
5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine,
N4-ethylcytosine, and 4-thiouracil.
13. The method according to claim 8, wherein the non-naturally
occurring sugar is selected from arabinose and arabinose
analogs.
14. The method according to claim 1 or 6, wherein the other
non-natural purine nucleoside has the structure (II): ##STR00164##
wherein: D is a hydrogen bond donor; D' is selected from the group
consisting of hydrogen, hydrogen bond donor, and hydrophilic group;
A is a hydrogen bond acceptor or a hydrophilic group; X is carbon
or nitrogen; each L is independently selected from the group
consisting of C, O, N and S; and S' is a pentose or hexose sugar
ring, or a non-naturally occurring sugar.
15. The method according to claim 14, wherein the sugar ring is
derivatized with a phosphate moiety, modified phosphate moiety, or
other linker moiety suitable for linking the pyrimidine nucleoside
to another nucleoside or nucleoside analog.
16. The method according to claim 14, wherein the hydrogen bond
donors are selected from the group consisting of --NH--,
--NH.sub.2, --SH and --OH.
17. The method according to claim 14, wherein the hydrogen bond
acceptors are selected from the group consisting of C.dbd.O,
C.dbd.S, --N.dbd. and the ring nitrogen atoms of an aromatic
heterocycle.
18. The method according to claim 14, wherein the non-naturally
occurring purine is 6-thioguanine or 7-deazaguanine
19. The method according to claim 1, wherein the non-nucleotidic
linker is selected from the group consisting of a linker from about
2 angstroms to about 200 angstroms in length, a metal, a soluble or
insoluble biodegradable polymer bead, an organic moiety having
functional groups that permit attachment to the 3'-terminal
nucleoside of the oligonucleotide, a biomolecule, a cyclic or
acyclic small molecule, an aliphatic or aromatic hydrocarbon,
either of which optionally can include, either in the linear chain
connecting the oligonucleotides or appended to it, one or more
functional groups selected from the group consisting of hydroxy,
amino, thiol, thioether, ether, amide, thioamide, ester, urea, and
thiourea; amino acids, carbohydrates, cyclodextrins, adamantane,
cholesterol, haptens antibiotics, glycerol or a glycerol homolog of
the formula HO--(CH.sub.2).sub.o--CH(OH)--(CH.sub.2).sub.p--OH,
wherein o and p independently are integers from 1 to about 6, and a
derivative of 1,3-diamino-2-hydroxypropane.
20. A method for synergistically stimulating an immune response in
a patient comprising administering to the patient at least one
immunomer compound and IL-2, wherein the immunomer compound has the
following structure (SEQ ID NO: 48): ##STR00165## wherein the
immunomer compound has internucleoside linkages selected from the
group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate, wherein the immunomer and the IL-2 are
synergistic in generating secretion of IL-6, IL-12 and
IFN-.gamma..
21. The method of claim 20, further comprising administering an
antigen to the patient.
22. The method of claim 21 wherein said antigen is an antigen
associated with cancer, infectious disease or allergy.
23. The method according to claim 21 or 22, wherein the antigen is
conjugated to the immunomer at a position other than the accessible
5' end.
24. A method for synergistically stimulating an immune response in
a patient comprising administering to the patient at least one
immunomer compound and IL-2, wherein the immunomer compound
comprises two oligonucleotides linked together at their 3' ends, an
internucleotide linkage, or a functionalized nucleobase or sugar by
a non-nucleotidic linker, wherein at least one of the
oligonucleotides is an immunostimulatory oligonucleotide having an
accessible 5' end and comprising an immunostimulatory dinucleotide
C*pG*, wherein C* is arabinocytosine or 2'-deoxy-2-substituted
arabincytosine, G* is arabinoguanosine or 2'-deoxy-2'-substituted
arabinguanosine, or 2'-deoxy-7-deazaguanosine or
2'-deoxy-6-thioguanosine, or 2'-deoxyinosine, and p is an
internucleoside linkage selected from the group consisting of
phosphodiester, phosphorothioate and phosphorodithioate, wherein
the immunomer and the IL-2 are synergistic in generating secretion
of IL-6, IL-12 and IFN-.gamma..
25. A method for synergistically stimulating an immune response in
a patient comprising administering to the patient at least one
immunomer compound and IL-2, wherein the immunomer compound has the
following structure (SEQ ID NO: 48): ##STR00166## wherein the
immunomer compound has internucleoside linkages selected from the
group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate, and wherein administration of said immunomer
compound and IL-2 synergistically stimulate the production of
cytokines.
26. The method according to claim 25, wherein the cytokines are one
or more cytokines selected from the group consisting of IL-12 and
IFN-.gamma., IFN-.alpha., IFN-.beta. or combinations thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/892,550, filed Jul. 15, 2004, which claims the benefit of
U.S. Provisional Application No. 60/487,529, filed Jul. 15, 2003,
and U.S. Provisional Application No. 60/503,242, Sep. 15, 2003,
which are incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION,
[0002] 1. Field of the Invention
[0003] The invention relates to the use of immunomer compounds and
immunostimulatory oligonucleotides as therapeutic agents.
[0004] 2. Summary of the Related Art
[0005] Recently, several researchers have demonstrated the validity
of the use of oligonucleotides as immunostimulatory agents in
immunotherapy applications. The observation that phosphodiester and
phosphorothioate oligonucleotides can induce immune stimulation has
created interest in developing these compounds as a therapeutic
tool. These efforts have focused on phosphorothioate
oligonucleotides containing the natural dinucleotide CpG. Kuramoto
et al., Jpn. J. Cancer Res. 83:1128-1131 (1992) teaches that
phosphodiester oligonucleotides containing a palindrome that
includes a CpG dinucleotide can induce interferon-alpha and gamma
synthesis and enhance natural killer activity. Krieg et al., Nature
371:546-549 (1995) discloses that phosphorothioate CpG-containing
oligonucleotides are immunostimulatory. Liang et al., J. Clin.
Invest. 98:1119-1129 (1996) discloses that such oligonucleotides
activate human B cells. Moldoveanu et al., Vaccine 16:1216-124
(1998) teaches that CpG-containing phosphorothioate
oligonucleotides enhance immune response against influenza virus.
McCluskie and Davis, J. Immunol. 161:4463-4466 (1998) teaches that
CpG-containing oligonucleotides act as potent adjuvants, enhancing
immune response against hepatitis B surface antigen.
[0006] Other modifications of CpG-containing phosphorothioate
oligonucleotides can also affect their ability to act as modulators
of immune response. See, e.g., Zhao et al., Biochem. Pharmacol.
(1996) 51:173-182; Zhao et al., Biochem Pharmacol. (1996)
52:1537-1544; Zhao et al., Antisense Nucleic Acid Drug Dev. (1997)
7:495-502; Zhao et al., Bioorg. Med. Chem. Lett. (1999)
9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000)
10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000)
10:2585-2588; Yu et al., Bioorg. Med. Chem. Lett. (2001)
11:2263-2267; and Kandimalla et al., Bioorg. Med. Chem. (2001)
9:807-813. U.S. Pat. No. 6,426,334 shows the promise of these
compounds as anti-cancer agents.
[0007] Another means by which an immune response may be modulated
is through the therapeutic use of cytokines. Cytokines are soluble
molecules that cells of the immune system produce to control
reactions between other cells. Thus, cytokines are regulators of
humoral and cellular immunity. An understanding of how T cells
mediate the immune response is critical in order to modulate the
response. CD4+ T helper (Th) cells differentiate along either the
Th1 or Th2 pathway. The Th1 pathway is important for the generation
of cell-mediated immunity and is characterized by the production
of, for example, .gamma.-interferon and interleukin-2 (IL-2). The
Th2 response is important for the generation of humoral immunity
and is characterized by the production of, for example, IL-4 and
IL-5. The Th1 response is known to be critical for immune system
defense against infections, e.g., viral infections, and immune
system surveillance of the body for the removal of neoplastic
cells.
[0008] Krieg, A., M. et al. (U.S. Pat. No. 6,429,199) and Krieg,
A., M. et al. (U.S. Pat. No. 6,218,371) purport to teach the
co-administration of immunostimulatory CpG oligonucleotides and
cytokines, particularly GM-CSF. Decker et al. (Experimental
Hematology 28:558-565 (2000)), demonstrate that the
co-adminstration of IL-2 with CpG oligonucleotides increases
TNF-.alpha. and IL-6 production in B-chronic lymphocytic (B-CLL)
cells but not in normal B-cells.
[0009] These reports make clear that there remains a need to be
able to further optimize the therapeutic effectiveness of
immunostimulatory oligonucleotides for the treatment of disease and
enhance the anticancer activity of immunostimulatory
oligonucleotides.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides optimized methods, compositions and
treatment regimens for enhancing the immune response caused by
immunostimulatory compounds used for the treatment of disease such
as, but not limited to, treatment of cancer, autoimmune disorders,
asthma, respiratory allergies, food allergies and infectious
diseases in a patient. The optimized methods according to the
invention provide synergy between the therapeutic effects of
immunostimulatory oligonucleotides in accordance with the
invention, and the therapeutic effect of cytokine immunotherapy
and/or chemotherapeutic agents. Modification of an
immunostimulatory oligonucleotide to optimally present 5' ends
dramatically enhances its anti-cancer activity. Such an
oligonucleotide is referred to herein as an "immunomer", which may
contain one or more immunostimulatory oligonucleotide.
[0011] In a first aspect, therefore, the invention provides methods
for treating cancer in a cancer patient comprising administering to
the patient an immunostimulatory oligonucleotide and/or immunomer
compound in combination with a chemotherapeutic agent, wherein the
immunostimulatory oligonucleotide and/or immunomer compound and the
chemotherapeutic agent create a synergistic therapeutic effect.
[0012] In a further aspect, the invention provides a method for
synergistically stimulating an immune response in a patient. The
method comprises administering to a patient a combination of a
therapeutically effective synergistic amount of at least one
immunomer compound or immunostimulatory oligonucleotide in
accordance with the invention and a therapeutically effective
synergistic amount of IL-2 (and/or an agent that induces IL-2
production in situ, such as a DNA vaccine or expression vector
expressing IL-2), wherein administration of said combination
synergistically stimulates the production of cytokines in a
patient. Preferred cytokines that are synergistically stimulated in
accordance with the invention are selected from the group
consisting of, IL-12 and interferon-.gamma. (IFN-.gamma.),
IFN-.alpha., IFN-.beta. or combinations thereof.
[0013] In accordance with the invention, an "immunomer" refers to
any compound comprising at least two oligonucleotides linked
directly at their 3' ends, or directly via internucleoside
linkages, or directly at a functionalized nucleobase or sugar, or
that are indirectly linked together via a non-nucleotidic linker,
wherein at least one of the oligonucleotides, in the context of the
immunomer compound, is an immunostimulatory oligonucleotide having
an accessible 5' end. In the context of the invention, an
immunostimulatory oligonucleotide is an oligonucleotide that
comprises at least one of an immunostimulatory CpG dinucleotide, an
immunostimulatory domain, or other immunostimulatory moiety. As
used herein, the term "accessible 5' end" means that the 5' end of
the oligonucleotide is sufficiently available such that the factors
that recognize and bind to immunomer compounds or immunostimulatory
oligonucleotides and stimulate the immune system have access to the
5' end. Such immunostimulatory oligonucleotides may include
secondary structures, provided that the 5' end remains
accessible.
[0014] In some embodiments, the immunostimulatory oligonucleotide
and/or immunomer compound used in the method according to the
invention comprises an immunostimulatory dinucleotide selected from
the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C is
cytidine or 2'-deoxycytidine, C* is 2'-deoxythymidine.
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other
non-natural pyrimidine nucleosides, or
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
G is guanosine or 2'-deoxyguanosine, G* is 2'
deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, or other non-natural purine
nucleoside, and p is an internucleoside linkage selected from the
group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate. In certain preferred embodiments, the
immunostimulatory dinucleotide is not CpG.
[0015] In some embodiments, the immunostimulatory oligonucleotide
and/or immunomer compound used in the method according to the
invention comprises an immunostimulatory domain of formula
(III):
5'-Nn-N1-Y-Z-N1-Nn-3' (III)
[0016] wherein:
[0017] Y is cytidine, 2'-deoxythymidine, 2'-deoxycytidine,
arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine,
2'-O-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other
non-natural pyrimidine nucleosides, or
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
[0018] Z is guanosine or 2'-deoxyguanosine, is 2'
deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other
non-natural purine nucleoside
[0019] N1, at each occurrence, is preferably a naturally occurring
or a synthetic nucleoside or an immunostimulatory moiety selected
from the group consisting of abasic nucleosides,
arabinonucleosides, 2'-deoxyuridine, .alpha.-deoxyribonucleosides,
.beta.-L-deoxyribonucleosides, and nucleosides linked by a
phosphodiester or modified internucleoside linkage to the adjacent
nucleoside on the 3' side, the modified internucleotide linkage
being selected from, without limitation, a linker having a length
of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl
linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol
linker, glyceryl linker, 2'-5' internucleoside linkage, and
phosphorothioate, phosphorodithioate, or methylphosphonate
internucleoside linkage;
[0020] Nn, at each occurrence, is independently a naturally
occurring nucleoside or an immunostimulatory moiety, preferably
selected from the group consisting of abasic nucleosides,
arabinonucleosides, 2'-deoxyuridine, a-deoxyribonucleosides,
2'-O-substituted ribonucleosides, and nucleosides linked by a
modified internucleoside linkage to the adjacent nucleoside on the
3' side, the modified internucleotide linkage being selected from
the group consisting of amino linker, C2-C18 alkyl linker,
poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker,
glyceryl linker, 2'-5' internucleoside linkage, and
methylphosphonate internucleoside linkage;
[0021] provided that at least one N1 or Nn is an immunostimulatory
moiety;
[0022] wherein n is a number from 0-30;
[0023] wherein the 3'nucleoside is optionally linked directly or
via a non-nucleotidic linker to another oligonucleotide, which may
or may not be immunostimulatory.
[0024] In a second aspect, the invention provides a method for
treating cancer in a cancer patient comprising administering an
immunostimulatory oligonucleotide and/or immunomer conjugate, which
comprises an immunostimulatory oligonucleotide and/or immunomer
compound, as described above, and a cancer antigen conjugated to
the immunostimulatory oligonucleotide and/or immunomer compound at
a position other than the accessible 5' end, in combination with a
chemotherapeutic agent.
[0025] In a third aspect, the invention provides pharmaceutical
formulations comprising an immunostimulatory oligonucleotide or
immunostimulatory oligonucleotide conjugate and/or an immunomer
compound or immunomer conjugate according to the invention, a
chemotherapeutic agent and a physiologically acceptable
carrier.
[0026] In a fourth aspect, the invention provides a method for
sensitizing cancer cells to ionizing radiation. The method
according to this aspect of the invention comprises administering
to a mammal an immunostimulatory oligonucleotide or an immunomer
compound according to the invention and treating the animal with
ionizing radiation.
[0027] In a fifth aspect, the invention provides a method for
synergistically stimulating an immune response in a patient
comprising administering to a patient a therapeutically effective
synergistic amount of at least one immunomer compound or
immunostimulatory oligonucleotide in combination with a
therapeutically effective synergistic amount of IL-2, (and
optionally an antigen), wherein administration of said combination
synergistically stimulates the production cytokines in a patient.
Preferred cytokines that are synergistically stimulated in
accordance with the invention are selected from the group
consisting of IL-12 and interferon-.gamma., IFN-.alpha., IFN-.beta.
or combinations thereof. In certain embodiments of this second
aspect of the invention, the antigen is operationally associated
with the immunomer compound at a position other than the accessible
5' end.
[0028] In a sixth aspect of the invention, at least one
immunostimulatory oligonucleotide that is not an immunomer compound
is used in combination with a therapeutically effective amount of
IL-2 to selectively and synergistically stimulate the production
cytokines in a patient. Preferred cytokines that are
synergistically stimulated in accordance with the invention are
selected from the group consisting of IL-12 and IFN-.gamma.,
IFN-.alpha., IFN-.beta. or combinations thereof. In accordance with
the present invention, preferred immunostimulatory oligonucleotides
that are not immunomer compounds include those containing at least
one immunostimulatory CpG dinucleotide wherein C is not cytosine or
deoxycytosine and/or G is not guanosine or 2-deoxyguanosine. Other
preferred immunostimulatory oligonucleotides of the invention that
are not immunomer compounds are those that include alternative
immunostimulatory moieties that are not CpG. Examples of such
alternative immunostimulatory moieties include but are not limited
to nucleosides comprising non-naturally occurring bases and/or
sugar and secondary structures of the oligonucleotide itself such
as hairpin structures that stabilize the oligonucleotide.
[0029] In a seventh aspect, the invention provides therapeutic
compositions comprising a therapeutically effective synergistic
amount of at least one immunomer compound, or immmunostimulatory
oligonucleotide, a therapeutically effective synergistic amount of
IL-2 (and/or an agent that induces IL-2 production in situ, such as
a DNA vaccine or expression vector expressing IL-2) and optionally
an antigen wherein administration of said combination
synergistically stimulates the production of cytokines in a
patient. Preferred cytokines that are synergistically stimulated in
accordance with the invention are selected from the group
consisting of IL-12 and IFN-.gamma., IFN-.alpha., IFN-.beta. or
combinations thereof.
[0030] The methods and compositions according to all aspects of the
invention are useful in therapeutic approaches to human or
veterinary diseases involving immune system modulation and
immune-based therapies. Particularly preferred disease targets
include cancer, infectious diseases, asthma and allergies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is a schematic representation of representative
immunomer compounds of the invention.
[0032] FIG. 2 depicts several representative immunomer compounds of
the invention.
[0033] FIG. 3 depicts a group of representative small molecule
linkers suitable for linear synthesis of immumomers of the
invention.
[0034] FIG. 4 depicts a group of representative small molecule
linkers suitable for parallel synthesis of immunomer compounds of
the invention.
[0035] FIG. 5 is a synthetic scheme for the linear synthesis of
immunomer compounds of the invention. DMTr=4,4'-dimethoxytrityl;
CE=cyanoethyl.
[0036] FIG. 6 is a synthetic scheme for the parallel synthesis of
immunomer compounds of the invention. DMTr=4,4'-dimethoxytrityl;
CE=cyanoethyl.
[0037] FIG. 7A is a graphic representation of the induction of
IL-12 by Oligonucleotide (Oligo) 1 and Immunomers 2-3 in BALB/c
mouse spleen cell cultures. These data suggest that Immunomer 2,
which has accessible 5'-ends, is a stronger inducer of IL-12 than
monomeric Oligo 1, and that Immunomer 3, which does not have
accessible 5'-ends, has equal or weaker ability to produce immune
stimulation compared with Oligo 1.
[0038] FIG. 7B is a graphic representation of the induction of IL-6
(top to bottom, respectively) by Oligo 1 and Immunomers 2-3 in
BALB/c mouse spleen cells cultures. These data suggest that
Immunomer 2, which has accessible 5'-ends, is a stronger inducer of
IL-6 than monomeric Oligo 1, and that Immunomer 3, which does not
have accessible 5'-ends, has equal or weaker ability to induce
immune stimulation compared with Oligo 1.
[0039] FIG. 7C is a graphic representation of the induction of
IL-10 by Oligo 1 and Immunomers 2-3 (top to bottom, respectively)
in BALB/c mouse spleen cell cultures.
[0040] FIG. 8A is a graphic representation of the induction of
BALB/c mouse spleen cell proliferation in cell cultures by
different concentrations of Immunomers 5 and 6, which have
inaccessible and accessible 5'-ends, respectively.
[0041] FIG. 8B is a graphic representation of BALB/c mouse spleen
enlargement by Oligo 4 and Immunomers 5-6, which have an
immunogenic chemical modification in the 5'-flanking sequence of
the CpG motif Again, the immunomer compound, which has accessible
5'-ends (6), has a greater ability to increase spleen enlargement
compared with Immunomer 5, which does not have accessible 5'-end
and with monomeric Oligo 4.
[0042] FIG. 9A is a graphic representation of induction of IL-12 by
different concentrations of Oligo 4 and Immunomers 7 and 8 in
BALB/c mouse spleen cell cultures.
[0043] FIG. 9B is a graphic representation of induction of IL-6 by
different concentrations of Oligo 4 and Immunomers 7 and 8 in
BALB/c mouse spleen cell cultures.
[0044] FIG. 9C is a graphic representation of induction of IL-10 by
different concentrations of Oligo 4 and Immunomers 7 and 8 in
BALB/c mouse spleen cell cultures.
[0045] FIG. 10A is a graphic representation of the induction of
cell proliferation by Immunomers 14, 15, and 16 in BALB/c mouse
spleen cell cultures.
[0046] FIG. 10B is a graphic representation of the induction of
cell proliferation by IL-12 by different concentrations of
Immunomers 14 and 16 in BALB/c mouse spleen cell cultures.
[0047] FIG. 10C is a graphic representation of the induction of
cell proliferation by IL-6 by different concentrations of
Immunomers 14 and 16 in BALB/c mouse spleen cell cultures.
[0048] FIG. 11A is a graphic representation of the induction of
cell proliferation by Oligo 4 and 17 and Immunomers 19 and 20 in
BALB/c mouse spleen cell cultures.
[0049] FIG. 11B is a graphic representation of the induction of
cell proliferation IL-12 by different concentrations of Oligo 4 and
17 and Immunomers 19 and 20 in BALB/c mouse spleen cell
cultures.
[0050] FIG. 11C is a graphic representation of the induction of
cell proliferation IL-6 by different concentrations of Oligo 4 and
17 and Immunomers 19 and 20 in BALB/c mouse spleen cell
cultures.
[0051] FIG. 12 is a graphic representation of BALB/c mouse spleen
enlargement using Oligo 4 and Immunomers 14, 23, and 24.
[0052] FIG. 13 shows the effect of a method according to the
invention on tumor growth in a nude mouse model for prostate
cancer.
[0053] FIG. 14 shows the effect of a method according to the
invention on body weight of the mice used in the study.
[0054] FIG. 15A is a graphic representation demonstrating the
synergistic effect on IL-12 production after BALB/c spleenocytes
were treated with Oligo 1 and IL-2.
[0055] FIG. 15B is a graphic representation demonstrating the
synergistic effect on IL-12 production after BALB/c spleenocytes
were treated with Oligo 2 and IL-2.
[0056] FIG. 15C is a graphic representation demonstrating the
synergistic effect on IL-12 production after BALB/c spleenocytes
were treated with Oligo 3 and IL-2.
[0057] FIG. 15D is a graphic representation demonstrating the
synergistic effect on IL-12 production after BALB/c spleenocytes
were treated with Oligo 4 and IL-2.
[0058] FIG. 16A is a graphic representation demonstrating the
effect on IL-6 production after BALB/c spleenocytes were treated
with Oligo 1 and IL-2.
[0059] FIG. 16B is a graphic representation demonstrating the
effect on IL-6 production after BALB/c spleenocytes were treated
with Oligo 2 and IL-2.
[0060] FIG. 16C is a graphic representation demonstrating the
effect on IL-6 production after BALB/c spleenocytes were treated
with Oligo 3 and IL-2.
[0061] FIG. 16D is a graphic representation demonstrating the
effect on IL-6 production after BALB/c spleenocytes were treated
with Oligo 4 and IL-2.
[0062] FIG. 17 is a graphic representation demonstrating the
synergistic effect on IL-12 production after BALB/c spleenocytes
were treated with Oligo 5 and IL-2.
[0063] FIG. 18A is a graphic representation demonstrating the
effect on IFN-.gamma. production after BALB/c spleenocytes were
treated with Oligo 1 and IL-2.
[0064] FIG. 18B is a graphic representation demonstrating the
effect on IFN-.gamma. production after BALB/c spleenocytes were
treated with Oligo 2 and IL-2.
[0065] FIG. 18C is a graphic representation demonstrating the
effect on IFN-.gamma. production after BALB/c spleenocytes were
treated with Oligo 3 and IL-2.
[0066] FIG. 18D is a graphic representation demonstrating the
effect on IFN-.gamma. production after BALB/c spleenocytes were
treated with Oligo 4 and IL-2.
[0067] FIG. 19 is a graphic representation demonstrating the effect
on IFN-.gamma. production after BALB/c spleenocytes were treated
with Oligo 5 and IL-2.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0068] The invention relates to optimized methods and compositions
for enhancing the immune response caused by immunostimulatory
compounds used in immune-based therapies. The optimized methods
according to the invention result in synergy between the
therapeutic effect of immunostimulatory compounds such as
immunostimulatory oligonucleotides and immunomer compounds and the
therapeutic effect of cytokine immunotherapy and/or
chemotherapeutic agents. The issued patents, patent applications,
and references that are cited herein are hereby incorporated by
reference to the same extent as if each was specifically and
individually indicated to be incorporated by reference. In the
event of inconsistencies between any teaching of any reference
cited herein and the present specification, the latter shall
prevail for purposes of the invention.
[0069] The invention provides methods for enhancing the anti-cancer
effect caused by immunostimulatory compounds used for immunotherapy
applications for the treatment of cancer. In the methods according
to the invention, immunostimulatory oligonucleotides and/or
immunomer compounds provide a synergistic therapeutic effect when
use in combination with chemotherapeutic agents. This result is
surprising in view of the fact that immunostimulatory
oligonucleotides and immunomer compounds cause cell division of
immune system cells, whereas chemotherapeutic agents normally kill
actively dividing cells.
[0070] In a first aspect, the invention provides a method for
treating cancer in a cancer patient comprising administering, in
combination with chemotherapeutic agents, immunostimulatory
oligonucleotides and/or immunomer compounds, the latter comprising
at least two oligonucleotides linked together, such that the
immunomer compound has more than one accessible 5' end, wherein at
least one of the oligonucleotides is an immunostimulatory
oligonucleotide. As used herein, the term "accessible 5' end" means
that the 5' end of the oligonucleotide is sufficiently available
such that the factors that recognize and bind to immunomer
compounds and stimulate the immune system have access to it.
Optionally, the 5' OH can be linked to a phosphate,
phosphorothioate, or phosphorodithioate moiety, an aromatic or
aliphatic linker, cholesterol, or another entity which does not
interfere with accessibility. Immunostimulatory oligonucleotides
and immunomer compounds induce an immune response when administered
to a vertebrate. When used in combination with chemotherapeutic
agents, a synergistic therapeutic effect is obtained.
[0071] Preferred chemotherapeutic agents used in the method
according to the invention include, without limitation Gemcitabine,
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, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP
845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317,
Picibanil/OK-432, AD 32Nalrubicin, 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, iodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCl, 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.
[0072] In the methods according to this aspect of the invention,
administration of immunostimulatory oligonucleotides and/or
immunomer compounds can be by any suitable route, including,
without limitation, parenteral, oral, sublingual, transdermal,
topical, intranasal, aerosol, intraocular, intratracheal,
intrarectal, vaginal, by gene gun, dermal patch or topical cream or
in eye drop or mouthwash form. Administration of the therapeutic
compositions of immunostimulatory oligonucleotides and/or immunomer
compounds can be carried out using known procedures at dosages and
for periods of time effective to reduce symptoms or surrogate
markers of the disease. When administered systemically, the
therapeutic composition is preferably administered at a sufficient
dosage to attain a blood level of immunostimulatory oligonucleotide
and/or immunomer compound from about 0.0001 micromolar to about 10
micromolar. For localized administration, much lower concentrations
than this may be effective, and much higher concentrations may be
tolerated. Preferably, a total dosage of immunostimulatory
oligonucleotide and/or immunomer compound ranges from about 0.0001
mg per patient per day to about 200 mg per kg body weight per day.
It may be desirable to administer simultaneously, or sequentially a
therapeutically effective amount of one or more of the therapeutic
compositions of the invention to an individual as a single
treatment episode.
[0073] For purposes of this aspect of the invention, the term "in
combination with" means in the course of treating the same disease
in the same patient, and includes administering the
immunostimulatory oligonucleotide and/or immunomer compound and/or
the chemotherapeutic agent in any order, including simultaneous
administration, as well as temporally spaced order of up to several
days apart. Such combination treatment may also include more than a
single administration of the immunostimulatory oligonucleotide
and/or immunomer compound, and/or independently the
chemotherapeutic agent. The administration of the immunostimulatory
oligonucleotide and/or immunomer compound and/or chemotherapeutic
agent may be by the same or different routes.
[0074] In some embodiments, the immunomer compound used in the
method according to the invention comprises two or more
immunostimulatory oligonucleotides, (in the context of the
immunomer) which may be the same or different. Preferably, each
such immunostimulatory oligonucleotide has at least one accessible
5' end.
[0075] In certain embodiments of the method according to the
invention, in addition to the immunostimulatory oligonucleotide(s),
the immunomer compound also comprises at least one oligonucleotide
that is complementary to a gene. As used herein, the term
"complementary to" means that the oligonucleotide hybridizes under
physiological conditions to a region of the gene. In some
embodiments, the oligonucleotide downregulates expression of a
gene. Such downregulatory oligonucleotides preferably are selected
from the group consisting of antisense oligonucleotides, ribozyme
oligonucleotides, small inhibitory RNAs and decoy oligonucleotides.
As used herein, the term "downregulate a gene" means to inhibit the
transcription of a gene or translation of a gene product. Thus, the
immunomer compounds used in the method according to the invention
can be used to target one or more specific disease targets, while
also stimulating the immune system.
[0076] In certain embodiments, the immunostimulatory
oligonucleotide and/or immunomer compound used in the method
according to the invention includes a ribozyme or a decoy
oligonucleotide. As used herein, the term "ribozyme" refers to an
oligonucleotide that possesses catalytic activity. Preferably, the
ribozyme binds to a specific nucleic acid target and cleaves the
target. As used herein, the term "decoy oligonucleotide" refers to
an oligonucleotide that binds to a transcription factor in a
sequence-specific manner and arrests transcription activity.
Preferably, the ribozyme or decoy oligonucleotide exhibits
secondary structure, including, without limitation, stem-loop or
hairpin structures. In certain embodiments, at least one
oligonucleotide comprises poly(I)-poly(dC). In certain embodiments,
at least one set of Nn includes a string of 3 to 10 dGs and/or Gs
or 2'-substituted ribo or arabino Gs.
[0077] For purposes of the invention, the term "oligonucleotide"
refers to a polynucleoside formed from a plurality of linked
nucleoside units. Such oligonucleotides can be obtained from
existing nucleic acid sources, including genomic or cDNA, but are
preferably produced by synthetic methods. In preferred embodiments
each nucleoside unit includes a heterocyclic base and a
pentofuranosyl, trehalose, arabinose, 2'-deoxy-2'-substituted
arabinose, 2'-O-substituted arabinose or hexose sugar group. The
nucleoside residues can be coupled to each other by any of the
numerous known internucleoside linkages. Such internucleoside
linkages include, without limitation, phosphodiester,
phosphorothioate, phosphorodithioate, alkylphosphonate,
alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane,
carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano,
thioether, bridged phosphoramidate, bridged methylene phosphonate,
bridged phosphorothioate, and sulfone internucleoside linkages. The
term "oligonucleotide" also encompasses polynucleosides having one
or more stereospecific internucleoside linkage (e.g., (R.sub.P)- or
(S.sub.P)-phosphorothioate, alkylphosphonate, or phosphotriester
linkages). As used herein, the terms "oligonucleotide" and
"dinucleotide" are expressly intended to include polynucleosides
and dinucleosides having any such internucleoside linkage, whether
or not the linkage comprises a phosphate group. In certain
preferred embodiments, these internucleoside linkages may be
phosphodiester, phosphorothioate, phosphorodithioate,
methylphosphonate linkages, or combinations thereof.
[0078] In some embodiments, the immunomer compound comprises
oligonucleotides each having from about 3 to about 35 nucleoside
residues, preferably from about 4 to about 30 nucleoside residues,
more preferably from about 4 to about 20 nucleoside residues. In
some embodiments, the oligonucleotides have from about 5 or 6 to
about 18, or from about 5 or 6 to about 14, nucleoside residues. As
used herein, the term "about" implies that the exact number is not
critical. Thus, the number of nucleoside residues in the
oligonucleotides is not critical, and oligonucleotides having one
or two fewer nucleoside residues, or from one to several additional
nucleoside residues are contemplated as equivalents of each of the
embodiments described above, for purposes of this invention. In
some embodiments, one or more of the oligonucleotides have 11
nucleotides.
[0079] The term "oligonucleotide" also encompasses polynucleosides
having additional substituents including, without limitation,
protein groups, lipophilic groups, intercalating agents, diamines,
folic acid, cholesterol and adamantane. The term "oligonucleotide"
also encompasses any other nucleobase containing polymer,
including, without limitation, peptide nucleic acids (PNA), peptide
nucleic acids with phosphate groups (PHONA), locked nucleic acids
(LNA), morpholino-backbone oligonucleotides, and oligonucleotides
having backbone sections with alkyl linkers or amino linkers.
[0080] The immunostimulatory oligonucleotides and/or immunomer
compounds used in the method according to the invention can include
naturally occurring nucleosides, modified nucleosides, or mixtures
thereof. As used herein, the term "modified nucleoside" is a
nucleoside that includes a modified heterocyclic base, a modified
sugar moiety, or a combination thereof. In some embodiments, the
modified nucleoside is a non-natural pyrimidine or purine
nucleoside, as herein described. In some embodiments, the modified
nucleoside is a 2'-substituted ribonucleoside an arabinonucleoside
or a 2'-deoxy-2'-fluoroarabinoside.
[0081] For purposes of the invention, the term "2'-substituted
ribonucleoside" includes ribonucleosides in which the hydroxyl
group at the 2' position of the pentose moiety is substituted to
produce a 2'-O-substituted ribonucleoside. Preferably, such
substitution is with a lower alkyl group containing 1-6 saturated
or unsaturated carbon atoms, or with an aryl group having 6-10
carbon atoms, wherein such alkyl, or aryl group may be
unsubstituted or may be substituted, e.g., with halo, hydroxy,
trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,
carboalkoxy, or amino groups. Examples of such 2'-O-substituted
ribonucleosides include, without limitation
2'-O-methylribonucleosides and
2'-O-methoxyethylribonucleosides.
[0082] The term "2'-substituted ribonucleoside" also includes
ribonucleosides in which the 2'-hydroxyl group is replaced with a
lower alkyl group containing 1-6 saturated or unsaturated carbon
atoms, or with an amino or halo group. Examples of such
2'-substituted ribonucleosides include, without limitation,
2'-amino, 2'-fluoro, 2'-allyl, and 2'-propargyl
ribonucleosides.
[0083] The term "oligonucleotide" includes hybrid and chimeric
oligonucleotides. A "chimeric oligonucleotide" is an
oligonucleotide having more than one type of internucleoside
linkage. One preferred example of such a chimeric oligonucleotide
is a chimeric oligonucleotide comprising a phosphorothioate,
phosphodiester or phosphorodithioate region and non-ionic linkages
such as alkylphosphonate or alkylphosphonothioate linkages (see
e.g., Pederson et al. U.S. Pat. Nos. 5,635,377 and 5,366,878).
[0084] A "hybrid oligonucleotide" is an oligonucleotide having more
than one type of nucleoside. One preferred example of such a hybrid
oligonucleotide comprises a ribonucleotide or 2'-substituted
ribonucleotide region, and a deoxyribonucleotide region (see, e.g.,
Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and
6,143,881).
[0085] For purposes of the invention, the term "immunostimulatory
oligonucleotide" refers to an oligonucleotide as described above
that induces an immune response when administered to a vertebrate,
such as a fish, bird, or mammal As used herein, the term "mammal"
includes, without limitation rats, mice, cats, dogs, horses,
cattle, cows, pigs, rabbits, non-human primates, and humans. Useful
immunostimulatory oligonucleotides can be found described in
Agrawal et al., WO 98/49288, published Nov. 5, 1998; WO 01/12804,
published Feb. 22, 2001; WO 01/55370, published Aug. 2, 2001;
PCT/US01/13682, filed Apr. 30, 2001; and PCT/US01/30137, filed Sep.
26, 2001. Preferably, the immunostimulatory oligonucleotide
comprises at least one phosphodiester, phosphorothioate,
methylphosphonate, or phosphordithioate internucleoside
linkage.
[0086] In a further aspect, the invention provides a method for
synergistically stimulating an immune response in a patient. The
method comprises administering to a patient, a combination of a
therapeutically effective synergistic amount of at least one
immunomer compound or immunostimulatory oligonucleotide in
accordance with the invention and a therapeutically effective
synergistic amount of IL-2 (and/or an agent that induces IL-2
production in situ, such as a DNA vaccine or expression vector
expressing IL-2), wherein administration of said combination
synergistically stimulates the production of cytokines in a
patient. Preferably, the cytokines that are synergistically
stimulated in accordance with the invention are selected from the
group consisting of, IL-12 and interferon-.gamma. (IFN-.gamma.),
IFN-.alpha., IFN-.beta. or combinations thereof.
[0087] The term "effective synergistic amount" is used herein to
denote known concentrations of immunomer compound or
immunostimulatory oligonucleotide and of IL-2 administered for an
effective period of time such that the combined stimulatory effect
of the immunomer compound or immunostimulatory oligonucleotide and
IL-2 are more than additive, i.e. the combined stimulatory effect
is greater than the expected total stimulatory effect calculated on
the basis of the sum of the individual stimulatory effects.
[0088] As used herein, the term "cytokine" refers to any of many
soluble molecules that cells of the immune system produce to
control reactions between other cells. The term "cytokine"
includes, for example, interleukins (e.g., IL-1, IL-2, IL-3, IL-6,
IL-10, IL12, etc.), interferons (e.g., IFN-.alpha., IFN-.beta.,
IFN-.gamma.), chemokines, hematopoietic growth factors (e.g.
erythropoietin), tumor necrosis factors, colony stimulating factors
(e.g., G-CSF, M-CSF, GM-CSF) and transforming growth factors
(TGF-alpha).
[0089] In accordance with the invention, an "immunomer" refers to
any compound comprising at least two oligonucleotides linked
directly at their 3' ends, or directly via internucleoside
linkages, or directly at a functionalized nucleobase or sugar, or
that are indirectly linked together via a non-nucleotidic linker,
wherein at least one of the oligonucleotides, in the context of the
immunomer compound, is an immunostimulatory oligonucleotide having
an accessible 5' end. In the context of the invention, an
immunostimulatory oligonucleotide is an oligonucleotide that
comprises at least one of an immunostimulatory "CpG" dinucleotide,
an immunostimulatory domain, or other immunostimulatory moiety. As
used herein, the term "accessible 5' end" means that the 5' end of
the oligonucleotide is sufficiently available such that the factors
that recognize and bind to immunomer compounds and
immunostimulatory oligonucleotides and stimulate the immune system
have access to the 5' end.
[0090] In some embodiments, at least one immunostimulatory
oligonucleotide of the immunomer compound comprises an
immunostimulatory dinucleotide of formula 5'-Pyr-Pur-3', wherein
Pyr is a natural or synthetic pyrimidine nucleoside and Pur is a
natural or synthetic purine nucleoside. As used herein, the term
"pyrimidine nucleoside" refers to a nucleoside wherein the base
component of the nucleoside is a pyrimidine base. Similarly, the
term "purine nucleoside" refers to a nucleoside wherein the base
component of the nucleoside is a purine base. For purposes of the
invention, a "synthetic" pyrimidine or purine nucleoside includes a
non-naturally occurring pyrimidine or purine base, a non-naturally
occurring sugar moiety, or a combination thereof.
[0091] Preferred pyrimidine nucleosides in the immunostimulatory
oligonucleotides and/or immunomer compounds used in the method
according to the invention have the structure (I):
##STR00001##
wherein:
[0092] D is a hydrogen bond donor;
[0093] D' is selected from the group consisting of hydrogen,
hydrogen bond donor, hydrogen bond acceptor, hydrophilic group,
hydrophobic group, electron withdrawing group and electron donating
group;
[0094] A is a hydrogen bond acceptor or a hydrophilic group;
[0095] A' is selected from the group consisting of hydrogen bond
acceptor, hydrophilic group, hydrophobic group, electron
withdrawing group and electron donating group;
[0096] X is carbon or nitrogen; and
[0097] S' is a pentose or hexose sugar ring, or a non-naturally
occurring sugar.
[0098] Preferably, the sugar ring is derivatized with a phosphate
moiety, modified phosphate moiety, or other linker moiety suitable
for linking the pyrimidine nucleoside to another nucleoside or
nucleoside analog.
[0099] Preferred hydrogen bond donors include, without limitation,
--NH--, --NH.sub.2, --SH and --OH. Preferred hydrogen bond
acceptors include, without limitation, C.dbd.O, C.dbd.S, and the
ring nitrogen atoms of an aromatic heterocycle, e.g., N3 of
cytosine.
[0100] In some embodiments, the base moiety in (I) is a
non-naturally occurring pyrimidine base. Examples of preferred
non-naturally occurring pyrimidine bases include, without
limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine,
N4-alkylcytosine, preferably N4-ethylcytosine, and 4-thiouracil. In
some embodiments, the sugar moiety S' in (I) is a non-naturally
occurring sugar moiety. For purposes of the present invention, a
"naturally occurring sugar moiety" is a sugar moiety that occurs
naturally as part of nucleic acid, e.g., ribose and 2'-deoxyribose,
and a "non-naturally occurring sugar moiety" is any sugar that does
not occur naturally as part of a nucleic acid, but which can be
used in the backbone for an oligonucleotide, e.g, hexose. Arabinose
and arabinose derivatives are examples of preferred sugar
moieties.
[0101] Preferred purine nucleoside analogs in immunostimulatory
oligonucleotides and/or immunomer compounds used in the method
according to the invention have the structure (II):
##STR00002##
[0102] wherein:
[0103] D is a hydrogen bond donor;
[0104] D' is selected from the group consisting of hydrogen,
hydrogen bond donor, and hydrophilic group;
[0105] A is a hydrogen bond acceptor or a hydrophilic group;
[0106] X is carbon or nitrogen;
[0107] each L is independently selected from the group consisting
of C, O, N and S; and
[0108] S' is a pentose or hexose sugar ring, or a non-naturally
occurring sugar.
[0109] Preferably, the sugar ring is derivatized with a phosphate
moiety, modified phosphate moiety, or other linker moiety suitable
for linking the pyrimidine nucleoside to another nucleoside or
nucleoside analog.
[0110] Preferred hydrogen bond donors include, without limitation,
--NH--, --NH.sub.2, --SH and --OH. Preferred hydrogen bond
acceptors include, without limitation, C.dbd.O, C.dbd.S, --NO.sub.2
and the ring nitrogen atoms of an aromatic heterocycle, e.g., N1 of
guanine.
[0111] In some embodiments, the base moiety in (II) is a
non-naturally occurring purine base. Examples of preferred
non-naturally occurring purine bases include, without limitation,
6-thioguanine and 7-deazaguanine. In some embodiments, the sugar
moiety S' in (II) is a naturally occurring sugar moiety, as
described above for structure (I).
[0112] In preferred embodiments, the immunostimulatory dinucleotide
in the immunostimulatory oligonucleotides and/or immunomer compound
used in the method according to the invention is selected from the
group consisting of CpG, C*pG, CpG*, and C*pG*, wherein C is
cytidine or 2'-deoxycytidine, C* is 2'-deoxythymidine,
arabinocytidine, 2'-deoxythymidine,
2'-deoxy-2'-substitutedarabinocytidine,
2'-O-substitutedarabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other
non-natural pyrimidine nucleosides, or
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
G is guanosine or 2'-deoxyguanosine, G* is 2'
deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other
non-natural purine nucleoside, and p is an internucleoside linkage
selected from the group consisting of phosphodiester,
phosphorothioate, and phosphorodithioate. In certain preferred
embodiments, the immunostimulatory dinucleotide is not CpG.
[0113] The immunostimulatory oligonucleotides may include
immunostimulatory moieties on one or both sides of the
immunostimulatory dinucleotide. Thus, in some embodiments, the
immunostimulatory oligonucleotide comprises an immunostimulatory
domain of structure (III):
5'-Nn-N1-Y-Z-N1-Nn-3' (III)
[0114] wherein:
[0115] Y is cytidine, 2'deoxythymidine, 2' deoxycytidine
arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine,
2'-deoxythymidine, 2'-O-substitutedarabinocytidine,
2'-deoxy-5-hydroxycytidine, 2'-deoxy-N4-alkyl-cytidine,
2'-deoxy-4-thiouridine, other non-natural pyrimidine nucleosides,
orl-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;
[0116] Z is guanosine or 2'-deoxyguanosine, 2'
deoxy-7-deazaguanosine, 2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'deoxyinosine, or other
non-natural purine nucleoside;
[0117] N1, at each occurrence, is preferably a naturally occurring
or a synthetic nucleoside or an immunostimulatory moiety selected
from the group consisting of abasic nucleosides,
arabinonucleosides, 2'-deoxyuridine, .alpha.-deoxyribonucleosides,
.beta.-L-deoxyribonucleosides, and nucleosides linked by a
phosphodiester or modified internucleoside linkage to the adjacent
nucleoside on the 3' side, the modified internucleotide linkage
being selected from, without limitation, a linker having a length
of from about 2 angstroms to about 200 angstroms, C2-C18 alkyl
linker, poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol
linker, glyceryl linker, 2'-5' internucleoside linkage, and
phosphorothioate, phosphorodithioate, or methylphosphonate
internucleoside linkage;
[0118] Nn, at each occurrence, is preferably a naturally occurring
nucleoside or an immunostimulatory moiety selected from the group
consisting of abasic nucleosides, arabinonucleosides,
2'-deoxyuridine, .alpha.-deoxyribonucleosides, 2'-O-substituted
ribonucleosides, and nucleosides linked by a modified
internucleoside linkage to the adjacent nucleoside on the 3' side,
the modified internucleoside linkage preferably being selected from
the group consisting of amino linker, C2-C18 alkyl linker,
poly(ethylene glycol) linker, 2-aminobutyl-1,3-propanediol linker,
glyceryl linker, 2'-5' internucleoside linkage, and
methylphosphonate internucleoside linkage;
[0119] provided that at least one N1 or Nn is an immunostimulatory
moiety;
[0120] wherein each n is independently a number from 0 to 30;
and
[0121] wherein, in the case of an immunomer compound, the 3'end is
linked directly or via a non-nucleotidic linker to another
oligonucleotide, which may or may not be immunostimulatory.
[0122] In some preferred embodiments, YZ is arabinocytidine or
2'-deoxy-2'-substituted arabinocytidine and arabinoguanosine or
2'deoxy-2'-substituted arabinoguanosine. Preferred
immunostimulatory moieties include modifications in the phosphate
backbones, including, without limitation, methylphosphonates,
methylphosphonothioates, phosphotriesters, phosphothiotriesters,
phosphorothioates, phosphorodithioates, triester prodrugs,
sulfones, sulfonamides, sulfamates, formacetal,
N-methylhydroxylamine, carbonate, carbamate, morpholino,
boranophosphonate, phosphoramidates, especially primary
amino-phosphoramidates, N3 phosphoramidates and N5
phosphoramidates, and stereospecific linkages (e.g., (R.sub.P)- or
(S.sub.P)-phosphorothioate, alkylphosphonate, or phosphotriester
linkages).
[0123] Preferred immunostimulatory moieties according to the
invention further include nucleosides having sugar modifications,
including, without limitation, 2'-substituted pentose sugars
including, without limitation, 2'-O-methylribose,
2'-O-methoxyethylribose, 2'-O-propargylribose, and
2'-deoxy-2'-fluororibose; 3'-substituted pentose sugars, including,
without limitation, 3'-O-methylribose; 1',2'-dideoxyribose;
arabinose; substituted arabinose sugars, including, without
limitation, 1'-methylarabinose, 3'-hydroxymethylarabinose,
4'-hydroxymethylarabinose, and 2'-substituted arabinose sugars;
hexose sugars, including, without limitation, 1,5-anhydrohexitol;
and alpha-anomers. In embodiments in which the modified sugar is a
3'-deoxyribonucleoside or a 3'-O-substituted ribonucleoside, the
immunostimulatory moiety is attached to the adjacent nucleoside by
way of a 2'-5' internucleoside linkage.
[0124] Preferred immunostimulatory moieties in immunostimulatory
oligonucleotides and/or immunomer compounds used in the method
according to the invention further include oligonucleotides having
other carbohydrate backbone modifications and replacements,
including peptide nucleic acids (PNA), peptide nucleic acids with
phosphate groups (PHONA), locked nucleic acids (LNA), morpholino
backbone oligonucleotides, and oligonucleotides having backbone
linker sections having a length of from about 2 angstroms to about
200 angstroms, including without limitation, alkyl linkers or amino
linkers. The alkyl linker may be branched or unbranched,
substituted or unsubstituted, and chirally pure or a racemic
mixture. Most preferably, such alkyl linkers have from about 2 to
about 18 carbon atoms. In some preferred embodiments such alkyl
linkers have from about 3 to about 9 carbon atoms. Some alkyl
linkers include one or more functional groups selected from the
group consisting of hydroxy, amino, thiol, thioether, ether, amide,
thioamide, ester, urea, and thioether. Some such functionalized
alkyl linkers are poly(ethylene glycol) linkers of formula
--O--(CH.sub.2--CH.sub.2--O--).sub.n (n=1-9). Some other
functionalized alkyl linkers are peptides or amino acids.
[0125] Preferred immunostimulatory moieties in immunostimulatory
oligonucleotides and/or immunomer compounds used in the method
according to the invention further include DNA isoforms, including,
without limitation, .beta.-L-deoxyribonucleosides and
.alpha.-deoxyribonucleosides. Preferred immunostimulatory moieties
incorporate 3' modifications, and further include nucleosides
having unnatural internucleoside linkage positions, including,
without limitation, 2'-5', 2'-2', 3'-3' and 5'-5' linkages.
[0126] Preferred immunostimulatory moieties in immunostimulatory
oligonucleotides and/or immunomer compounds used in the method
according to the invention further include nucleosides having
modified heterocyclic bases, including, without limitation,
5-hydroxycytosine, 5-hydroxymethylcytosine, N4-alkylcytosine,
preferably N4-ethylcytosine, 4-thiouracil, 6-thioguanine,
7-deazaguanine, inosine, nitropyrrole, C5-propynylpyrimidine, and
diaminopurines, including, without limitation,
2,6-diaminopurine.
[0127] By way of specific illustration and not by way of
limitation, for example, in the immunostimulatory domain of
structure (III), a methylphosphonate internucleoside linkage at
position N1 or Nn is an immunostimulatory moiety, a linker having a
length of from about 2 angstroms to about 200 angstroms, C2-C18
alkyl linker at position X1 is an immunostimulatory moiety, and a
.beta.-L-deoxyribonucleoside at position X1 is an immunostimulatory
moiety. See Table 1 below for representative positions and
structures of immunostimulatory moieties. It is to be understood
that reference to a linker as the immunostimulatory moiety at a
specified position means that the nucleoside residue at that
position is substituted at its 3'-hydroxyl with the indicated
linker, thereby creating a modified internucleoside linkage between
that nucleoside residue and the adjacent nucleoside on the 3' side.
Similarly, reference to a modified internucleoside linkage as the
immunostimulatory moiety at a specified position means that the
nucleoside residue at that position is linked to the adjacent
nucleoside on the 3' side by way of the recited linkage.
TABLE-US-00001 TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES
N1 Naturally-occurring nucleosides, abasic nucleoside, arabino-
nucleoside, 2'-deoxyuridine, .beta.-L-deoxyribonucleoside C2- C18
alkyl linker, poly(ethylene glycol) linkage, 2-aminobutyl-
1,3-propanediol linker (amino linker), 2'-5' internucleoside
linkage, methylphosphonate internucleoside linkage Nn
Naturally-occurring nucleosides, abasic nucleoside, arabino-
nucleosides, 2'-deoxyuridine, 2'-O-substituted ribonucleoside,
2'-5' internucleoside linkage, methylphosphonate internucleoside
linkage, provided that N1 and N2 cannot both be abasic linkages
[0128] Table 2 shows representative positions and structures of
immunostimulatory moieties within an immunostimulatory
oligonucleotide having an upstream potentiation domain. As used
herein, the term "Spacer 9" refers to a poly(ethylene glycol)
linker of formula --O--(CH.sub.2CH.sub.2--O).sub.n--, wherein n is
3. The term "Spacer 18" refers to a poly(ethylene glycol) linker of
formula --O--(CH.sub.2CH.sub.2--O).sub.n--, wherein n is 6. As used
herein, the term "C2-C18 alkyl linker refers to a linker of formula
--O--(CH.sub.2).sub.q--O--, where q is an integer from 2 to 18.
Accordingly, the terms "C3-linker" and "C3-alkyl linker" refer to a
linker of formula --O--(CH.sub.2).sub.3--O--. For each of Spacer 9,
Spacer 18, and C2-C18 alkyl linker, the linker is connected to the
adjacent nucleosides by way of phosphodiester, phosphorothioate,
phosphorodithioate or methylphosphonate linkages.
TABLE-US-00002 TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5'
N2 Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol
linker 5' N1 Naturally-occurring nucleosides,
.beta.-L-deoxyribonucleoside, C2- C18 alkyl linker, poly(ethylene
glycol), abasic linker, 2- aminobutyl-1,3-propanediol linker 3' N1
Naturally-occurring nucleosides, 1',2'-dideoxyribose, 2'-O-
methyl-ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18 3'
N2 Naturally-occurring nucleosides, 1',2'-dideoxyribose, 3'-
deoxyribonucleoside, .beta.-L-deoxyribonucleoside, 2'-O-propargyl-
ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18,
methylphosphonate internucleoside linkage 3' N 3
Naturally-occurring nucleosides, 1',2'-dideoxyribose, C2-C18 alkyl
linker, Spacer 9, Spacer 18, methylphosphonate internucleoside
linkage, 2'-5' internucleoside linkage, d(G)n, polyI-polydC 3'N 2 +
1',2'-dideoxyribose, .beta.-L-deoxyribonucleoside, C2-C18 alkyl 3'N
3 linker, d(G)n, polyI-polydC 3'N3 +
2'-O-methoxyethyl-ribonucleoside, methylphosphonate 3' N 4
internucleoside linkage, d(G)n, polyI-polydC 3'N5 +
1',2'-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyI-polydC 3' N
6 5'N1 + 1',2'-dideoxyribose, d(G)n, polyI-polydC 3' N 3
[0129] Table 3 shows representative positions and structures of
immunostimulatory moieties within an immunostimulatory
oligonucleotide having a downstream potentiation domain.
TABLE-US-00003 TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5'
N2 methylphosphonate internucleoside linkage 5' N1
methylphosphonate internucleoside linkage 3' N1
1',2'-dideoxyribose, methylphosphonate internucleoside linkage,
2'-O-methyl 3' N2 1',2'-dideoxyribose,
.beta.-L-deoxyribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer
18, 2-aminobutyl-1,3-propanediol linker, methylphosphonate
internucleoside linkage, 2'-O-methyl 3' N3 3'-deoxyribonucleoside,
3'-O-substituted ribonucleoside, 2'-O-propargyl-ribonucleoside 3'N2
+ 1',2'-dideoxyribose, .beta.-L-deoxyribonucleoside 3' N3
[0130] The immunomer compounds used in the method according to the
invention comprise at least two oligonucleotides linked directly or
via a non-nucleotidic linker. For purposes of the invention, a
"non-nucleotidic linker" is any moiety that can be linked to the
oligonucleotides by way of covalent or non-covalent linkages.
Preferably such linker is from about 2 angstroms to about 200
angstroms in length. Several examples of preferred linkers are set
forth below. Non-covalent linkages include, but are not limited to,
electrostatic interaction, hydrophobic interactions, .pi.-stacking
interactions, and hydrogen bonding. The term "non-nucleotidic
linker" is not meant to refer to an internucleoside linkage, as
described above, e.g., a phosphodiester, phosphorothioate, or
phosphorodithioate functional group, that directly connects the
3'-hydroxyl groups of two nucleosides. For purposes of this
invention, such a direct 3'-3' linkage is considered to be a
"nucleotidic linkage."
[0131] In some embodiments, the non-nucleotidic linker is a metal,
including, without limitation, gold particles. In some other
embodiments, the non-nucleotidic linker is a soluble or insoluble
biodegradable polymer bead.
[0132] In yet other embodiments, the non-nucleotidic linker is an
organic moiety having functional groups that permit attachment to
the oligonucleotide. Such attachment preferably is by any stable
covalent linkage.
[0133] In some embodiments, the non-nucleotidic linker is a
biomolecule, including, without limitation, polypeptides,
antibodies, lipids, antigens, allergens, and oligosaccharides. In
some other embodiments, the non-nucleotidic linker is a small
molecule. For purposes of the invention, a small molecule is an
organic moiety having a molecular weight of less than 1,000 Da. In
some embodiments, the small molecule has a molecular weight of less
than 750 Da.
[0134] In some embodiments, the small molecule is an aliphatic or
aromatic hydrocarbon, either of which optionally can include,
either in the linear chain connecting the oligonucleotides or
appended to it, one or more functional groups selected from the
group consisting of hydroxy, amino, thiol, thioether, ether, amide,
thioamide, ester, urea, and thiourea. The small molecule can be
cyclic or acyclic. Examples of small molecule linkers include, but
are not limited to, amino acids, carbohydrates, cyclodextrins,
adamantane, cholesterol, haptens and antibiotics. However, for
purposes of describing the non-nucleotidic linker, the term "small
molecule" is not intended to include a nucleoside.
[0135] In some embodiments, the small molecule linker is glycerol
or a glycerol homolog of the formula
HO--(CH.sub.2).sub.o--CH(OH)--(CH.sub.2).sub.p--OH, wherein o and p
independently are integers from 1 to about 6, from 1 to about 4, or
from 1 to about 3. In some other embodiments, the small molecule
linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such
derivatives have the formula
HO--(CH.sub.2).sub.m--C(O)NH--CH.sub.2--CH(OH)--CH.sub.2--NHC(O)--(CH.sub-
.2).sub.m--OH, wherein m is an integer from 0 to about 10, from 0
to about 6, from 2 to about 6, or from 2 to about 4.
[0136] Some non-nucleotidic linkers in immunomer compounds used in
the method according to the invention permit attachment of more
than two oligonucleotides, as schematically depicted in FIG. 1. For
example, the small molecule linker glycerol has three hydroxyl
groups to which oligonucleotides may be covalently attached. Some
immunomer compounds according to the invention, therefore, comprise
more than two oligonucleotides linked at their 3' ends to a
non-nucleotidic linker. Some such immunomer compounds comprise at
least two immunostimulatory oligonucleotides, each having an
accessible 5' end.
[0137] The immunostimulatory oligonucleotides and/or immunomer
compounds used in the method according to the invention may
conveniently be synthesized using an automated synthesizer and
phosphoramidite approach as schematically depicted in FIGS. 5 and
6, and further described in the Examples. In some embodiments, the
immunostimulatory oligonucleotides and/or immunomer compounds are
synthesized by a linear synthesis approach (see FIG. 5). As used
herein, the term "linear synthesis" refers to a synthesis that
starts at one end of the immunomer compound and progresses linearly
to the other end. Linear synthesis permits incorporation of either
identical or un-identical (in terms of length, base composition
and/or chemical modifications incorporated) monomeric units into
the immunostimulatory oligonucleotides and/or immunomer
compounds.
[0138] An alternative mode of synthesis for immunomer compounds is
"parallel synthesis", in which synthesis proceeds outward from a
central linker moiety (see FIG. 6). A solid support attached linker
can be used for parallel synthesis, as is described in U.S. Pat.
No. 5,912,332. Alternatively, a universal solid support, such as
phosphate attached to controlled pore glass support, can be
used.
[0139] Parallel synthesis of immunomer compounds has several
advantages over linear synthesis: (1) parallel synthesis permits
the incorporation of identical monomeric units; (2) unlike in
linear synthesis, both (or all) the monomeric units are synthesized
at the same time, thereby the number of synthetic steps and the
time required for the synthesis is the same as that of a monomeric
unit; and (3) the reduction in synthetic steps improves purity and
yield of the final immunomer product.
[0140] At the end of the synthesis by either linear synthesis or
parallel synthesis protocols, the immunostimulatory
oligonucleotides or immunomer compounds used in the method
according to the invention may conveniently be deprotected with
concentrated ammonia solution or as recommended by the
phosphoramidite supplier, if a modified nucleoside is incorporated.
The product immunostimulatory oligonucleotides and/or immunomer
compound is preferably purified by reversed phase HPLC,
detritylated, desalted and dialyzed.
[0141] Immunostimulatory oligonucleotides suitable for use as a
component of an immunomer compound, or in accordance with the
fourth aspect of the invention, are described in the following U.S.
patents and pending U.S. patent applications and are incorporated
herein by reference: U.S. Pat. Nos. 6,426,334 and 6,476,000; and
U.S. patent application Ser. Nos. 09/770,602, 09/845,623,
09/965,116, 60/440,587, 10/361,111, 60/471,247, 60/477. Preferred
immunostimulatory oligonucleotides and immunomer compounds of the
invention are described in pending U.S. patent application Ser. No.
10/279,684. Table 4 shows representative immunomer compounds used
in the method according to the invention. Additional immunomer
compounds are found described in the Examples and in U.S. patent
application Ser. No. 10/279,684.
TABLE-US-00004 TABLE 4 Examples of Immunomer Sequences Oligo or
Immu- nomer No. Sequences and Modification (5'-3') 1
5'-GAGAACGCTCGACCTT-3' 2
5'-GAGAACGCTCGACCTT-3'-3'-TTCCAGCTCGCAAGAG-5' 3
3'-TTCCAGCTCGCAAGAG-5'-5'-GAGAACGCTCGACCTT-3' 4
5'-CTATCTGACGTTCTCTGT-3' 5 ##STR00003## 6 ##STR00004## 7
##STR00005## 8 ##STR00006## 9 ##STR00007## 10 ##STR00008## 11
##STR00009## 12 ##STR00010## 13 5'-CTGACGTTCTCTGT-3' 14
##STR00011## 15 ##STR00012## 16 ##STR00013## 17
5'-XXTGACGTTCTCTGT-3' 18 ##STR00014## 19 ##STR00015## 20
##STR00016## 21 5'-TCTGACGTTCT-3' 22 ##STR00017## 23 ##STR00018##
24 ##STR00019## 191 5'-CTGTCRTTCTC-X.sub.1-CTCTTRCTGTC-5' 192
5'-TCRTCRTTG-X.sub.1-GTTRCTRCT-5' 193
5'-TCRTCRTTCTG-X.sub.1-GTCTTRCTRCT-5' 194
5'-TCGTTG-Y.sub.1-X.sub.2-Y.sub.1-GTTGCT-5' 195
5'-TCGTT-Y.sub.1X.sub.2-Y.sub.1-TTGCT-5' ##STR00020## ##STR00021##
L = C3-alkyl liner; X = 1',2'-dideoxyriboside, Y = .sup.5OHdC; R =
7-deaza-dG R = arabinoguamosine; X.sub.1 = glycerol linker;
##STR00022## ##STR00023##
[0142] A further aspect of the invention provides an
immunostimulatory nucleic acid comprising at least two
oligonucleotides, wherein the immunostimulatory nucleic acid has a
secondary structure. In certain embodiments, the immunostimulatory
nucleic acid has a 3'-end stem loop secondary structure by way of
hydrogen bonding with a complementary sequence. In certain
embodiments the nucleic acid that has reduced immunostimulatory
activity forms a 5'-end stem loop secondary structure by way of
hydrogen bonding with a complementary sequence. In this aspect,
immunostimulatory nucleic acid comprises a structure as detailed in
formula (I).
Domain A-Domain B-Domain C (I)
[0143] Domains may be from about 2 to about 12 nucleotides in
length. Domain A may be 5'-3' or 3'-5' or 2'-5' DNA, RNA, RNA-DNA,
DNA-RNA having or not having a palindromic or self-complementary
domain containing or not containing at least one dinucleotide
selected from the group consisting of CpG, C*pG, C*pG* and CpG*,
wherein C is cytidine or 2'-deoxycytidine, G is guanosine or
2'-deoxyguanosine, C* is 2'-deoxythymidine,
1-(2'-deoxy-B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2'-dideoxy-5-halocytosine, 2'-deoxy-5-nitrocytosine,
arabinocytidine, 2'-deoxy-2'-substitutedarabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, or other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'- deoxyinosine, or other
purine nucleoside analogs, and p is an internucleoside linkage
selected from the group consisting of phosphodiester,
phosphorothioate, and phosphorodithioate. In certain preferred
embodiments, the immunostimulatory dinucleotide is not CpG.
[0144] In certain embodiments, Domain A will have more than one
dinucleotide selected from the group consisting of CpG, C*pG, C*pG*
and CpG*.
[0145] Domain B, as depicted by an "X" below, is a linker joining
Domains A and C that may be a 3'-'5' linkage, a 2'-5' linkage, a
3'-3' linkage, a phosphate group, a nucleoside, or a non-nucleoside
linker that may be aliphatic, aromatic, aryl, cyclic, chiral,
achiral, a peptide, a carbohydrate, a lipid, a fatty acid, mono-
tri- or hexapolyethylene glycol, or a heterocyclic moiety.
[0146] Domain C may be 5'-3' or 3'-5', 2'-5' DNA, RNA, RNA-DNA,
DNA-RNA Poly I-Poly C having or not having a palindromic or
self-complementary sequence, which can or cannot have a
dinucleotide selected from the group consisting of CpG, C*pG,
C*pG*, CpG*, wherein C is cytidine or 2'-deoxycytidine, G is
guanosine or 2'-deoxyguanosine, C* is 2'-deoxythymidine,
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine,
2' dideoxy-5-halocytosine, 2'-deoxy-5-halocytosine,
arabinocytidine, 2'-deoxy-2'-substituted arabinocytidine,
2'-O-substituted arabinocytidine, 2'-deoxy-5-hydroxycytidine,
2'-deoxy-N4-alkyl-cytidine, 2'-deoxy-4-thiouridine, other
pyrimidine nucleoside analogs, G* is 2'-deoxy-7-deazaguanosine,
2'-deoxy-6-thioguanosine, arabinoguanosine,
2'-deoxy-2'substituted-arabinoguanosine,
2'-O-substituted-arabinoguanosine, 2'-deoxyinosine, or other purine
nucleoside analogs, and p is an internucleoside linkage selected
from the group consisting of phosphodiester, phosphorothioate, and
phosphorodithioate. In certain preferred embodiments, the
immunostimulatory dinucleotide is not CpG. In some embodiments,
Domain B is preferably a non-nucloetidic linker connecting
oligonucleotides of Domain A and Domain C, which are referred to as
"immunomers." In certain preferred embodiments, Domain C does not
have the dinucleotide CpG, C*pG, C*pG* or CpG*.
[0147] By way of non-limiting example, in certain embodiments of
this aspect the immunostimulatory nucleic acid will have a
structure as detailed in formula (II).
##STR00024##
[0148] As one skilled in the art would recognize, there is a
secondary structure element in the terminal end of the molecule in
the form of an intramolecular stem-loop.
[0149] By way of non-limiting example, in certain embodiments of
this aspect the immunostimulatory nucleic acid will have a
structure as detailed in formula (III)
##STR00025##
The structure depicted in formula (III) is referred to herein as a
"terminal dimmer," since the ends of the two molecules are blocked
because the sequences of the two ends are complementary allowing
for intermolecular hydrogen bonding. In addition, domains A and A'
may or may not be identical, domains B and B' may or may not be
identical and domains C and C' may or may not be identical.
[0150] By way of non-limiting example, in certain embodiments of
this aspect the immunostimulatory nucleic acid will have a
structure as detailed in formula (IV).
##STR00026##
[0151] As would be recognized by one skilled in the art, the
terminal end of the depicted molecule has a secondary structure
because the complementary sequence of its end is hydrogen bonded to
this region. In certain embodiments, a molecule such as a ligand
may be attached to the terminal end in order to facilitate cellular
uptake or improve stability of the molecule.
[0152] Non-limiting examples of some nucleic acid molecules of the
invention are presented in Table 5.
TABLE-US-00005 TABLE 5 SEQ ID NO: Sequence* Structure 91
5'-CTGTCTGACGTTCTCTG-3' ##STR00027## 92
5'-CTGTCTGACGTTCTCTG-GAA-CAGAG-3' ##STR00028## 93
5'-CTGTCTGACGTTCTCTG-GAA-CAGAGAACGTC-3' ##STR00029## 94
5'-CTGTCTGACGTTCTCTG-GAA-CAGAGAACGTCAGACAG-3' ##STR00030## 95
5'-GACAG-GAA-CTGTCTGACGTTCTCTG-3' ##STR00031## 96
5'-AACGTCAGACAG-GAA-CTGTCTGACGTTCTCTG-3' ##STR00032## 97
5'-CAGAGAACGTCAGACAG-GAA-CTGTCTGACGTTCTCTG-3' ##STR00033## 98
5'-CTATCTGACGTTCTCTGT-3' ##STR00034## 99
5'-CTATCTGACGTTCTCTGT-gtgatcac-3' ##STR00035## 100
5'-gtgatcac-CTATCTGACGTTCTCTGT-3' ##STR00036## 101
5'-CTGTCTGTCGTTCTCTG-3' ##STR00037## 102
5'-CTGTCTGTCGTTCTCTG-GAA-CAGAG-3' ##STR00038## 103
5'-CTGTCTGTCGTTCTCTG-GAA-CAGAGAACGAC-3' ##STR00039## 104
5'-CTGTCTGTCGTTCTCTG-GAA-CAGAGAACGACAGACAG-3' ##STR00040## 105
5'-GACAG-GAA-CTGTCTGTCGTTCTCTG-3' ##STR00041## 106
5'-AACGACAGACAG-GAA-CTGTCTGACGTTCTCTG-3' ##STR00042## 107
5'-CAGAGAACGACAGACAG-GAA-CTGTCTGTCGTTCTCTG-3' ##STR00043## 108
5'-TCGTCGTT-GAGCTCT-GAA-AGAGCTC-3' ##STR00044## 109
5'-TCGTCGTT-GTGAGCTCTGT-GAA-ACAGAGCTCAC-3' ##STR00045## 110
5'-TCGTCGTT-GCACAGAGCTCTGCT-GAA- AGCAGAGCTCTGTGC-3' ##STR00046##
111 5'-TCGTCGTT-GCTGACAGAGCTCTGCTAT-GAA- ATAGCAGAGCTCTGTCAGC-3'
##STR00047## 112 5'-TCGTCGTT-GTGCTCT-GAA-CTTGCTC-3' ##STR00048##
113 5'-TCGTCGTT-GTGTGCTCTGT-GAA-CATCAGTCTAC-3' ##STR00049## 114
5'-TCGTCGTT-gagctct-GAA-agagctc-3' ##STR00050## 115
5'-TCGTCGTT-gtgagctctgt-GAA-acagagctcac-3' ##STR00051## 116
5'-TCGTCGTT-GAGCTCT-GAA-AGAGCTC-3' ##STR00052## 117
5'-TCGTCGTT-GTGAGCTCTGT-GAA-ACAGAGCTCAC-3' ##STR00053## 118
5'-TCGTCGTT-GAGCTCT-GAA-AGAGCTC-3' ##STR00054## 119
5'-TCGTCGTT-GAGCTCT-GAA-AGAGCTC-3' ##STR00055## 120
5'-TGCTGCTT-GAGCTCT-GAA-AGAGCTC-3' ##STR00056## 121
5'-TCTTGACGTTCTCTCT-3' ##STR00057## 122
5'-TCTTGACGTTCTCTCT-GAA-AGAGAG-3' ##STR00058## 123
5'-TCTTGACGTTCTCTCT-GAA-agagag-3' ##STR00059## 124
5'-tcttgacgttctctct-GAA-AGAGAG-3' ##STR00060## 125
5'-tcttgacgttctctct-GAA-agagag-3' ##STR00061## 126
5'-tcttgacgttctctct-gaa-agagag-3' ##STR00062## 127
5'-TCTTGACGTTCTCTCT-X-AGAGAG-3' ##STR00063## 128
5'-tcttgacgttctctct-X-agagag-3' ##STR00064## *upper case-PS; lower
case-PO; Bold-2'-O-methyl-ribonucleotides (in 116 and 117);
G-2'-deoxy-7-deaza-G (in 118); G-araG (in 119); X-C3-linker (in 127
and 128).
[0153] Alternatively, the nucleic acid molecule of the invention
can be two immunomers linked by way of a non-nucleotidic linker.
Non-limiting representative examples of these molecules are
presented in Table 6.
TABLE-US-00006 TABLE 6 SEQ ID NO: Sequence* Structure 129
5'-TCGTCGTT-X-GTCTCGAGAC-5' ##STR00065## 130
5'-TCGTCGTT-XX-GTCTCGAGAC-5' ##STR00066## 131
5'-TCGTCGTT-XXX-GTCTCGAGAC-5' ##STR00067## 132
5'-TCGTCGTT-Y-GTCTCGAGAC-5' ##STR00068## 133
5'-TCGTCGTT-Z-GTCTCGAGAC-5' ##STR00069## 134
5'-TCGTCGTT-XXX-GUCUCGAGAC-5' ##STR00070## 135
5'-TCGTCGTT-XXX-GTCTCGAGAC-5' ##STR00071## 136
5'-TTGTGCTT-XXX-GTCTCGAGAC-5' ##STR00072## 137
5'-TCGTCGTT-XXX-GTCTCCACAC-5' ##STR00073## 138
5'-TCGTCGTT-XXX-ccgtagctacGG-5' ##STR00074## 139
5'-TCGTCGTT-XX-ccgtagctacGG-5' ##STR00075## 140
5'-TCGTCGTT-X-ccgtagctacGG-5' ##STR00076## 141
5'-TCGTCGTT-3'-ccgtagctacGG-5' ##STR00077## 142
5'-TCGTCGTT-Y-ccgtagctacGG-5' ##STR00078## 143
5'-TCGTCGTT-Z-ccgtagctacGG-5' ##STR00079## 144
5'-TCGTCGTT-XXX-ctcgag-5' ##STR00080## 145
5'-TCGTCGTT-XXX-ctgtctcgagacag-5' ##STR00081## 146
5'-TCGTCGTT-XXX-cgactgtctcgagacagtcg-5' ##STR00082## 147
5'-TCGTCGTT-XXX-gucucgagac-5' ##STR00083## 148
5'-TCGTCGTTG-X-tgatcgatgca-3'-X-3'-GTTGCTGCT-5' ##STR00084## 149
5'-TCGTCGTTG-3'-X-3'-tgcatcgatgca-X-GTTGCTGCT-5' ##STR00085## 150
5'-TCGTCGTTG-X-TGCATCGATGCA-3'-X-3'-GTTGCTGCT-5' ##STR00086## 151
5'-TCGTCGTTG-3'-X-3'-TGCATCGATGCA-X-GTTGCTGCT-5' ##STR00087## 152
5'-tcgtcgttg-X-TGCATCGATGCA-3'-X-3'-gttgctgct-5' ##STR00088## 153
5'-tcgtcgttg-3'-X-3'-TGCATCGATGCA-X-gttgctgct-5' ##STR00089## 154
5'-tcgtcgtt-XXX-gtctcgagac-5' ##STR00090## 155
5'-TCGTCGTT-XXX-gtctcgagac-5' ##STR00091## 156
5'-TCGTCGTTG-X-tgcatcgatgca-3' ##STR00092## 157
5'-TCGTCGTTGtgcatcgatgca-3' ##STR00093## 158
5'-tcgtcgttgTGCATCGATGCA-3' ##STR00094## *Upper case-PS; lower
case-PO, X-C3-linker; Y-tetraethyleneglycol linker;
Z-hexaethyleneglycol linker, bold-2'-O-methylribonucleotides (in
134 and 147); G-2'-deoxy-7-deaza-G (in 135).
Alternatively, further, non-limiting, representatives are presented
in Table 7.
TABLE-US-00007 TABLE 7 159 ##STR00095## 160 ##STR00096## 161
##STR00097## 162 ##STR00098## 163 ##STR00099## 164 ##STR00100## 165
5'-TCRTCRTT-XXX-GTCTCGAGAC-5' 166 5'-TCRTCRTT-XXX-GUCUCGAGAC-5' 167
5'-TCG, TCG, TT-XXX-GUCUCGAGAC-5' 168 5'-TCG, TCG,
TT-XXX-GTCTCCACTC-5' 169 5'-TCG, TCG, TT-XXX-GUCUCCACUC-5' 170
##STR00101## 171 ##STR00102## 172
TCGTCGTT-gtgagctctgtg-GAA-acagagcucac Italic phase represents a
phosphodiester linkage, other linkages are phosphorothioate unless
otherwise indicated Underline = 2'-OMe-nucleosie, X = C3 linker R =
2'-deoxy-7-deazaguanosine G.sub.1 = 2'-deoxy-7-deazaguanoise
[0154] Another aspect of the invention provides an
immunostimulatory nucleic acid wherein the sequence of the
immunostimulatory oligonucleotide and/or immunomer is at least
partially self-complementary. A self-complementary sequence as used
herein refers to a base sequence which, upon suitable alignment,
may form intramolecular or, more typically, intermolecular
basepairing between G-C, A-T, A-U and/or G-U wobble pairs. In one
embodiment the extent of self-complementarity is at least 50
percent. For example an 8-mer that is at least 50 percent
self-complementary may have a sequence capable of forming 4, 5, 6,
7, or 8 G-C, A-T, A-U and/or G-U wobble basepairs. Such basepairs
may but need not necessarily involve bases located at either end of
the self-complementary immunostimulatory oligonucleotide and/or
immunomer. Where nucleic acid stabilization may be important to the
immunostimulatory oligonucleotide and/or immunomer, it may be
advantageous to "clamp" together one or both ends of a
double-stranded nucleic acid, either by basepairing or by any other
suitable means. The degree of self-complementarity may depend on
the alignment between immunostimulatory oligonucleotide and/or
immunomer, and such alignment may or may not include single- or
multiple-nucleoside overhangs. In other embodiments, the degree of
self-complementarity is at least 60 percent, at least 70 percent,
at least 80 percent, at least 90 percent, or even 100 percent.
[0155] By way of non-limiting example, in certain embodiments of
this aspect the immunostimulatory nucleic acid will have a
structure as detailed in formula (V)
##STR00103##
As would be recognized by one skilled in the art, the depicted
immunomer compounds have secondary structure because the sequences
of the domains are complementary allowing for intermolecular
hydrogen bonding. Domains A and A' may or may not be identical,
domains A and C may or may not be identical, domains A and C' may
or may not be identical, domains A' and C may or may not be
identical, domains A' and C' may or may not be identical, domains B
and B' may or may not be identical and domains C and C' may or may
not be identical. Moreover, additional immunomers can bind through
intermolecular hydrogen bonding thereby creating a chain, or
multimers, of immunomers according to the invention. n can be any
number of continuous self complementary immunomer compounds.
[0156] As used herein, the term "complementary" means having the
ability to hybridize to a nucleic acid. Such hybridization is
ordinarily the result of hydrogen bonding between complementary
strands, preferably to form Watson-Crick or Hoogsteen base pairs,
although other modes of hydrogen bonding, as well as base stacking
can also lead to hybridization.
[0157] As used herein, the term "secondary structure" refers to
intermolecular hydrogen bonding. Intermolecular hydrogen bonding
results in the formation of a duplexed nucleic acid molecule.
[0158] Non-limiting representative nucleic acid molecules are
presented in Table 8.
TABLE-US-00008 TABLE 8 173
5'-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5'
174 5'-TCG.sub.1AACG.sub.1TTCG-X-GCTTG.sub.1CAAG.sub.1CT-5' 175
5'-TCTCACCTTCT-X-TCTTCCACTCT-5' 176
5'-TCG.sub.2AACG.sub.2TTCG.sub.2-X-G.sub.2CTTG.sub.2CAAG.sub.2CT-5'
177 5'-TCG.sub.2AACG.sub.2TTCG-X-GCTTG.sub.2CAAG.sub.2CT-5' 178
5'-TCG.sub.1TCG.sub.1AACG.sub.1TTCGAGATGAT-3' 179
5'-TCG.sub.2TCG.sub.2AACG.sub.2TTCG.sub.2AGATGAT-3' 180
5'-TCG.sub.3TCG.sub.3AACG.sub.3TTCG.sub.3AGATGAT-3' 181
5'-TC.sub.1GTC.sub.1GAAC.sub.1GTTC.sub.1GAGATGAT-3' 182
5'-TC.sub.2GTC.sub.2GAAC.sub.2GTTC.sub.2GAGATGAT-3' 183
5'-TC.sub.3GTC.sub.3GAAC.sub.3GTTC.sub.3GAGATGAT-3' 184
5'-TCG.sub.1AACG.sub.1TTC-X-CTTG.sub.1CAAG.sub.1CT-5' 185
5'-TCG.sub.1TTCG.sub.1AACG.sub.1-X-G.sub.1CAAG.sub.1CTTG.sub.1CT-5'
186 5'-TCCAACCTTCG-X-GCTTCCAACCT-5' 187
5'-TCG.sub.1TTG.sub.1CAACG.sub.1-X-G.sub.1CAACG.sub.1TTG.sub.1CT-5'
188 5'-TCG.sub.2AACG.sub.2TTCT-X-TCTTG.sub.2CAAG.sub.2CT-5' 189
5'-TCG.sub.1AACG.sub.2TTCG.sub.1-X-G.sub.1CTTG.sub.2CAAG.sub.1CT-5'
190
5'-TCG.sub.1AAC.sub.1GTTCG.sub.1-X-G.sub.1CTTGC.sub.1AAG.sub.1CT-5'
Normal phase represents a phosphorothioate linkage G.sub.1 =
2'-deoxy-7-deazaguanosine G.sub.2 = Arabinoguanosine G.sub.3 =
2'-deoxyinosine C.sub.1 =
1-(2'-deoxy-.beta.-D-ribofuranosyl)-2-oxo-7-deaza-8-methylpurin- e
C.sub.2 = Arabinocytidine C.sub.3 = 2'-deoxy-5-hydroxycytidine X =
C3 Linker
[0159] A particularly preferred immunomer compound for use in the
methods of the invention has the following structure.
##STR00104##
[0160] The methods and compositions according to all aspects of the
invention are useful in therapeutic approaches to treating diseases
wherein the treatment involves immune system modulation and
immune-based therapies. Particularly preferred disease targets
include cancer, infectious diseases and allergies.
[0161] In certain embodiments, the therapeutic method is for the
treatment of 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;
pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin
cancer; testicular cancer; thyroid cancer; and renal cancer, as
well as other carcinomas and sarcomas.
[0162] In some embodiments, the therapeutic method is for the
treatment of an infection. By way of non-limiting example, viruses
that have been found to infect humans include but are not limited
to: Retroviridae (e.g. human immunodeficiency viruses, such as
HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or
HIV-III), and other isolates, such as HIV-LP; Picornaviridae (e.g.
polio viruses, hepatitis A virus; enteroviruses, human Coxsackie
viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains
that cause gastroenteritis); Togaviridae (e.g. equine encephalitis
viruses, rubella viruses); Flaviridae (e.g. dengue viruses,
encephalitis viruses, yellow fever viruses); Coronoviridae (e.g.
coronaviruses); Rhabdoviradae (e.g. vesicular stomatitis viruses,
rabies viruses); Coronaviridae (e.g. coronaviruses); Rhabdoviridae
(e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae
(e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses,
mumps virus, measles virus, respiratory syncytial virus);
Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.
Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses);
Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.
reoviruses, orbiviurses and rotaviruses); Birnaviridae;
Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);
Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae
(most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1
and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus;
Poxviridae (variola viruses, vaccinia viruses, pox viruses); and
Iridoviridae (e.g. African swine fever virus); and unclassified
viruses (e.g. the etiological agents of Spongiform
encephalopathies, the agent of delta hepatitis (thought to be a
defective satellite of hepatitis B virus), the agents of non-A,
non-B hepatitis (class 1=internally transmitted; class
2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related
viruses, and astroviruses).
[0163] In certain embodiments, therapeutic methods of the invention
are directed to the treatment of an allergy. 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 (Fells 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). Specific allergens may be purchased commercially
(e.g., INDOOR Biotechnologies Inc., Charlottesville, Va.
22903).
[0164] In a second aspect, the invention provides a method for
treating cancer in a cancer patient comprising administering to the
patient a chemotherapeutic agent in combination with an
immunostimulatory oligonucleotide and/or immunomer conjugate, which
comprises an immunostimulatory oligonucleotide and/or immunomer
compound, as described above, and an antigen conjugated to the
immunostimulatory oligonucleotide and/or immunomer compound at a
position other than the accessible 5' end. In some embodiments, the
non-nucleotidic linker comprises an antigen associated with cancer,
which is conjugated to the oligonucleotide. In some other
embodiments, the antigen is conjugated to the oligonucleotide at a
position other than its 3' end. In some embodiments, the antigen
produces a vaccine effect. For purposes of the invention, the term
"associated with" means that the antigen is present when the
cancer, is present, but either is not present, or is present in
reduced amounts, when the cancer is absent.
[0165] The immunostimulatory oligonucleotides and/or immunomer
compound is covalently linked to the antigen, or it is otherwise
operatively associated with the antigen. As used herein, the term
"operatively associated with" refers to any association that
maintains the activity of both immunostimulatory oligonucleotide
and/or immunomer compound and antigen. Nonlimiting examples of such
operative associations include being part of the same liposome or
other such delivery vehicle or reagent. Additionally, a nucleic
acid molecule encoding the antigen can be cloned into an expression
vector and administered in combination with the immunostimulatory
oligonucleotide and/or immunomer compound. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid to which it has been linked. Preferred vectors
are those capable of autonomous replication and expression of
nucleic acids to which they are linked (e.g., an episome). Vectors
capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression vectors."
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of "plasmids" which refer
generally to circular double stranded DNA loops which, in their
vector form, are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression
vectors which serve equivalent functions and which become known in
the art subsequently hereto.
[0166] In embodiments wherein the immunostimulatory oligonucleotide
and/or immunomer compound is covalently linked to the antigen, such
covalent linkage preferably is at any position on the
immunostimulatory oligonucleotide and/or immunomer compound other
than an accessible 5' end of an immunostimulatory oligonucleotide.
For example, the antigen may be attached at an internucleoside
linkage or may be attached to the non-nucleotidic linker.
Alternatively, the antigen may itself be the non-nucleotidic
linker.
[0167] In a third aspect, the invention provides pharmaceutical
formulations comprising an immunostimulatory oligonucleotide and/or
immunostimulatory oligonucleotide conjugate and/or immunomer
compound or immunomer conjugate according to the invention, a
chemotherapeutic agent and a physiologically acceptable carrier. As
used herein, the term "physiologically acceptable" refers to a
material that does not interfere with the effectiveness of the
immunomer compound and is compatible with a biological system such
as a cell, cell culture, tissue, or organism. Preferably, the
biological system is a living organism, such as a vertebrate.
Preferred chemotherapeutic agents include, without limitation
Gemcitabine 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 32Nalrubicin, 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, iodine
seeds, CDK4 and CDK2 inhibitors, PARP inhibitors,
D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,
Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD
9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane
Analog, nitrosoureas, alkylating agents such as melphelan and
cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan,
Carboplatin, Chlorombucil, Cytarabine HCl, 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.
[0168] In yet another embodiment, the formulations include a cancer
vaccine selected from the group consisting of EFG, Anti-idiotypic
cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV
ganglioside conjugate vaccine, Her2/new, Ovarex, M-Vax, O-Vax,
L-Vax, STn-KHL theratope, BLP25 (MUC-1), liposomal idiotypic
vaccine, Melacine, peptide antigen vaccines, toxin/antigen
vaccines, MVA-vased vaccine, PACIS, BCG vaccine, TA-HPV, TA-CIN,
DISC-virus and ImmunCyst/TheraCys.
[0169] In a further aspect, the invention provides a method for
treating cancer in a cancer patient comprising administering to the
patient a monoclonal antibody in combination with an
immunostimulatory oligonucleotide and/or immunomer compound, as
described herein. Passive immunotherapy in the form of antibodies,
and particularly monoclonal antibodies, has been the subject of
considerable research and development as anti-cancer agents. The
term "monoclonal antibody" as used herein refers to an antibody
molecule of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions derived from
human germline immunoglobulin sequences. Examples of anti-cancer
agents include, but are not limited to, Panorex (Glaxo-Welcome),
Rituxan (IDEC/Genentech/Hoffman la Roche), Mylotarg (Wyeth),
Campath (Millennium), Zevalin (IDEC and Schering AG), Bexxar
(Corixa/GSK), Erbitux (Imclone/BMS), Avastin (Genentech) and
Herceptin (Genentech/Hoffman la Roche). Antibodies may also be
employed in active immunotherapy utilising anti-idiotype antibodies
which appear to mimic (in an immunological sense) cancer antigens.
Monoclonal antibodies can be generated by methods known to those
skilled in the art of recombinant DNA technology.
[0170] As used herein, the term "carrier" encompasses any
excipient, diluent, filler, salt, buffer, stabilizer, solubilizer,
lipid, or other material well known in the art for use in
pharmaceutical formulations. It will be understood that the
characteristics of the carrier, excipient, or diluent will depend
on the route of administration for a particular application. The
preparation of pharmaceutically acceptable formulations containing
these materials is described in, e.g., Remington's Pharmaceutical
Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co.,
Easton, Pa., 1990.
[0171] Toll-like receptors (TLRs) function as sensors of infection
and induce the activation of innate and adaptive immune responses.
TLRs recognize a wide variety of ligands, called
pathogen-associated molecular patterns (PAMPs). Upon recognizing
conserved pathogen-associated molecular products, TLRs activate
host defense responses through their intracellular signalling
domain, the Toll/interleukin-1 receptor (TIR) domain, and the
downstream adaptor protein MyD88. Dendritic cells and macrophages
normally respond to Toll-like receptor (TLR) ligands and cytokines
(for example, interleukin-1.beta.; IL-6 and tumour necrosis factor,
TNF), which they also produce; natural killer (NK) cells and T
cells are also involved. After TLR stimulation by bacterial
compounds, innate immune cells release a range of cytokines. Some
examples of TLR ligands include, but are not limited to,
lipoproteins; peptidoglycan, zymosan (TLR2), double-stranded RNA,
polyI:polyC (TLR3), lipopolysaccharide, heat shock proteins, taxol
(TLR4), flagellin (TLRS), and imidazoquinolines-R848, resiquimod,
imiquimod; ssRNA (TLR7/8).
[0172] In a fourth aspect, the invention provides a method for
sensitizing cancer cells to ionizing radiation. The method
according to this aspect of the invention comprises administering
to a mammal an immunostimulatory oligonucleotide or an immunomer
compound according to the invention and treating the animal with
ionizing radiation. In certain preferred embodiments,
.gamma.-Irradiation is administered at 1.56 Gy/min. In certain
preferred embodiments, radiation therapy is administered from about
0.1 to about 10.0 Gy, preferably from about 0.25 to about 8.0 Gy,
more preferably from about 0.5 to about 5.0 Gy, or as 3.0 Gy of
radiation either twice for one week, four times for one week, or
three times on Days 2, 4, and 9. In certain embodiments
pre-treatment with an immunostimulatory oligonucleotide or an
immunomer compound is from about 2 to about 6 h prior to
.gamma.-irradiation.
[0173] In a fifth aspect, the invention provides a method for
synergistically stimulating an immune response in a patient
comprising administering to a patient a therapeutically effective
synergistic amount of an immunomer compound in combination with a
therapeutically effective synergistic amount of IL-2, and an
antigen, wherein administration of said combination synergistically
stimulates the production of cytokines in a patient. Preferred
cytokines stimulated in accordance with the invention include but
are not limited to one or more of, IL-12, interferon-.gamma.,
IFN-.alpha. and IFN-.beta..
[0174] In certain embodiments, the method is for the treatment of
cancer and the antigen is one specific to or associated with a
cancer. In some embodiments, the method is for the treatment of an
infection and the antigen is an antigen associated with the
infection. In certain embodiments, the method is for the treatment
of an allergy and the antigen is associated with the allergy. As
used herein, the term "associated with" means that the antigen is
present when the cancer, allergen or infectious disease is present,
but either is not present, or is present in reduced amounts, when
the cancer, allergen or infectious disease is absent.
[0175] As used herein, the term "antigen" means a substance that is
recognized and bound specifically by an antibody or by a T cell
antigen receptor. Antigens can include peptides, proteins,
glycoproteins, polysaccharides, gangliosides and lipids; portions
thereof and combinations thereof. The antigens can be those found
in nature or can be synthetic. Haptens are included within the
scope of "antigen." A hapten is a low molecular weight compound
that is not immunogenic by itself but is rendered immunogenic when
conjugated with an immunogenic molecule containing antigenic
determinants.
[0176] In certain embodiments, antigens useful in methods and
compositions of the invention are tumor-associated and/or
tumor-specific antigens. Non-limiting examples include: Prostate
Specific Antigen (PSA) and Prostatic Acid Phosphatase (PAP), which
are markers normally present in the blood in small amounts that can
be elevated in the presence of prostate cancer; Cancer Antigen 125
(CA-125), which is at elevated levels in patients with ovarian
cancer and is sometimes elevated in the presence of other cancers;
CA 15-3 and CA 27-29, which are useful in following the course of
breast cancer and its response to treatment; CA 19-9, which is
commonly used as a check for the spread of pancreatic cancer and is
also elevated in patients with colorectal, stomach and bile duct
cancer; Carcinoembryonic Antigen (CEA), which is normally present
in small amounts but can be elevated in the blood of patients with
a wide variety of cancers; Alpha-Fetoprotein, which is a marker for
hepatocellular and germ cell (nonseminoma) carcinoma; and
Galactosyl Transferase II, an isozyme of galactosyl transferase,
that has been shown to be elevated in a variety of malignancies,
predominantly gastrointestinal. As known by one skilled in the art,
tumor-associated and tumor-specific antigens are available
commercially. Also contemplated by the invention are those antigens
that can be made by recombinant nucleic acid technologies and/or
synthetic antigens, e.g., peptides produced by methods known in the
art.
[0177] In certain embodiments of the fifth aspect of the invention,
the invention provides a method for treating cancer in a cancer
patient comprising administering to the patient a therapeutically
effective synergistic amount of IL-2 in combination with an
immunomer conjugate, which comprises an immunomer compound, as
described above, and an antigen. In certain embodiments, the
antigen is conjugated to the immunomer compound at a position other
than the accessible 5' end. In some embodiments, the
non-nucleotidic linker of the immunomer compound comprises an
antigen associated with cancer. In some embodiments, the antigen is
conjugated to the immunomer compound at a position other than its
5' end. In some embodiments, the antigen produces a vaccine effect.
For purposes of the invention, the term "associated with" means
that the antigen is present when the cancer is present, but either
is not present, or is present in reduced amounts, when the cancer
is absent.
[0178] In some embodiments of the fifth aspect of the invention,
the immunomer compound is covalently linked to the antigen, or it
is otherwise operatively associated with the antigen. As used
herein, the term "operatively associated with" refers to any
association that maintains the activity of the immunomer compound
and antigen. Nonlimiting examples of such operative associations
include being part of the same liposome or other such delivery
vehicle or reagent. In embodiments wherein the immunomer compound
is covalently linked to the antigen, such covalent linkage
preferably is at any position on the immunomer compound other than
at an accessible 5' end of the immunomer compound. For example, the
antigen may be attached at an internucleoside linkage or may be
attached to the non-nucleotidic linker. Alternatively, the antigen
may itself be the non-nucleotidic linker.
[0179] In a sixth aspect of the invention, at least one
immunostimulatory oligonucleotide that is not an immunomer compound
is used in combination with a therapeutically effective amount of
IL-2 to selectively and synergistically stimulate the production of
cytokines in a patient. Preferred cytokines synergistically
stimulated in accordance with the invention are selected from the
group consisting of, IL-12 and IFN-.gamma., IFN-.alpha., IFN-.beta.
or combinations thereof. In accordance with the present invention,
preferred immunostimulatory oligonucleotides that are not immunomer
compounds include those containing at least one immunostimulatory
CpG dinucleotide wherein C is not cytosine or deoxycytosine and/or
G is not guanosine or 2-deoxyguanosine. Other preferred
immunostimulatory oligonucleotides of the invention that are not
immunomer compounds are those that include alternative
immunostimulatory moieties that are not CpG. Examples of such
alternative immunostimulatory moieties include but are not limited
to nucleosides comprising non-naturally occurring bases and/or
sugar and secondary structures of the oligonucleotide itself such
as hairpin structures that stabilize the oligonucleotide, as
described in the following U.S. patents and pending U.S. patent
applications and are incorporated herein by reference: U.S. Pat.
Nos. 6,426,334 and 6,476,000; and U.S. patent application Ser. Nos.
09/770,602, 09/845,623, 09/965,116, 60/440,587, 10/361,111,
60/471,247, 60/477,608.
[0180] In certain embodiments of the invention, each of the
immunomer compound or immunostimulatory oligonucleotide and IL-2 is
admixed with a pharmaceutically acceptable carrier prior to
administration to the patient. In certain embodiments, the
immunomer compound or immunostimulatory oligonucleotide are mixed
together with a pharmaceutically acceptable carrier prior to
administration, or combined as part of a pharmaceutical composition
as described in the fourth aspect of the invention. As used herein,
the term "carrier" encompasses any excipient, diluent, filler,
salt, buffer, stabilizer, solubilizer, lipid, or other material
well known in the art for use in pharmaceutical formulations. It
will be understood that the characteristics of the carrier,
excipient, or diluent will depend on the route of administration
for a particular application. The preparation of pharmaceutically
acceptable formulations containing these materials is described in,
e.g., Remington: The Science and Practice of Pharmacy, 20.sup.th
Edition, ed. A. L. Gennaro, Lippincott Williams & Wilkins
Publishing Co., Philadelphia, Pa., 19106 (ISBN: 0683306472).
[0181] In a seventh aspect, the invention provides therapeutic
compositions comprising a pharmaceutically acceptable carrier, a
therapeutically effective synergistic amount of an immunomer
compound or immunostimulotory oligonucleotide, a therapeutically
effective synergistic amount of IL-2 and optionally, an antigen,
wherein administration of said therapeutic composition
synergistically stimulates the production of cytokines in a
patient. Preferred cytokines that are synergistically stimulated in
accordance with the invention are selected from the group
consisting of IL-12 and interferon-.gamma., IFN-.alpha., IFN-.beta.
or combinations thereof.
[0182] All aspects of the invention are useful in the treatment of
disease, and are particularly useful in immune-based therapies for
treating cancer, infectious diseases and allergies. As used herein
the term "treating" or "treatment" of disease includes: prevention
of disease; dimunition or eradication of signs or symptoms of
disease after onset; and prevention of relapse of disease.
[0183] In the methods according to the invention, administration of
an immunomer compound or immmumostimulatory oligonucleotide in
combination with IL-2 can be by any suitable route including,
without limitation, parenteral, oral, sublingual, transdermal,
topical, intranasal, aerosol, intraocular, intratracheal,
intrarectal, vaginal, by gene gun, dermal patch or in eye drop or
mouthwash form. Administration of immunomer compounds,
immunostimulatory oligonucleotides, IL-2 or therapeutic
compositions thereof can be carried out using known procedures
using therapeutically effective synergistic amounts and for periods
of time effective to treat disease.
[0184] The term "in combination with" means in the course of
treating the same disease in the same patient, and includes
administering the immunomer compound and /or immunostimulatory
oligonucleotide and/or IL-2 in any order, including simultaneous
administration, as well as temporally spaced order of up to several
days apart. Such combination treatment may also include more than a
single administration of the immunomer compound and /or
immunostimulatory oligonucleotide, and/or IL-2, independently. The
administration of the immunomer compound and IL-2 may be by the
same or different routes.
[0185] One of skill in the art will appreciate that such
synergistic effect of either the immunomer compound or
immunostimulatory oligonucleotide, IL-2 or both may vary
considerably depending on the tissue, organ, the particular disease
or the patient to be treated in accordance with the invention.
Furthermore, one of skill in the art will appreciate that the
therapeutically effective synergistic amount of either the
immunomer compound or immunostimulatory oligonucleotide or IL-2 may
be lowered or increased by fine tuning and altering the amount of
the other component.
[0186] When administered systemically, the immunomer compound is
preferably administered at a sufficient dosage to attain a blood
level of immunomer compound from about 0.0001 micromolar to about
10 micromolar. For localized administration, much lower
concentrations than this may be effective, and much higher
concentrations may be tolerated. Preferably, a total dosage of
immunostimulatory oligonucleotide and/or immunomer compound ranges
from about 0.0001 mg per patient per day to about 200 mg per kg
body weight per day. It may be desirable to administer
simultaneously, or sequentially, a therapeutically effective
synergistic amount of each of the immunomer compound or IL-2 to an
individual as a single treatment episode. Preferably, IL-2 is
administered in an amount of about 750 to about 75,000 units.
[0187] The invention provides a kit comprising a cytokine and or
chemotherapeutic agent, and immunostimulatory oligonucleotides
and/or immunomer compounds, the latter comprising at least two
oligonucleotides linked together, such that the immunomer compound
has more than one accessible 5' end, wherein at least one of the
oligonucleotides is an immunostimulatory oligonucleotide. In
another aspect, the kit comprises an immunostimulatory
oligonucleotide and/or immunostimulatory oligonucleotide conjugate
and/or immunomer compound or immunomer conjugate according to the
invention, a cytokine and/or chemotherapeutic agent and a
physiologically acceptable carrier. The kit will generally also
include a set of instructions for use.
[0188] The examples below are intended to further illustrate
certain preferred embodiments of the invention, and are not
intended to limit the scope of the invention.
EXAMPLES
Example 1
Synthesis of Oligonucleotides Containing Immunomodulatory
Moieties
[0189] Oligonucleotides were synthesized on a 1 .mu.mol scale using
an automated DNA synthesizer (Expedite 8909; PerSeptive Biosystems,
Framingham, Mass.), following the linear synthesis or parallel
synthesis procedures outlined in FIGS. 5 and 6.
[0190] Deoxyribonucleoside phosphoramidites were obtained from
Applied Biosystems (Foster City, Calif.). 1',2'-dideoxyribose
phosphoramidite, propyl-1-phosphoramidite, 2-deoxyuridine
phosphoramidite,
1,3-bis-[5-(4,4'-dimethoxytrityl)pentylamidyl]-2-propanol
phosphoramidite and methyl phosponamidite were obtained from Glen
Research (Sterling, Va.). .beta.-L-2'-deoxyribonucleoside
phosphoramidite, .alpha.-2'-deoxyribonucleoside phosphoramidite,
mono-DMT-glycerol phosphoramidite and di-DMT-glycerol
phosphoramidite were obtained from ChemGenes (Ashland, Mass.).
(4-Aminobutyl)-1,3-propanediol phosphoramidite was obtained from
Clontech (Palo Alto, Calif.). Arabinocytidine phosphoramidite,
arabinoguanosine, arabinothymidine and arabinouridine were obtained
from Reliable Pharmaceutical (St. Louis, Mo.). Arabinoguanosine
phosphoramidite, arabinothymidine phosphoramidite and
arabinouridine phosphoramidite were synthesized at Hybridon, Inc.
(Cambridge, Mass.) (Noronha et al. (2000) Biochem.,
39:7050-7062).
[0191] All nucleoside phosphoramidites were characterized by
.sup.31P and .sup.1H NMR spectra. Modified nucleosides were
incorporated at specific sites using normal coupling cycles. After
synthesis, oligonucleotides were deprotected using concentrated
ammonium hydroxide and purified by reverse phase HPLC, followed by
dialysis. Purified oligonucleotides as sodium salt form were
lyophilized prior to use. Purity was tested by CGE and MALDI-TOF
MS.
Example 2
Analysis of Spleen Cell Proliferation
[0192] In vitro analysis of splenocyte proliferation was carried
out using standard procedures as described previously (see, e.g.,
Zhao et al., Biochem Pharma 51:173-182 (1996)). The results are
shown in FIG. 8A. These results demonstrate that at the higher
concentrations, Immunomer 6, having two accessible 5' ends results
in greater splenocyte proliferation than does Immunomer 5, having
no accessible 5' end or Oligonucleotide 4, with a single accessible
5' end Immunomer 6 also causes greater splenocyte proliferation
than the LPS positive control.
Example 3
In Vivo Splenomegaly Assays
[0193] To test the applicability of the in vitro results to an in
vivo model, selected oligonucleotides were administered to mice and
the degree of splenomegaly was measured as an indicator of the
level of immunostimulatory activity. A single dose of 5 mg/kg was
administered to BALB/c mice (female, 4-6 weeks old, Harlan Sprague
Dawley Inc, Baltic, Conn.) intraperitoneally. The mice were
sacrificed 72 hours after oligonucleotide administration, and
spleens were harvested and weighed. The results are shown in FIG.
8B. These results demonstrate that Immunomer 6, having two
accessible 5' ends, has a far greater immunostimulatory effect than
do Oligonucleotide 4 or Immunomer 5.
Example 4
Cytokine Analysis
[0194] The secretion of IL-12 and IL-6 in vertebrate cells,
preferably BALB/c mouse spleen cells or human PBMC, was measured by
sandwich ELISA. The required reagents including cytokine antibodies
and cytokine standards were purchased form PharMingen, San Diego,
Calif. ELISA plates (Costar) were incubated with appropriate
antibodies at 5 .mu.g/mL in PBSN buffer (PBS/0.05% sodium azide, pH
9.6) overnight at 4.degree. C. and then blocked with PBS/1% BSA at
37.degree. C. for 30 minutes. Cell culture supernatants and
cytokine standards were appropriately diluted with PBS/10% FBS,
added to the plates in triplicate, and incubated at 25.degree. C.
for 2 hours. Plates were overlaid with 1 .mu.g/mL appropriate
biotinylated antibody and incubated at 25.degree. C. for 1.5 hours.
The plates were then washed extensively with PBS-T Buffer
(PBS/0.05% Tween 20) and further incubated at 25.degree. C. for 1.5
hours after adding streptavidin conjugated peroxidase (Sigma, St.
Louis, Mo.). The plates were developed with Sure Blue.TM.
(Kirkegaard and Perry) chromogenic reagent and the reaction was
terminated by adding Stop Solution (Kirkegaard and Perry). The
color change was measured on a Ceres 900 HDI Spectrophotometer
(Bio-Tek Instruments). The results are shown in Table 5A below.
[0195] Human peripheral blood mononuclear cells (PBMCs) were
isolated from peripheral blood of healthy volunteers by
Ficoll-Paque density gradient centrifugation (Histopaque-1077,
Sigma, St. Louis, Mo.). Briefly, heparinized blood was layered onto
the Histopaque-1077 (equal volume) in a conical centrifuge and
centrifuged at 400.times.g for 30 minutes at room temperature. The
buffy coat, containing the mononuclear cells, was removed carefully
and washed twice with isotonic phosphate buffered saline (PBS) by
centrifugation at 250.times.g for 10 minutes. The resulting cell
pellet was then resuspended in RPMI 1640 medium containing
L-glutamine (MediaTech, Inc., Herndon, Va.) and supplemented with
10% heat inactivated FCS and penicillin-streptomycin (100 U/ml).
Cells were cultured in 24 well plates for different time periods at
1.times.10.sup.6 cells/ml/well in the presence or absence of
oligonucleotides. At the end of the incubation period, supernatants
were harvested and stored frozen at -70.degree. C. until assayed
for various cytokines including IL-6 (BD Pharmingen, San Diego,
Calif.), IL-10 (BD Pharmingen), 1L-12 (BioSource International,
Camarillo, Calif.), IFN-.alpha. (BioSource International) and
-.gamma. (BD Pharmingen) and TNF-.alpha. (BD Pharmingen) by
sandwich ELISA. The results are shown in Tables 9 and 9A below.
[0196] In all instances, the levels of IL-12 and IL-6 in the cell
culture supernatants were calculated from the standard curve
constructed under the same experimental conditions for IL-12 and
IL-6, respectively. The levels of IL-10, IFN-gamma and TNF-.alpha.
in the cell culture supernatants were calculated from the standard
curve constructed under the same experimental conditions for IL-10,
IFN-gamma and TNF-.alpha., respectively.
TABLE-US-00009 TABLE 9 Immunomer Structure and Immunostimulatory
Activity in Human PBMC Cultures Oligo Oligo Length/ IL-12 (pg/mL)
IL-6 (pg/mL) No. Sequences and Modification (5'-3') or Each Chain
D1 D2 D1 D2 25 5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 184 332 3077
5369 26 ##STR00105## 11mer (PS) 237 352 3724 4892 Oligo Oligo
Length/ Il-10 (pg/mL) IFN-.gamma. (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain D1 D2 D1 D2 25
5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 37 88 125 84 26 ##STR00106##
11mer (PS) 48 139 251 40 Oligo Oligo Length/ TNF-.alpha. (pg/mL)
No. Sequences and Modification (5'-3') or Each Chain D1 D2 25
5'-CTATCTGTCGTTCTCTGT-3' 18mer (PS) 537 nt 26 ##STR00107## 11mer
(PS) 681 nt D1 and D2 are donors 1 and 2.
TABLE-US-00010 TABLE 9A Immunomer Structure and Immunostimulatory
Activity in BALB/c Mouse Spleen Cell Cultures Oligo Oligo Length/
IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and Modification (5'-3')
or Each Chain 3 .mu.g/mL 10 .mu.g/mL 26 ##STR00108## 11mer (PS) 870
10670 27 ##STR00109## 11mer (PS) 1441 7664 28 ##STR00110## 11mer
(PS) 1208 1021 29 ##STR00111## 11mer (PS) 162 1013 30 ##STR00112##
14mer (PO) 264 251 31 ##STR00113## 14mer (PO) 149 119 32
##STR00114## 11mer (PS) 2520 9699 33 ##STR00115## 11mer (PS) 2214
16881 34 ##STR00116## 11mer (PS) 3945 10766 35 ##STR00117## 11mer
(PS) 2573 19411 36 ##STR00118## 14mer (PO) 2699 408 37 ##STR00119##
14mer (PO) 839 85 38 ##STR00120## 14mer (PO) 143 160
Italic phase represents a phosphodiester linkage.
##STR00121##
[0197] In addition, the results shown in FIGS. 7A-C demonstrate
that Immunomer 2, with two accessible 5' ends elevates IL-12 and
IL-6, but not IL-10 at lower concentrations than Oligonucleotide 1
or Immunomer 3, with one or zero accessible 5' ends,
respectively.
Example 5
Immunostimulatory Activity of Immunomer Compounds Containing a
Non-Natural Pyrimidine or Non-Natural Purine Nucleoside
[0198] As shown in Tables 10-12, immunostimulatory activity was
maintained for immunomer compounds of various lengths having a
non-natural pyrimidine nucleoside or non-natural purine nucleoside
in the immunostimulatory dinucleotide motif.
TABLE-US-00011 TABLE 10 Immunomer Structure and Immunostimulatory
Acivity Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain @3 .mu.g/mL @3 .mu.g/mL 51
5'-CTCACTTTCGTTCTCTGT-3' 18mer 404 348 57 ##STR00122## 11mer 591
365 58 ##STR00123## 11mer 303 283 59 ##STR00124## 8mer 55 66 60
##STR00125## 8mer 242 143 ##STR00126## ##STR00127##
TABLE-US-00012 TABLE 11 Immonomer Structure and Immunostimulatory
Activity Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain 3 .mu.g/mL 3 .mu.g/mL 25
5'-CTATCTGTCGTTCTCTGT-3' 18mer 379 339 61 ##STR00128## 11mer 1127
470 62 ##STR00129## 11mer 787 296 63 ##STR00130## 8mer 64 126 64
##STR00131## 8mer 246 113 ##STR00132## ##STR00133##
TABLE-US-00013 TABLE 12 Immunomer Structure and Immunostimulatory
Activity Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain 3 .mu.g/mL 3 .mu.g/mL 4
5'-CTATCTGACGTTCTCTGT-3' 18mer 1176 1892 65 ##STR00134## 18mer 443
192 66 ##STR00135## 18mer 627 464 67 ##STR00136## 14mer 548 152 68
##STR00137## 14mer 1052 1020 69 ##STR00138## 11mer 2050 2724 70
##STR00139## 11mer 1780 1741 71 ##STR00140## 8mer 189 55 72
##STR00141## 8mer 308 212 ##STR00142## ##STR00143##
Example 6
Effect of the Linker on Immunostimulatory Activity
[0199] In order to examine the effect of the length of the linker
connecting the two oligonucleotides, immunomer compounds that
contained the same oligonucleotides, but different linkers were
synthesized and tested for immunostimulatory activity. The results
shown in Table 13 suggest that linker length plays a role in the
immunostimulatory activity of immunomer compounds. The best
immunostimulatory effect was achieved with C3- to C6-alkyl linkers
or abasic linkers having interspersed phosphate charges.
TABLE-US-00014 TABLE 13 Immunomer Structure and Immunostimulatory
Activity Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain 0.3 .mu.g/mL 1 .mu.g/mL 4
5'-CTATCTGACGTTCTCTGT-3' 18mer 257 635 73 ##STR00144## 10mer 697
1454 74 ##STR00145## 10mer 1162 669 75 ##STR00146## 10mer 1074 1375
76 ##STR00147## 10mer 563 705 77 ##STR00148## 10mer 264 543 78
##STR00149## 10mer 1750 2258 79 ##STR00150## 10mer 2255 2034 80
##STR00151## 10mer 1493 1197 81 ##STR00152## 10mer 3625 2642 82
##STR00153## 10mer 4248 2988 83 ##STR00154## 10mer 1241 1964
##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159##
##STR00160##
Example 7
Effect of Oligonucleotide Backbone on Immunostimulatory
Activity
[0200] In general, immunostimulatory oligonucleotides that contain
natural phosphodiester backbones are less immunostimulatory than
are the same length oligonucleotides with a phosphorothioate
backbones. This lower degree of immunostimulatory activity could be
due in part to the rapid degradation of phosphodiester
oligonucleotides under experimental conditions. Degradation of
oligonucleotides is primarily the result of 3'-exonucleases, which
digest the oligonucleotides from the 3' end. The immunomer
compounds of this example do not contain a free 3' end. Thus,
immunomer compounds with phosphodiester backbones should have a
longer half life under experimental conditions than the
corresponding monomeric oligonucleotides, and should therefore
exhibit improved immunostimulatory activity. The results presented
in Table 14 demonstrate this effect, with Immunomers 84 and 85
exhibiting immunostimulatory activity as determined by cytokine
induction in BALB/c mouse spleen cell cultures.
TABLE-US-00015 TABLE 14 Immunomer Structure and Immunostimulatory
Activity Oligo Length/ IL-12 (pg/mL) IL-6 (pg/mL) No. Sequences and
Modification (5'-3') or Each Chain 0.3 .mu.g/mL 1 .mu.g/mL 4
5'-CTATCTGACGTTCTCTGT-3' 18mer 225 1462 84 ##STR00161## 14mer 1551
159 85 ##STR00162## 14mer 466 467 L = C3-linker
Example 8
In Vivo Anti-Cancer Activity of Immunomer Compounds in Combination
with Chemotherapeutic Agents
[0201] PC3 cells were cultured in 90% Ham's, F12K Medium with 10%
Fetal Bovine Serum (FBS), in presence of 100 U/ml Penicillin and
100 .mu.g/ml Streptomycin to establish the Human Prostate cancer
model (PC3). Male athymic nude mice, 4-6 weeks old (Frederick
Cancer Research and Development Center, Frederick, Md.), were
accommodated for 6 days for environmental adjustment prior to the
study. Cultured PC3 cells were harvested from the monolayer
cultures, washed twice with Ham's, F12K Medium (10% FBS),
resuspended in FBS-free Ham's, F12K Medium: Matrigel basement
membrane matrix (Becton Dickinson Labware, Bedford, Mass.) (5:1;
V/V), and injected subcutaneously (5.times.10.sup.6 cells, total
volume 0.2 ml) into the left inguinal area of each of the mice. The
animals were monitored by general clinical observation, body
weight, and tumor growth. Tumor growth was monitored by the
measurement, with calipers, of two perpendicular diameters of the
implant. Tumor mass (weight in grams) was calculated by the
formula, 1/2a.times.b.sup.2, where `a` is the long diameter (cm)
and `b` is the short diameter (cm). When the mean tumor sizes
reached .about.80 mg, the animals bearing human cancer xenografts
were randomly divided into the treatment and control groups (5
animals/group). The control group received sterile physiological
saline (0.9% NaCl) only Immunomers 26 or 194, aseptically dissolved
in physiological saline, was administered by subcutaneously
injection at dose of 0.5 or 1.0 mg/kg/day, 3 doses/week.
Gemcitabine HCl (Eli Lilly and Company, Indianapolis, Ind.) was
given twice by intraperitoneal injection at 160 mg/kg on Day 0 and
3. The detailed treatment schedule is shown as follows. [0202] G1:
Saline [0203] G2: Gemcitabine (160 mg/kg/day, IP, Day 0 and 3)
[0204] G3: 26 (1.0 mg/kg/day, SC, 3 doses/week, for 6 weeks) [0205]
G4: 26 (0.5 mg/kg/day, SC, 3 doses/week, for 6 weeks) [0206] G5:
194 (1.0 mg/kg/day, SC, 3 doses/week, for 6 weeks) [0207] G6: 194
(0.5 mg/kg/day, SC, 3 doses/week, for 6 weeks) [0208] G7: 26 (0.5
mg/kg/day, SC, 3 doses/week, for 6 weeks)+Gemcitabine (160
mg/kg/day, Day 0 and 3) [0209] G8: 194 (0.5 mg/kg/day, SC, 3
doses/week, for 6 weeks)+Gemcitabine (160 mg/kg/day, Day 0 and
3)
[0210] The tumor measurements after various treatments are
presented in Table 15 and FIG. 13. The tumor growth in all
Immunomer 26 and 194 treated animals was remarkably inhibited
compared with saline control (p<0.5). There was a tendency of
dose-response relationship in these treatment groups (FIG. 13).
There was no significant difference between 26 and 194 (Table
15).
TABLE-US-00016 TABLE 15 Tumor mass of tumor-bearing mice following
treatment of 26, 194, Gemcitabine or combination therapy
Gemcitabine 26 26 Day Saline SD SE 160 mg/kg SD SE 1 mg/kg SD SE
0.5 mg/kg SD SE 0 82.7 16.7 7.5 82.6 15.7 7.0 80.1 10.6 4.7 80.4
10.5 4.7 3 81.9 13.3 5.9 73.0 3.4 1.5 67.5 8.1 3.6 54.3 8.4 3.7 6
80.5 11.5 5.2 50.4 11.7 5.2 50.4 9.0 4.0 45.3 5.5 2.5 9 87.7 8.2
3.7 35.7 6.3 2.8 40.9 5.1 2.3 43.9 9.3 4.2 12 97.6 18.6 8.3 36.2
3.3 1.5 41.3 6.2 2.8 46.5 3.8 1.7 15 112.0 21.5 9.6 31.7 4.1 1.8
42.8 12.8 5.7 50.0 14.1 6.3 18 126.3 17.3 7.7 40.8 8.4 3.7 54.9 7.6
3.4 59.3 6.7 3.0 21 152.5 25.5 11.4 47.4 9.8 4.4 62.5 10.4 4.6 71.0
16.7 7.5 24 187.0 29.2 13.1 56.5 5.2 2.3 79.5 24.1 10.8 100.1 9.7
4.3 27 245.2 24.1 10.8 68.0 14.8 6.6 94.1 28.9 12.9 124.5 21.1 9.5
30 343.6 63.9 28.6 89.4 11.1 5.0 119.8 18.7 8.3 162.4 37.5 16.8 33
438.5 107.1 47.9 106.5 14.1 6.3 176.6 43.8 19.6 213.6 66.7 29.8 36
614.4 185.1 82.8 144.2 48.2 21.6 248.7 47.0 21.0 325.3 106.2 47.5
39 866.8 237.4 106.2 175.3 61.4 27.5 320.1 64.2 28.7 416.8 154.5
69.1 42 1136.9 205.9 92.1 269.1 78.8 35.2 417.8 78.7 35.2 546.9
139.1 62.2 45 383.8 146.4 65.5 550.8 134.2 60.0 667.6 284.9 127.4
48 538.6 260.1 116.3 736.0 197.3 88.2 852.8 399.3 178.6 194 194 26
+ GEM 194 + GEM Day 1 mg/kg SD SE 0.5 mg/kg SD SE 0.5/160 mg/kg SD
SE 0.5/160 mg/kg SD SE 0 80.4 11.0 4.9 79.9 10.3 4.6 79.4 10.1 4.5
78.7 12.0 5.4 3 52.3 9.3 4.2 64.7 9.0 4.0 45.1 8.2 3.7 44.6 8.7 3.9
6 38.8 4.6 2.1 46.9 14.7 6.6 31.2 5.9 2.6 34.7 4.4 2.0 9 34.5 9.5
4.3 43.5 13.6 6.1 22.1 4.8 2.1 23.0 3.2 1.5 12 35.8 9.4 4.2 43.0
15.9 7.1 15.0 3.8 1.7 11.9 2.2 1.0 15 36.6 8.7 3.9 48.6 15.4 6.9
18.0 3.1 1.4 12.4 3.5 1.6 18 45.1 14.6 6.5 62.0 20.2 9.0 17.9 3.1
1.4 15.5 1.7 0.8 21 53.5 12.3 5.5 73.6 20.5 9.2 18.3 2.8 1.2 14.8
2.1 1.0 24 72.6 22.7 10.1 93.6 23.0 10.3 23.6 4.5 2.0 23.0 1.5 0.7
27 86.5 13.7 6.1 119.3 17.3 7.8 27.8 4.1 1.8 25.9 3.7 1.7 30 114.5
22.8 10.2 157.1 49.0 21.9 33.6 5.0 2.2 36.9 6.5 2.9 33 161.4 44.1
19.7 218.1 81.2 36.3 43.8 10.9 4.9 47.7 16.1 7.2 36 198.3 43.5 19.4
313.2 104.6 46.8 50.3 13.6 6.1 46.4 16.4 7.3 39 249.8 77.9 34.9
420.2 199.4 89.2 67.3 29.4 13.2 59.4 28.7 12.9 42 366.5 110.5 49.4
527.5 219.0 98.0 77.2 28.0 12.5 82.1 29.1 13.0 45 490.2 122.2 54.7
620.3 258.1 115.4 104.9 57.9 25.9 110.7 46.3 20.7 48 683.4 144.6
64.7 759.1 223.0 99.7 128.2 77.7 34.7 133.4 62.6 28.0 51 177.9
109.6 49.0 177.3 68.0 30.4 54 233.1 143.5 64.2 224.0 79.8 35.7 57
297.7 190.7 85.3 289.7 121.9 54.5
[0211] The body weight measurements after treatments at various
times are presented in Table 16 and FIG. 14. There was no
significant difference in body weight gains among 26 or 194 alone
compared with controls. Gemcitabine treated animals had body weight
loss in the first week and recovered in a week afterwards.
Combination with 26 or 194 did not change the side effect profiles
of Gemcitabine. No other clinical abnormality or death was observed
in all the groups.
TABLE-US-00017 TABLE 16 Body weights of tumor-bearing mice
following treatment of 26, 194 or saline. Gemcitabine 26 26 Day
Saline SD SE 160 mg/kg SD SE 1 mg/kg SD SE 0.5 mg/kg SD SE 0 24.1
2.5 1.1 23.5 0.9 0.4 23.2 1.4 0.6 23.0 2.4 1.1 7 25.8 3.0 1.3 20.7
4.4 2.0 25.2 2.4 1.1 24.8 2.8 1.2 14 26.8 3.2 1.4 25.2 4.0 1.8 26.3
2.0 0.9 26.0 2.9 1.3 21 28.2 3.3 1.5 27.1 3.9 1.7 27.8 2.0 0.9 27.6
2.8 1.2 28 29.4 3.5 1.6 28.1 4.3 1.9 28.6 2.6 1.1 28.0 2.7 1.2 35
30.6 3.7 1.6 29.4 2.9 1.3 29.5 2.3 1.0 28.6 2.8 1.3 42 31.1 3.7 1.7
30.3 3.0 1.4 30.2 2.3 1.0 29.4 3.9 1.7 194 194 26 + GEM 194 + GEM
Day 1 mg/kg SD SE 0.5 mg/kg SD SE 0.5/160 mg/kg SD SE 0.5/160 mg/kg
SD SE 0 22.5 1.3 0.6 24.1 1.6 0.7 21.9 1.7 0.7 23.0 0.8 0.4 7 24.3
0.9 0.4 25.6 2.0 0.9 19.1 2.0 0.9 22.3 3.3 1.5 14 25.1 1.3 0.6 27.0
2.1 0.9 24.6 1.6 0.7 25.9 2.7 1.2 21 26.1 1.3 0.6 27.8 1.5 0.7 26.8
1.6 0.7 27.1 2.6 1.2 28 27.2 1.5 0.7 28.3 2.2 1.0 27.2 1.6 0.7 27.7
3.2 1.4 35 28.0 1.4 0.6 29.1 2.3 1.0 27.7 2.1 1.0 28.0 2.4 1.1 42
28.9 1.5 0.7 29.8 2.2 1.0 28.4 2.8 1.2 28.1 3.4 1.5
[0212] In summary, 26 and 194 significantly inhibited tumor growth
in nude mice bearing human prostate cancer PC3 xenografts with no
significant side effects. When 26 or 194 was given in combination
with Gemcitabine, each compound significantly increased the
therapeutic effect of Gemcitabine without changes in side effect
profiles. In addition, there was a tendency in dose dependent
response of 26 or 194 treatment.
Example 9
In Vivo Anti-Cancer Activity of Immunomer Compounds in Combination
with Chemotherapeutic Agents
[0213] The experiment of Example 8 was repeated using taxotere
instead of Gemcitabine. Taxotere was administered on days 0 and 7.
165 was administered 5 days per week. 26 and 194 were administered
on days 0, 2, 4, 7, 9 and 11. The results are shown in Table 17
below. These results clearly demonstrate synergy between the
immunomer compounds and taxotere.
TABLE-US-00018 TABLE 17 In vivo anti-cancer activity of immunomer
compounds in combination with other chemotherapeutic agents
Taxotere 165 26 Day Saline SD SE (15 mg/kg) SD SE (20 mg/kg) SD SE
(1 mg/kg) SD SE 0.00 56.93 7.92 3.54 56.64 7.94 3.55 57.93 5.56
2.49 56.74 7.79 3.48 3.00 196.42 22.48 10.05 128.51 20.83 9.32
95.79 16.04 7.18 87.12 6.64 2.97 6.00 708.85 32.64 14.60 320.63
136.80 61.18 285.71 68.70 30.72 250.36 52.58 23.51 9.00 1370.95
239.99 107.33 598.69 196.60 87.92 534.93 225.19 100.71 450.46 92.25
41.26 12.00 2222.96 300.65 134.45 924.91 297.89 133.22 994.10
474.89 212.38 814.21 197.16 88.17 15.00 3303.04 672.86 300.91
1589.08 578.38 258.66 1601.73 576.19 257.68 1465.87 348.37 155.80
Taxotere + Taxotere + 194 Day 165 SD SE 26 (** mg/kg) SD SE (1
mg/kg) SD SE 0.00 55.51 9.55 4.27 56.59 8.91 3.99 55.28 10.89 4.87
3.00 78.47 21.79 9.74 80.14 21.59 9.65 91.01 23.60 10.55 6.00
211.52 88.59 39.62 216.85 89.40 39.98 303.00 61.33 27.43 9.00
302.66 178.36 79.76 307.53 184.05 82.31 512.30 110.16 49.26 12.00
496.20 342.69 153.25 510.18 351.16 157.04 884.12 308.22 137.84
15.00 686.47 385.97 172.61 703.50 394.65 176.49 1479.21 416.64
186.33
Example 10
Administration of Immunostimulatory Oligonucleotides and IL-2
[0214] Splenocytes were isolated from BALB/c mice as described
above and were plated in 24-well dishes at a density of
5.times.10.sup.6 cells/mL. CpG oligonucleotides were dissolved in
TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA) was added to a final
concentration of 0.03, 0.1, 0.3, 1.0, 3.0, or 10.0 .mu.g/mL to
mouse spleen cell cultures. In order to study the role of IL-2 in
CpG oligonucleotide-induced time-dependent cytokine secretion,
recombinant human IL-2 (Sigma) was added at a concentration of 10
U/ml at the start of the experiment. The cells were then incubated
at 37.degree. C. for 4, 8, 24 and 48 h in the presence of test
oligonucleotides and the supernatants were collected for ELISA
assays. Untreated cells (only IL-2 addition) were taken as
controls.
[0215] The secretion of mouse IL-12, IL-6 and IFN-.gamma. was
measured by sandwich ELISA. The required regents, including
cytokine antibodies and standards were purchases from PharMingen.
ELISA plates (Costar) were incubated with appropriate capture
antibodies in PBSN (PBS/0.05% sodium azide, pH 9.6) buffer
overnight at 4.degree. C. and then blocked with PBS/1% BSA at
37.degree. C. for 30 min. Cell culture supernatants and cytokine
standards were appropriately diluted with PBS/1% BSA, added to the
plates in triplicate, and incubated at 25.degree. C. for 2 h.
Plates were washed and incubated with the appropriate biotinylated
antibody and incubated at 25.degree. C. for 1.5 h. The plates were
washed extensively with PBS/0.05% Tween 20 and then further
incubated at 25.degree. C. for 1.5 h. after addition of
streptavidine-conjugated peroxidase (Sigma). Plates were developed
with Sure Blue.TM. (Kirkegaard and Perry) chromogenic reagent and
the reaction was terminated by adding Stop Solution (Kirkegaard and
Perry). The color change was measured on a Ceres 900 HDI
Spectrophotometer (Bio-Tek Instruments) at 450 nm. The levels of
IL-12, IL6 and IFN-.gamma. in the cell culture supernatants were
calculated from the standard curve constructed under the same
experimental conditions for IL-12, IL-6 and IFN-.gamma.
respectively.
[0216] The oligonucleotides used in this study are presented in
Table 18.
TABLE-US-00019 TABLE 18 SEQ ID NO: Sequence Chemistry 86
5'-CTATCTGACGTTCTCTGT-3' PS-oligo 87 (5'-TCTGACRTTCT).sub.2S R =
7-deaza-dG, PS-oligo 88 (5'-TCTGACGTTCT).sub.2S PS-oligo 89
(5'-XXCTGACGTTCTCTGT).sub.2S PO-oligo 90 (5'-TCTGAYGTTCT).sub.2S Y
= R*, PS-oligo
The results are shown in FIGS. 15-19. Not shown is an assay
indicating that the use of SEQ ID NOs 86-90 alone stimulate
IFN-.gamma. production only negligibly. The results demonstrate
synergy between SEQ ID NOs 86-90 and IL-2 in generating secretion
of IL-6, IL-12 and IFN-.gamma..
Equivalents
[0217] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made
without departing from the true scope of the invention and appended
claims.
Sequence CWU 1
1
165116DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gagaacgctc gacctt 16216DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2gagaacgctc gacctt 16316DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3gagaacgctc gacctt 16418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4ctatctgacg ttctctgt 18513DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5tgacgttctc tgt 13613DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6tgacgttctc tgt 13718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7ctatctgacg ttctctgt 18818DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8ctatctgacg ttctctgt 18918DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9ctatctgang ttctctgt 181018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10ctatctgacn ttctctgt 181114DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11ctgangttct ctgt 141214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 12ctgacnttct ctgt 141314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13ctgacgttct ctgt 141414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 14ctgacgttct ctgt 141514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 15ctgangttct ctgt 141614DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 16ctgacnttct ctgt 141715DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 17nntgacgttc tctgt 151816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18nnntgacgtt ctctgt 161916DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 19nnntgangtt ctctgt 162016DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 20nnntgacntt ctctgt 162111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21tctgacgttc t 112214DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 22nnntctgacg ttct 142314DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 23nnntctgang ttct 142414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24nnntctgacn ttct 142518DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 25ctatctgtcg ttctctgt 182611DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 26tctgtcnttc t 112711DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 27tctgtcnttc t 112811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 28tctgtnnttc t 112913DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 29nntctgtcnt tct 133014DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 30ctgtcnttct ctgt 143114DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 31ctgtnnttct ctgt 143211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 32tctgacnttc t 113313DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 33nntctgacnt tct 133411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34tctgacnttc t 113511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35tctgannttc t 113614DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36ctgangttct ctgt 143714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 37ctgacnttct ctgt 143814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 38ctgannttct ctgt 14394DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 39ctat 4403DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 40cta
34111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 41ctgtcnttct c 11429DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 42tcntcnttg 94311DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 43tcntcnttct g
11446DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 44tcgttg 6455DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 45tcgtt 5466DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 46agagag
6476DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 47agagag 64811DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 48tctgtcnttc t 114910DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 49cagagctctg 105010RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 50cagagcucug 105118DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 51ctcactttcg ttctctgt 18528DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 52ttgtgctt 85312DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 53ggcatcgatg cc
12546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 54gagctc 65514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55gacagagctc tgtc 14568DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56tcgtcgtt 85711DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 57tctttngttc t
115811DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 58tctttcnttc t 11598DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59ttngttct 8608DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 60ttcnttct
86111DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 61tctgtngttc t 116211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 62tctgtcnttc t 11638DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 63gtngttct 8648DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 64gtcnttct
86518DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 65ctatctgang ttctctgt 186618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 66ctatctgacn ttctctgt 186714DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 67ctgangttct ctgt 146814DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 68ctgacnttct ctgt 146911DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 69tctgangttc t 117011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 70tctgacnttc t 11718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 71gangttct 8728DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 72gacnttct
87310DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 73ctgacgttct 107420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 74gctgacagag ctctgtcagc 207510RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 75cagagcucug 10769DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 76tcgtcgttg
97712DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 77tgcatcgatg ca 127812DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 78acgtagctac gt 127912DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 79tgcatcgatg ca 128012DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 80acgtagctac gt 12819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 81tcgtcgttg 9828DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 82tcgtcgtt
88310DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 83cagagctctg 108414DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 84ctgacgttct ctgt 148514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 85ctgacgttct ctgt 148618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 86ctatctgacg ttctctgt 188722DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 87tctgacnttc ttctgacntt ct 228822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 88tctgacgttc ttctgacgtt ct 228928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 89ctgacgttct ctgtctgacg ttctctgt
289022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 90tctgangttc ttctgangtt ct
229117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 91ctgtctgacg ttctctg 179225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 92ctgtctgacg ttctctggaa cagag 259331DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 93ctgtctgacg ttctctggaa cagagaacgt c
319437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 94ctgtctgacg ttctctggaa cagagaacgt
cagacag 379525DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 95gacaggaact gtctgacgtt ctctg
259632DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 96aacgtcagac aggaactgtc tgacgttctc tg
329737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 97cagagaacgt cagacaggaa ctgtctgacg
ttctctg 379818DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 98ctatctgacg ttctctgt
189926DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 99ctatctgacg ttctctgtgt gatcac
2610026DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 100gtgatcacct atctgacgtt ctctgt
2610117DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 101ctgtctgtcg ttctctg
1710225DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 102ctgtctgtcg ttctctggaa cagag
2510331DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 103ctgtctgtcg ttctctggaa cagagaacga c
3110437DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 104ctgtctgtcg ttctctggaa cagagaacga
cagacag 3710525DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 105gacaggaact gtctgtcgtt ctctg
2510632DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 106aacgacagac aggaactgtc tgacgttctc tg
3210737DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 107cagagaacga cagacaggaa ctgtctgtcg
ttctctg 3710825DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 108tcgtcgttga
gctctgaaag agctc 2510933DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 109tcgtcgttgt
gagctctgtg aaacagagct cac 3311041DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 110tcgtcgttgc
acagagctct gctgaaagca gagctctgtg c 4111149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 111tcgtcgttgc tgacagagct ctgctatgaa atagcagagc
tctgtcagc 4911225DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 112tcgtcgttgt gctctgaact tgctc
2511333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 113tcgtcgttgt gtgctctgtg aacatcagtc tac
3311425DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 114tcgtcgttga gctctgaaag agctc
2511533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 115tcgtcgttgt gagctctgtg aaacagagct cac
3311625DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 116tcgtcgttga gctctgaaag agctc
2511733DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 117tcgtcgttgt gagctctgtg aaacagagct cac
3311825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 118tcntcnttga gctctgaaag agctc
2511925DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 119tcntcnttga gctctgaaag agctc
2512025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 120tgctgcttga gctctgaaag agctc
2512116DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 121tcttgacgtt ctctct 1612225DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 122tcttgacgtt ctctctgaaa gagag 2512325DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 123tcttgacgtt ctctctgaaa gagag 2512425DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 124tcttgacgtt ctctctgaaa gagag 2512525DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 125tcttgacgtt ctctctgaaa gagag 2512625DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 126tcttgacgtt ctctctgaaa gagag 2512716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 127tcttgacgtt ctctct 1612816DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 128tcttgacgtt ctctct 161298DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 129tcgtcgtt 813021DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 130tcgtcgttgt
gcatcgatgc a 2113121DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 131tcgtcgttgt gcatcgatgc a
2113225DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 132tcgtcgttga gcucugaaag agcuc
2513329DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 133tcgtcgttga gcucucugaa
agagagcuc 2913433DNAArtificial SequenceDescription of Combined
DNA/RNA Molecule Synthetic oligonucleotide 134tcgtcgttga gcucucugug
aaacagagag cuc 3313533DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 135tcntcnttgt
gagctctgtg aaacagagct cac 331368DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 136tcntcntt
813725DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 137gtgagctctg tgaaacagag ctcac
2513833DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 138tcntcnttgt gagctctgtg
aaacagagcu cac 3313910DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 139cagagctctg
1014010RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 140cagagcucug 101418DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 141tcntcntt 814210RNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 142cagagcucug
1014310DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 143ctcacctctg 1014410RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 144cucaccucug 1014533DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 145tcntcnttgt gagctctgtg aaacagagcu cac
3314625RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 146gugagcucug ugaaacagag cucac
2514734DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 147tcgtcgttgt gagctctgtg
gaaacagagc ucac 3414811DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 148tcnaacnttc n
1114911DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 149tcnaacnttc g 1115011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 150tctcaccttc t 1115111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 151tcnaacnttc n 1115211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 152tcnaacnttc g 1115321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 153tcntcnaacn ttcnagatga t 2115421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 154tcntcnaacn ttcnagatga t 2115521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 155tcntcnaacn ttcnagatga t 2115621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 156tngtngaang ttngagatga t 2115721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 157tngtngaang ttngagatga t 2115821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 158tngtngaang ttngagatga t 2115910DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 159tcnaacnttc 1016011DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 160tcnttcnaac n 1116111DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 161tccaaccttc g 1116211DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 162tcnttncaac n 1116311DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 163tcnaacnttc t 1116411DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 164tcnaacnttc n 1116511DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 165tcnaangttc n 11
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